U.S. patent application number 17/583026 was filed with the patent office on 2022-05-19 for manufacturing of glycoproteins.
The applicant listed for this patent is Alexion Pharmaceuticals, Inc.. Invention is credited to Meghan DEWITT, Rahul GODAWAT, Saravanamoorthy RAJENDRAN, Siguang SUI.
Application Number | 20220154155 17/583026 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-19 |
United States Patent
Application |
20220154155 |
Kind Code |
A1 |
GODAWAT; Rahul ; et
al. |
May 19, 2022 |
MANUFACTURING OF GLYCOPROTEINS
Abstract
A method of producing recombinant alkaline phosphatase
comprising control of production parameters, particularly harvest
clarified culture fluid (HCCF) and filtration pool (UFDF), to
provide a defined total sialic acid content.
Inventors: |
GODAWAT; Rahul; (Woodbridge,
CT) ; DEWITT; Meghan; (Madison, CT) ; SUI;
Siguang; (South Glastonbury, CT) ; RAJENDRAN;
Saravanamoorthy; (Long Valley, NJ) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Alexion Pharmaceuticals, Inc. |
Boston |
MA |
US |
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Appl. No.: |
17/583026 |
Filed: |
January 24, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17043464 |
Sep 29, 2020 |
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PCT/US2019/022102 |
Mar 13, 2019 |
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17583026 |
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62650583 |
Mar 30, 2018 |
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International
Class: |
C12N 9/16 20060101
C12N009/16; C07K 1/22 20060101 C07K001/22; C12N 5/071 20060101
C12N005/071; C12P 21/00 20060101 C12P021/00 |
Claims
1. A method of producing recombinant alkaline phosphatase
comprising: (A) (a) inoculating Chinese Hamster Ovary (CHO) cells
expressing recombinant alkaline phosphatase in culture medium; (b)
culturing the CHO cells in the culture medium; (c) isolating the
recombinant alkaline phosphatase from the cell medium by at least
one purification step to form harvest clarified culture fluid
(HCCF) with a total sialic acid content (TSAC) of from about 2.1
mol/mol to about 4.3 mol/mol; (d) performing at least one
additional protein purification step to form a filtration pool
(UFDF), wherein the UFDF is held at a temperature of from about
13.degree. C. to about 27.degree. C. for from about 1 hour to about
60 hours, and at a protein concentration of from about 1.7 g/L to
about 5.3 g/L; and (e) subjecting the UFDF to at least one
chromatography step to obtain partially purified recombinant
alkaline phosphatase, wherein the recombinant alkaline phosphatase
has a TSAC of about 0.7 mol/mol to about 3.5 mol/mol; or (B) (a)
inoculating CHO cells expressing recombinant alkaline phosphatase
in culture medium; (b) culturing the CHO cells in the culture
medium; (c) adding a nutrient supplement(s) to the cell medium; (d)
isolating the recombinant alkaline phosphatase from the cell medium
by at least one purification step to form a filtration pool (UFDF);
and (C) recovering the recombinant alkaline phosphatase from the
UFDF, wherein the recombinant alkaline phosphatase in the UFDF has
a total sialic acid content (TSAC) of from about 2.1 mol/mol to
about 4.3 mol/mol.
2. The method of claim 1, wherein sialidase is selectively removed
from the cell culture, the HCCF, and/or the UFDF and/or an
exogenous sialyltransferase is added to the cell culture, the HCCF,
and/or the UFDF.
3. The method of claim 1, wherein: (i) the culturing of the CHO
cells in the culture medium is at a temperature of from about
36.degree. C. to about 38.degree. C.; (ii) the nutrient
supplement(s) is added to the cell culture at least one day after
inoculation of CHO cells into the culture medium; (iii) the
nutrient supplement(s) is added at more than 2 different times;
(iv) the culture medium is selected from the group consisting of
EX-CELL.RTM. 302 Serum-Free Medium; CD DG44 Medium; BD Select.TM.
Medium; SFM4CHO Medium; and combinations thereof; (v) the culturing
of the CHO cells is in a 0.25 L to a 25,000 L bioreactor; (vi) the
temperature of the cell culture is decreased from about 80 hours to
about 120 hours after the inoculation; (vii) step (d) of (A) or (B)
occurs about 10 to about 14 days after inoculation; (viii) the TSAC
of the HCCF is from about 2.2 mol/mol to about 3.6 mol/mol; and/or
(ix) the at least one additional purification step comprises at
least one of harvest clarification, filtration, ultrafiltration,
diafiltration, viral inactivation, affinity capture, and
combinations thereof.
4. The method of claim 3, wherein: (a) the culturing of the CHO
cells in the culture medium is at a temperature of about 37.degree.
C.; (b) the culturing of the CHO cells is in a 100 L to 25,000 L
bioreactor; (c) the TSAC of the HCCF is from about 2.2 mol/mol to
about 3.4 mol/mol; and/or (d) the at least one additional
purification step comprises ultrafiltration and/or
diafiltration.
5. The method of claim 4, wherein the culturing of the CHO cells is
in a 2000 L to 20,000 L bioreactor.
6. The method of claim 1, wherein: (i) the UFDF is held at a
temperature of about 14.degree. C. to about 26.degree. C.; (ii) the
UFDF is held for about 10 hours to about 50 hours; (iii) the UFDF
has a protein concentration from about 2.0 g/L to about 4.3 g/L;
(iv) the UFDF has an alkaline phosphatase concentration from about
3.0 g/L to about 4.5 g/L; and/or (v) the at least one additional
chromatography step and/or the at least one additional protein
purification step is performed to obtain recombinant alkaline
phosphatase with a TSAC of about 0.9 mol/mol to about 3.9
mol/mol.
7. The method of claim 6, wherein: (a) the UFDF is held at a
temperature of about 15.degree. C. to about 26.degree. C.; (b) the
UFDF is held for about 12 hours to about 48 hours; (c) the UFDF has
a protein concentration of about 3.1 g/L; (d) the UFDF has an
alkaline phosphatase concentration from about 3.3 g/L to about 4.1
g/L; and/or (e) the at least one additional chromatography step
and/or the at least one additional protein purification step is
performed to obtain recombinant alkaline phosphatase with a TSAC of
about 1.1 mol/mol to about 3.2 mol/mol.
8. The method of claim 7, wherein: (i) the UFDF is held at a
temperature of about 15.degree. C. to about 25.degree. C.; (ii) the
UFDF is held for about 14 hours to about 42 hours; (iii) the at
least one additional chromatography step and/or the at least one
additional protein purification step is performed to obtain
recombinant alkaline phosphatase with a TSAC of about 1.4 mol/mol
to about 2.6 mol/mol.
9. The method of claim 8, wherein: (a) the UFDF is held at a
temperature of about 19.degree. C. to about 25.degree. C.; (b) the
UFDF is held for about 17 hours to about 34 hours; (c) the at least
one additional chromatography step and/or the at least one
additional protein purification step is performed to obtain
recombinant alkaline phosphatase with a TSAC of about 1.2 mol/mol
to about 3.0 mol/mol.
10. The method of claim 9, wherein: (i) the UFDF is held for about
19 hours to about 33 hours; and/or (ii) the TSAC of the recombinant
alkaline phosphatase in the UFDF is from about 2.2 mol/mol to about
3.6 mol/mol.
11. The method of claim 10, wherein: (a) the UFDF is held for about
25 hours to about 38 hours; and/or (b) the TSAC of the recombinant
alkaline phosphatase in the UFDF is from about 2.2 mol/mol to about
3.4 mol/mol.
12. The method of claim 11, wherein the UFDF is held for about 29
hours to about 35 hours.
13. The method claim 1, wherein: (i) the at least one
chromatography step is protein affinity chromatography; (ii) the at
least one chromatography step is Protein A chromatography; (iii)
the at least one chromatography step comprises column
chromatography; (iv) the at least one chromatography step comprises
hydrophobic interaction chromatography; (v) the at least one
additional protein purification step of (A) comprises an additional
diafiltration; (vi) the at least one chromatography step and/or the
at least one additional protein purification step comprises
hydrophobic interaction chromatography and/or at least one
additional diafiltration step; (vii) step (d) of (A) further
comprises a viral inactivation step; and/or (viii) step (e) of (A)
or (B) further comprises at least one additional chromatography
and/or purification step.
14. The method of claim 1, wherein the recombinant alkaline
phosphatase comprises the structure of W-sALP--X-Fc-Y-Dn-Z, wherein
W is absent or is an amino acid sequence of at least one amino
acid; X is absent or is an amino acid sequence of at least one
amino acid; Y is absent or is an amino acid sequence of at least
one amino acid; Z is absent or is an amino acid sequence of at
least one amino acid; F c is a fragment crystallizable region; Dn
is a poly-aspartate, poly-glutamate, or combination thereof,
wherein n=10 or 16; and sALP is a soluble alkaline phosphatase.
15. The method of claim 14, wherein the recombinant alkaline
phosphatase comprises asfotase alfa (SEQ ID NO: 1).
16. The method of claim 13, wherein the recombinant alkaline
phosphatase obtained from the protein affinity chromatography is
stored at from about 2.degree. C. to about 8.degree. C.
17. The method of claim 1, wherein (A) further comprises: (i)
adding a nutrient supplement to the cell medium culture of (b)
after inoculation; and/or (ii) measuring recombinant alkaline
phosphatase activity.
18. A recombinant alkaline phosphatase produced in a mammalian cell
culture, wherein the recombinant alkaline phosphatase in a harvest
clarified culture fluid (HCCF) produced from the cell culture has a
total sialic acid content (TSAC) always greater than or equal to
about 1.2 mol/mol, and wherein the mammalian cell culture is about
100 L to about 25,000 L.
19. A filtration pool (UFDF) comprising recombinant alkaline
phosphatase, wherein the UFDF is produced from about 100 L to about
25,000 L cell culture, and wherein the UFDF is held at from about
19.degree. C. to about 25.degree. C., for from about 14 to about 42
hours, at a protein concentration of from about 2.0 to about 4.3
g/L.
20. A composition comprising a recombinant alkaline phosphatase
produced by the method of claim 1, wherein, optionally, the
composition further comprises at least one pharmaceutically
acceptable carrier, diluent, or excipient or a combination
thereof.
21. A method comprising administering the composition of claim 20
to a subject to increase cleavage of inorganic pyrophosphate (PPi)
or to treat a condition associated with an alkaline phosphatase
deficiency in the subject.
22. A method of controlling glycosidase activity or total sialic
acid content (TSAC) in a sialic acid-containing recombinant
protein, comprising culturing the recombinant protein in mammalian
cell culture and conducting at least one purification step to
provide a UFDF, wherein the temperature, protein concentration
and/or hold time is controlled in the UFDF.
Description
FIELD OF THE DISCLOSURE
[0001] In general, this disclosure relates to methods of
manufacturing recombinant polypeptides and recombinant
glycoproteins i.e., alkaline phosphatase, in particular to methods
of manufacturing recombinant fusion proteins, including but not
limited to asfotase alfa.
[0002] There is a need in the art to develop effective therapeutic
molecules and methods for treating diseases, particularly diseases
having skeletal manifestations, such as hypophosphatasia. For
recombinant polypeptides and recombinant glycoproteins, such as
asfotase alfa, control of the physical parameters of the product is
particularly important.
[0003] A method of producing recombinant alkaline phosphatase
comprising: (a) inoculating Chinese Hamster Ovary (CHO) cells
expressing recombinant alkaline phosphatase in culture medium, (b)
culturing the CHO cells in culture medium, (c) adding nutrient
supplements to the cell culture of (b) at least one day after
inoculation, (d) isolating the recombinant alkaline phosphatase
from the cell culture of (c) by at least one purification step to
form harvest clarified culture fluid (HCCF) with a total sialic
acid content (TSAC) of about 1.5 mol/mol to about 4.0 mol/mol, (e)
performing at least one additional protein purification step to
form a filtration pool (UFDF), wherein the UFDF is held at a
temperature of from about 13.degree. C. to about 27.degree. C. for
from about 1 hour to about 60 hours, and at a protein concentration
of from about 1.7 g/L to about 5.3 g/L; and (f) subjecting the UFDF
to at least one chromatography step to obtain partially purified
recombinant alkaline phosphatase, wherein the recombinant alkaline
phosphatase has a TSAC of about 0.9 mol/mol to about 3.0
mol/mol.
BACKGROUND
[0004] Hypophosphatasia (HPP) is a life-threatening, genetic, and
ultra-rare metabolic disorder that results in a failure to produce
functional tissue nonspecific alkaline phosphatase (TNSALP). It
leads to the accumulation of unmineralized bone matrix (e.g.
rickets, osteomalacia), characterized by hypo-mineralization of
bones and teeth. When growing bone does not mineralize properly,
impairment of growth disfigures joints and bones. This result in
turn impacts motor performance, respiratory function, and may even
lead to death. Different forms of HPP include perinatal, infantile,
juvenile (or childhood), and adult HPP. Recently, six clinical
forms have been defined, most based upon age at symptom onset,
including perinatal, benign prenatal, infantile, juvenile, adult,
and odonto-HPP. Asfotase alfa is an approved, first-in-class
targeted enzyme replacement therapy designed to address defective
endogenous TNSALP levels. For the first reports of treating HPP
with TNSALP, see Whyte et al., 2012 N Engl J Med. 366:904-13.
[0005] Asfotase alfa (STRENSIQ.RTM., Alexion Pharmaceuticals, Inc.)
is a soluble fusion glycoprotein comprised of the catalytic domain
of human TNSALP, a human immunoglobulin G1 Fc domain, and a
deca-aspartate peptide (i.e., D.sub.10) used as a bone-targeting
domain. In vitro, asfotase alfa binds with a greater affinity to
hydroxyapatite than does soluble TNSALP lacking the deca-aspartate
peptide, thus allowing the TNSALP moiety of asfotase alfa to
efficiently degrade excess local inorganic pyrophosphate (PPi) and
restore normal mineralization to bones. Pyrophosphate hydrolysis
promotes bone mineralization, and its effects were similar among
the species evaluated in nonclinical studies.
[0006] Production of commercial scale quantities of therapeutically
effective alkaline phosphatases (e.g., asfotase alfa) requires a
complicated, sensitive, and multi-step process. The present
disclosure provides for a method of manufacturing glycoproteins,
including alkaline phosphatases such as asfotase alfa, with
improved control of the final protein product characteristics.
BRIEF SUMMARY
[0007] Disclosed herein are improved manufacturing processes that
can be used to increase efficiency in the production of alkaline
phosphatases (e.g., asfotase alfa). Methods as described here can
also be used for maintaining, preserving, modulating and/or
improving the enzymatic activity of a recombinant protein, such as
alkaline phosphatases (e.g., asfotase alfa) produced by cultured
mammalian cells, particularly by cultured Chinese Hamster Ovary
(CHO) cells. Such alkaline phosphatases (e.g., asfotase alfa) are
suited for use in therapy, for example, for treatment of conditions
associated with decreased alkaline phosphatase protein levels
and/or function (e.g., insufficient cleavage of inorganic
pyrophosphate (PPi), HPP, etc.) in a subject, for example, a human
subject.
[0008] In one aspect, the present disclosure provides a method for
producing a recombinant polypeptide having alkaline phosphatase
function. In various embodiments, the alkaline phosphatase function
may include any functions of alkaline phosphatase known in the art,
such as enzymatic activity toward natural substrates including
phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and
pyridoxal 5'-phosphate (PLP). Such recombinant polypeptide can
comprise asfotase alpha (SEQ ID NO: 1).
[0009] In some embodiments, the disclosure is directed to a method
of producing recombinant alkaline phosphatase comprising: (a)
inoculating Chinese Hamster Ovary (CHO) cells expressing
recombinant alkaline phosphatase in culture medium; (b) culturing
the CHO cells in culture medium; (c) isolating the recombinant
alkaline phosphatase from the cell culture of (c) by at least one
purification step to form harvest clarified culture fluid (HCCF)
with a total sialic acid content (TSAC) of about 2.1 mol/mol to
about 4.3 mol/mol; (d) performing at least one additional protein
purification step to form a filtration pool (UFDF), wherein the
UFDF is held at a temperature of from about 13.degree. C. to about
27.degree. C. for from about 1 hour to about 60 hours, and at a
protein concentration of from about 1.7 g/L to about 5.3 g/L; and
(e) subjecting the UFDF to at least one chromatography step to
obtain partially purified recombinant alkaline phosphatase, wherein
the recombinant alkaline phosphatase has a TSAC of about 0.7
mol/mol to about 3.5 mol/mol.
[0010] In some embodiments, the disclosure is directed to a method
further comprising adding nutrient supplements to the cell culture
of (b) after inoculation.
[0011] In some embodiments, the disclosure provides a method of
producing recombinant alkaline phosphatase comprising: (a)
inoculating Chinese Hamster Ovary (CHO) cells expressing
recombinant alkaline phosphatase in culture medium; (b) culturing
the CHO cells in culture medium; (c) adding nutrient supplements to
the cell culture of (b) at least one day after inoculation; (d)
isolating the recombinant alkaline phosphatase from the cell
culture of (c) by at least one purification step to form a
filtration pool; and (e) recovering the recombinant alkaline
phosphatase from the filtration pool, wherein the recombinant
alkaline phosphatase in the filtration pool has a total sialic acid
content (TSAC) of from about 2.1 to about 4.3 mol/mol when
subjected to the recovery.
[0012] In some embodiments, the recombinant alkaline phosphatase in
the filtration pool has a TSAC of from about 2.2 mol/mol to about
3.6 mol/mol when subjected to the recovery. In some embodiments,
the recombinant alkaline phosphatase in the filtration pool has a
TSAC of from about 2.2 mol/mol to about 3.4 mol/mol when subjected
to the recovery.
[0013] In some embodiments, the sialidase is selectively removed
from the cell culture, the HCCF, and/or the UFDF. In some
embodiments, an exogenous sialyltransferase is added to the cell
culture, the HCCF, and/or the UFDF.
[0014] In some embodiments, the culturing of the CHO cells in the
culture media is at a temperature of from about 36.degree. C. to
about 38.degree. C. In some embodiments, the culturing of the CHO
cells in the culture media is at a temperature of about 37.degree.
C. In some embodiments, the nutrient supplements are added to the
cell culture at least one day after inoculation of CHO cells into
the culture medium. In some embodiments, the nutrient supplements
are added at more than 2 different times. In some embodiments, the
culture medium is selected from the group consisting of
EX-CELL.RTM. 302 Serum-Free Medium; CD DG44 Medium; BD Select.TM.
Medium; SFM4CHO Medium; and combinations thereof.
[0015] In some embodiments, the culturing of the CHO cells is in a
0.25 L to 25,000 L bioreactor. In some embodiments, the culturing
of the CHO cells is in a 100 L to 25,000 L bioreactor. In some
embodiments, the culturing of the CHO cells is in a 2000 L to
20,000 L bioreactor. In some embodiments, the temperature of the
cell culture is decreased from about 80 hours to about 120 hours
after the inoculation. In some embodiments, step (d) occurs about
10 to about 14 days after inoculation.
[0016] In some embodiments, the TSAC of the HCCF is from about 2.2
mol/mol to about 3.6 mol/mol. In some embodiments, the TSAC of the
HCCF is from about 2.2 mol/mol to about 3.4 mol/mol. In some
embodiments, the one additional purification step comprises at
least one of harvest clarification, filtration, ultrafiltration,
diafiltration, viral inactivation, affinity capture, and
combinations thereof. In some embodiments, the one additional
purification step comprises ultrafiltration and/or
diafiltration.
[0017] In some embodiments, the UFDF is held at a temperature of
about 14.degree. C. to about 26.degree. C. In some embodiments, the
UFDF is held at a temperature of about 15.degree. C. to about
26.degree. C. In some embodiments, the UFDF is held at a
temperature of about 15.degree. C. to about 25.degree. C. In some
embodiments, the UFDF is held at a temperature of about 19.degree.
C. to about 25.degree. C.
[0018] In some embodiments, the UFDF is held for about 10 hours to
about 50 hours. In some embodiments, the UFDF is held for about 12
hours to about 48 hours. In some embodiments, the UFDF is held for
about 14 hours to about 42 hours. In some embodiments, the UFDF is
held for about 17 hours to about 34 hours. In some embodiments, the
UFDF is held for about 19 hours to about 33 hours. In some
embodiments, the UFDF is held for about 25 hours to about 38 hours.
In some embodiments, the UFDF is held for about 29 hours to about
35 hours.
[0019] In some embodiments, the UFDF has a protein concentration
from about 2.0 g/L to about 4.3 g/L. In some embodiments, the UFDF
has a protein concentration from about 2.4 g/L to about 3.7 g/L. In
some embodiments, the UFDF has a protein concentration of about 3.1
g/L. In some embodiments, the UFDF has an alkaline phosphatase
concentration from about 3.0 g/L to about 4.5 g/L. In some
embodiments, the UFDF has an alkaline phosphatase concentration
from about 3.3 g/L to about 4.1 g/L. In some embodiments, the
alkaline phosphate is asfotase alfa.
[0020] In some embodiments, the at least one chromatography step is
protein affinity chromatography. In some embodiments, the at least
one chromatography step is Protein A chromatography. In some
embodiments, step (d) further comprises a viral inactivation step.
In some embodiments, step (e) further comprises at least one
additional chromatography step and/or purification step.
[0021] In some embodiments, the at least one additional
chromatography step comprises column chromatography. In some
embodiments, the column chromatography comprises hydrophobic
interaction chromatography. In some embodiments, the at least one
additional purification step comprises an additional diafiltration.
In some embodiments, the one additional chromatography and/or
purification step comprises hydrophobic interaction chromatography
and/or at least one additional diafiltration step. In some
embodiments, the at least one additional chromatography step is
performed to obtain recombinant alkaline phosphatase with a TSAC of
about 0.9 mol/mol to about 3.9 mol/mol. In some embodiments, the at
least one additional chromatography step is performed to obtain
recombinant alkaline phosphatase with a TSAC of about 1.1 mol/mol
to about 3.2 mol/mol. In some embodiments, the at least one
additional chromatography step is performed to obtain recombinant
alkaline phosphatase with a TSAC of about 1.4 mol/mol to about 3.0
mol/mol. In some embodiments, the at least one additional
chromatography step is performed to obtain recombinant alkaline
phosphatase with a TSAC of about 1.2 mol/mol to about 3.0
mol/mol.
[0022] In some embodiments, the recombinant alkaline phosphatase
comprises the structure of W-sALP--X-Fc-Y-D.sub.nZ, wherein (i) W
is absent or is an amino acid sequence of at least one amino acid;
(ii) X is absent or is an amino acid sequence of at least one amino
acid; (iii) Y is absent or is an amino acid sequence of at least
one amino acid; (iv) Z is absent or is an amino acid sequence of at
least one amino acid; (v) Fc is a fragment crystallizable region;
(vi) D.sub.n is a poly-aspartate, poly-glutamate, or combination
thereof, wherein n=10 or 16; and (vii) sALP is a soluble alkaline
phosphatase. In some embodiments, the recombinant alkaline
phosphatase comprises asfotase alfa (SEQ ID NO: 1).
[0023] In some embodiments, the recombinant alkaline phosphatase
obtained from the protein affinity chromatography is stored at from
about 2.degree. C. to about 8.degree. C.
[0024] In some embodiments, the disclosure provides a method
additionally comprising measuring recombinant alkaline phosphatase
activity.
[0025] In some embodiments, the disclosure provides a recombinant
alkaline phosphatase produced in a mammalian cell culture, wherein
the recombinant alkaline phosphatase in a filtration pool produced
from the cell culture has a total sialic acid content (TSAC)
greater than or equal to about 1.2 mol/mol, and wherein the
mammalian cell culture is about 100 L to about 25,000 L.
[0026] In some embodiments, the disclosure provides a filtration
pool (UFDF) comprising recombinant alkaline phosphatase, wherein
the UFDF is produced from about 100 L to about 25,000 L cell
culture, and wherein the UFDF is held at from about 19.degree. C.
to about 25.degree. C., for from about 14 to about 42 hours, at a
protein concentration of from about 2.0 to about 4.3 g/L.
[0027] In some embodiments, the disclosure provides a recombinant
alkaline phosphatase produced by the methods as described
herein.
[0028] In some embodiments, the disclosure provides a
pharmaceutical formulation comprising a recombinant alkaline
phosphatase produced by a method as described herein, and at least
one pharmaceutically acceptable carrier, diluent, excipient, or
combination thereof.
[0029] In some embodiments, the disclosure provides a method of
using the recombinant alkaline phosphatase made by the methods
described herein to increase cleavage of inorganic pyrophosphate
(PPi) in a subject.
[0030] In some embodiments, the disclosure is directed to a method
of treating a subject suffering from a condition associated with
alkaline phosphatase deficiency, comprising administering to the
subject a therapeutically effective amount of the recombinant
alkaline phosphatase produced by the methods described herein.
[0031] In some embodiments, the disclosure is directed to a method
of controlling total sialic acid content (TSAC) in a
TSAC-containing recombinant protein through mammalian cell
culturing, comprising at least one purification step and at least
one chromatography step.
[0032] In some embodiments, the disclosure is directed to a method
of controlling glycosidase activity in mammalian cell culture
producing a recombinant protein, comprising at least one
purification step and at least one chromatography step.
[0033] In some embodiments, the recombinant protein is a
recombinant enzyme. In some embodiments, the recombinant protein is
an alkaline phosphatase protein. In some embodiments, the
recombinant protein is asfotase alfa. In some embodiments, the
glycosidase is sialidase. In some embodiments, the at least one
purification step comprises ultrafiltration and/or diafiltration,
and the at least one chromatography step comprises protein A
chromatography.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] In the appended drawings:
[0035] FIG. 1 illustrates an embodiment of the methods described
herein for production of asfotase alfa.
[0036] FIG. 2 represents protein siaylation of cells grown with the
addition of various nutrient supplements, and with and without a
temperature shift. Brx-1=Control Process with temperature shift;
Brx-2=Control Process with temperature shift; Brx-3=Cell Boost 2+5
with temperature shift; Brx-4=Cell Boost 2+5 with temperature
shift; Brx 5=Cell Boost 6 with temperature shift; Brx-6=Cell Boost
6 without temperature shift; Brx-7=Cell Boost 7a+7b with
temperature shift; Brx-8=Cell Boost 7a+7b without temperature
shift. Day 14 values shown.
[0037] FIG. 3A and FIG. 3B represent the correlation between TSAC
decline and protein concentration (FIG. 3A) and temperature (FIG.
3B) during the HCCF post-harvest concentration/diafiltration
(UF/DF) hold. FIG. 3A suggests an increase in TSAC decline rate by
0.11 mol/mol/10 hr every 1 g/L increase in UF1 concentration
(Location 1 (LNH): mean: 2.1 g/L and Location 2 (LBH): mean: 3.3
g/L). FIG. 3B suggests an increase in TSAC decline rate by 0.11
mol/mol/10 hr for every 1.degree. C. increase in UF1 hold
temperature.
[0038] FIG. 4 is an overview of the the small-scale UF/DF1
operation and hold time performed for each of three 10 L clarified
harvest lots, as described in Example 2.
[0039] FIG. 5 represents the TSAC versus hold time, temperature,
and protein concentration at the Protein A chromatography pool step
for harvest batch #A. Three protein concentrations (1.88 g/L, 3.09
g/L, and 4.44 g/L); three hold temperatures (15.degree. C.,
19.degree. C., and 25.degree. C.); and five hold times (12, 24, 36,
48, and 60 hours) were tested.
[0040] FIG. 6 represents the TSAC versus hold time, temperature,
and protein concentration at the Protein A chromatography pool step
for harvest batch #B. Three protein concentrations (2.25 g/L, 3.69
g/L, and 5.25 g/L); three hold temperatures (15.degree. C.,
19.degree. C., and 25.degree. C.); and five hold times (12, 24, 36,
48, and 60 hours) were tested.
[0041] FIG. 7 represents the TSAC versus hold time, temperature,
and protein concentration at the Protein A chromatography pool step
for harvest batch #C. Three protein concentrations (1.77 g/L, 2.87
g/L, and 4.05 g/L); three hold temperatures (15.degree. C.,
19.degree. C., and 25.degree. C.); and five hold times (12, 24, 36,
48, and 60 hours) were tested.
[0042] FIG. 8 shows the TSAC measurement after the Protein A
chromatography step (ProA) and at the bulk drug substance fill step
(BDS) for 51 manufacturing batches.
[0043] FIG. 9 shows the TSAC measurement at the cell culture fluid
(CCF) and the harvest clarified culture fluid (HCCF) for 39
manufacturing batches.
[0044] FIG. 10 shows the TSAC measurement at the harvest clarified
culture fluid, (HCCF), the Protein A chromatography step (ProA),
and the bulk drug substance fill step (BDS) for 39 manufacturing
batches.
[0045] FIG. 11 shows the TSAC drop from HCCF to using a JMP model
created by performing fit model analysis. Also included are the
actual experimental results.
[0046] FIG. 12 includes the JMP model prediction profiler outputs
for hold time, protein concentration, and hold temperature as a
function of TSAC drop from HCCF.
DETAILED DESCRIPTION
Definitions
[0047] "About", "Approximately": As used herein, the terms "about"
and "approximately", as applied to one or more particular cell
culture conditions or numerical values, refer to a range of values
that are similar to the stated reference value for that culture
condition, conditions, or numerical values. In certain embodiments,
the term "about" refers to a range of values that fall within 25,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
or 1 percent or less of the stated reference value for that culture
condition, conditions, or numerical values.
[0048] "Amino acid": The term "amino acid," as used herein, refers
to any of the twenty naturally occurring amino acids that are
normally used in the formation of polypeptides, or analogs or
derivatives of those amino acids Amino acids of the present
disclosure can be provided in medium to cell cultures. The amino
acids provided in the medium may be provided as salts or in hydrate
form.
[0049] "Batch culture": The term "batch culture," as used herein,
refers to a method of culturing cells in which all of the
components that will ultimately be used in culturing the cells,
including the medium (see definition of "medium" below) as well as
the cells themselves, are provided at the beginning of the
culturing process. A batch culture is typically stopped at some
point and the cells and/or components in the medium are harvested
and optionally purified. In some embodiments, the methods described
here are used in a batch culture.
[0050] "Bioreactor": The term "bioreactor" as used herein refers to
any vessel used for the growth of a cell culture (e.g., a mammalian
cell culture). The bioreactor can be of any size so long as it is
useful for the culturing of cells. Typically, the bioreactor will
be at least 1 liter and may be 10, 100, 250, 500, 1000, 2500, 5000,
8000, 10,000, 12,0000, 20,000 liters or more, or any volume in
between. In some embodiments, the bioreactor is 100 liters to
25,000 liters, 500 liters to 20,000 liters, 1,000 liters to 20,000
liters, 2,000 liters to 20,000 liters, 5,000 liters to 20,000
liters, or 10,000 liters to 20,000 liters. The internal conditions
of the bioreactor, including, but not limited to pH and
temperature, are typically controlled during the culturing period.
The bioreactor can be composed of any material that is suitable for
holding mammalian or other cell cultures suspended in media under
the culture conditions of the present disclosure, including glass,
plastic or metal. The term "production bioreactor" as used herein
refers to the final bioreactor used in the production of the
polypeptide or protein of interest. The volume of the large-scale
cell culture production bioreactor is typically at least 500 liters
and may be 1000, 2500, 5000, 8000, 10,000, 12,0000, 20,000 liters
or more, or any volume in between. One of ordinary skill in the art
will be aware of and will be able to choose suitable bioreactors
for use in practicing the present disclosure.
[0051] "Cell density": The term "cell density," as used herein,
refers to the number of cells present in a given volume of
medium.
[0052] "Cell viability": The term "cell viability," as used herein,
refers to the ability of cells in culture to survive under a given
set of culture conditions or experimental variations. The term as
used herein also refers to that portion of cells which are alive at
a particular time in relation to the total number of cells, living
and dead, in the culture at that time.
[0053] "Culture" and "cell culture": These terms, as used herein,
refer to a cell population that is suspended in a medium (see
definition of "medium" below) under conditions suitable for
survival and/or growth of the cell population. As will be clear to
those of ordinary skill in the art, these terms as used herein may
refer to the combination comprising the cell population and the
medium in which the population is suspended.
[0054] "Fed-batch culture": The term "fed-batch culture," as used
herein, refers to a method of culturing cells in which additional
components are provided to the culture at some time subsequent to
the beginning of the culture process. The provided components
typically comprise nutritional supplements for the cells, which
have been depleted during the culturing process. A fed-batch
culture is typically stopped at some point and the cells and/or
components in the medium are harvested and optionally purified.
Fed-batch culture may be performed in the corresponding fed-batch
bioreactor. In some embodiments, the method comprises a fed-batch
culture.
[0055] "Fragment": The term "fragment," as used herein, refers to a
polypeptide and is defined as any discrete portion of a given
polypeptide that is unique to or characteristic of that
polypeptide. The term as used herein also refers to any discrete
portion of a given polypeptide that retains at least a fraction of
the activity of the full-length polypeptide. In some embodiments
the fraction of activity retained is at least 10% of the activity
of the full-length polypeptide. In various embodiments the fraction
of activity retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,
or 90% of the activity of the full-length polypeptide. In other
embodiments the fraction of activity retained is at least 95%, 96%,
97%, 98%, or 99% of the activity of the full-length polypeptide. In
one embodiment, the fraction of activity retained is 100% of the
activity of the full-length polypeptide. The term as used herein
also refers to any portion of a given polypeptide that includes at
least an established sequence element found in the full-length
polypeptide. In some embodiments, the sequence element spans at
least 4-5 amino acids of the full-length polypeptide. In some
embodiments, the sequence element spans at least about 10, 15, 20,
25, 30, 35, 40, 45, 50 or more amino acids of the full-length
polypeptide.
[0056] "Glycoprotein" or "glycoproteins: These terms, as used
herein, refer to a protein or polypeptide with carbohydrate groups
(such as sialic acid) attached to the polypeptide chain.
[0057] "Medium", "media", "cell culture medium", and "culture
medium": These terms, as used herein, refer to a solution
containing nutrients which nourish growing mammalian cells.
Typically, these solutions provide essential and non-essential
amino acids, vitamins, energy sources, lipids, and trace elements
required by the cell for minimal growth and/or survival. The
solution may also contain components that enhance growth and/or
survival above the minimal rate, including hormones and growth
factors. The solution is, e.g., formulated to a pH and salt
concentration optimal for cell survival and proliferation. In some
embodiments, a culture medium may be a "defined media"--a
serum-free media that contains no proteins, hydrolysates or
components of unknown composition. Defined media are free of
animal-derived components and all components have a known chemical
structure. In some embodiments, the culture medium is a basal
medium, i.e., an undefined medium containing a carbon source,
water, salts, a source of amino acids and nitrogen (e.g., animal,
e.g., beef, or yeast extracts). Various mediums are commercially
available and are known to those in the art. In some embodiments,
the culture medium is selected from EX-CELL.RTM. 302 Serum-Free
Medium (Sigma Aldrich, St. Louis, Mo.), CD DG44 Medium
(ThermoFisher Scientific, Waltham, Mass.), BD Select Medium (BD
Biosciences, San Jose, Calif.), or a mixture thereof. a mixture of
BD Select Medium with SFM4CHO Medium (Hyclone, Logan Utah). In some
embodiments, the culture medium comprises a combination of SFM4CHO
Medium and BD Select.TM. Medium. In some embodiments, the culture
medium comprises a combination of SFM4CHO Medium and BD Select.TM.
Medium at a ratio selected from 90/10, 80/20, 75/25, 70/30, 60/40,
or 50/50. In some embodiments, the culture medium comprises a
combination of SFM4CHO Medium and BD Select.TM. Medium at a ratio
of 70/30 to 90/10. In some embodiments, the culture medium
comprises a combination of SFM4CHO Medium and BD Select.TM. Medium
at a ratio 75/25.
[0058] "Osmolality" and "osmolarity": Osmolality is a measure of
the osmotic pressure of dissolved solute particles in an aqueous
solution. The solute particles include both ions and non-ionized
molecules. Osmolality is expressed as the concentration of
osmotically active particles (i.e., osmoles) dissolved in 1 kg of
solution (1 mOsm/kg H.sub.2O at 38.degree. C. is equivalent to an
osmotic pressure of 19 mm Hg). "Osmolarity," by contrast, refers to
the number of solute particles dissolved in 1 liter of solution.
When used herein the abbreviation "mOsm" means "milliosmoles/kg
solution".
[0059] "Perfusion culture": The term "perfusion culture," as used
herein, refers to a method of culturing cells in which additional
components are provided continuously or semi-continuously to the
culture subsequent to the beginning of the culture process. The
provided components typically comprise nutritional supplements for
the cells, which have been depleted during the culturing process. A
portion of the cells and/or components in the medium are typically
harvested on a continuous or semi-continuous basis and are
optionally purified. In some embodiments, the nutritional
supplements as described herein are added in a perfusion culture,
i.e., they are provided continuously over a defined period of
time.
[0060] "Polypeptide": The term "polypeptide," as used herein,
refers a sequential chain of amino acids linked together via
peptide bonds. The term is used to refer to an amino acid chain of
any length, but one of ordinary skill in the art will understand
that the term is not limited to lengthy chains and can refer to a
minimal length chain comprising two amino acids linked together via
a peptide bond.
[0061] "Protein": The term "protein," as used herein, refers to one
or more polypeptides that function as a discrete unit. If a single
polypeptide is the discrete functioning unit and does not require
permanent physical association with other polypeptides in order to
form the discrete functioning unit, the terms "polypeptide" and
"protein" as used herein are used interchangeably.
[0062] "Recombinantly-expressed polypeptide" and "recombinant
polypeptide": These terms, as used herein, refer to a polypeptide
expressed from a host cell that has been genetically engineered to
express that polypeptide. The recombinantly-expressed polypeptide
can be identical or similar to a polypeptide that is normally
expressed in the mammalian host cell. The recombinantly-expressed
polypeptide can also be foreign to the host cell, i.e.,
heterologous to peptides normally expressed in the host cell.
Alternatively, the recombinantly-expressed polypeptide can be
chimeric in that portions of the polypeptide contain amino acid
sequences that are identical or similar to polypeptides normally
expressed in the mammalian host cell, while other portions are
foreign to the host cell.
[0063] "Seeding": The term "seeding," as used herein, refers to the
process of providing a cell culture to a bioreactor or another
vessel. The cells may have been propagated previously in another
bioreactor or vessel. Alternatively, the cells may have been frozen
and thawed immediately prior to providing them to the bioreactor or
vessel. The term refers to any number of cells, including a single
cell. In various embodiments, alkaline phosphatase (e.g., asfotase
alfa) is produced by a process in which cells are seeded in a
density of about 1.0.times.10.sup.5 cells/mL, 1.5.times.10.sup.5
cells/mL, 2.0.times.10.sup.5 cells/mL, 2.5.times.10.sup.5 cells/mL,
3.0.times.10.sup.5 cells/mL, 3.5.times.10.sup.5 cells/mL,
4.0.times.10.sup.5 cells/mL, 4.5.times.10.sup.5 cells/mL,
5.0.times.10.sup.5 cells/mL, 5.5.times.10.sup.5 cells/mL,
6.0.times.10.sup.5 cells/mL, 6.5.times.10.sup.5 cells/mL,
7.0.times.10.sup.5 cells/mL, 7.5.times.10.sup.5 cells/mL,
8.0.times.10.sup.5 cells/mL, 8.5.times.10.sup.5 cells/mL,
9.0.times.10.sup.5 cells/mL, 9.5.times.10.sup.5 cells/mL,
1.0.times.10.sup.6 cells/mL, 1.5.times.10.sup.6 cells/mL,
2.0.times.10.sup.6 cells/mL, or a higher density. In one particular
embodiment, in such process cells are seeded in a density of about
4.0.times.10.sup.5 cells/mL, 5.5.times.10.sup.5 cells/mL or
8.0.times.10.sup.5 cells/mL.
[0064] "Total Sialic Acid Content" or "TSAC": The term as used
herein refers to the amount of sialic acid (a carbohydrate) on a
particular protein molecule. It is expressed as moles TSAC per mole
of protein, or, "mol/mol." TSAC concentration is measured during
the purification process. For example, one method of TSAC
quantitation is where TSAC is released from asfotase alfa using
acid hydrolysis, and the released TSAC is subsequently detected via
electrochemical detection using high-performance anion-exchange
chromatography with pulsed amperometric detection technique
("HPAE-PAD").
[0065] "Titer": The term "titer," as used herein, refers to the
total amount of recombinantly-expressed polypeptide or protein
produced by a cell culture divided by a given amount of medium
volume. Titer is typically expressed in units of milligrams of
polypeptide or protein per milliliter of medium.
[0066] Acronyms used herein include, e.g., HCCF: Harvest Clarified
Culture Fluid; UF: ultrafiltration, DF: diafiltration; VCD: Viable
Cell Density; IVCC: Integral of Viable Cell Concentration; TSAC:
Total Sialic Acid Content; HPAE-PAD: High-Performance Anion
Exchange Chromatography with Pulsed Amperometric Detection; SEC:
Size Exclusion Chromatography; AEX: Anion Exchange Chromatography;
LoC: Lab-on-Chip; and MALDI-TOF: Matrix Assisted Laser
Desorption/Ionization--Time of Flight.
[0067] As used herein, the term "hydrophobic interaction
chromatography (HIC) column" refers to a column containing a
stationary phase or resin and a mobile or solution phase in which
the hydrophobic interaction between a protein and hydrophobic
groups on the stationary phase or resin separates a protein from
impurities including fragments and aggregates of the subject
protein, other proteins or protein fragments and other contaminants
such as cell debris, or residual impurities from other purification
steps. The stationary phase or resin comprises a base matrix or
support such as a cross-linked agarose, silica or synthetic
copolymer material to which hydrophobic ligands are attached.
Examples of such stationary phase or resins include phenyl-,
butyl-, octyl-, hexyl- and other alkyl substituted agarose, silica,
or other synthetic polymers. Columns may be of any size containing
the stationary phase, or may be open and batch processed. In some
embodiments, the recombinant alkaline phosphatase is isolated from
the cell culture using HIC.
[0068] As used herein, the term "preparation" refers to a solution
comprising a protein of interest (e.g., a recombinant alkaline
phosphatase described herein) and at least one impurity from a cell
culture producing such protein of interest and/or a solution used
to extract, concentrate, and/or purify such protein of interest
from the cell culture. For example, a preparation of a protein of
interest (e.g., a recombinant alkaline phosphatase described
herein) may be prepared by homogenizing cells, which grow in a cell
culture and produce such protein of interest, in a homogenizing
solution. In some embodiments, the preparation is then subjected to
one or more purification/isolation process, e.g., a chromatography
step.
[0069] As used herein, the term "solution" refers to a homogeneous,
molecular mixture of two or more substances in a liquid form.
Specifically, in some embodiments, the proteins to be purified,
such as the recombinant alkaline phosphatases or their fusion
proteins (e.g., asfotase alfa) in the present disclosure represent
one substance in a solution. The term "buffer" or "buffered
solution" refers to solutions which resist changes in pH by the
action of its conjugate acid-base range. Examples of buffers that
control pH at ranges of about pH 5 to about pH 7 include HEPES,
citrate, phosphate, and acetate, and other mineral acid or organic
acid buffers, and combinations of these. Salt cations include
sodium, ammonium, and potassium. As used herein the term "loading
buffer/solution" or "equilibrium buffer/solution" refers to the
buffer/solution containing the salt or salts which is mixed with
the protein preparation for loading the protein preparation onto a
chromatography column, e.g., HIC column. This buffer/solution is
also used to equilibrate the column before loading, and to wash to
column after loading the protein. The "elution buffer/solution"
refers to the buffer/solution used to elute the protein from the
column. As used herein, the term "solution" refers to either a
buffered or a non-buffered solution, including water.
[0070] The term "sialic acid" refers generally to N- or
O-substituted derivatives of neuraminic acid, a monosaccharide with
a nine-carbon backbone. Sialic acid may also refer specifically to
the compound N-acetylneuraminic acid and is sometimes abbreviated
as NeuSAc or NANA. Presence of sialic acid may affect absorption,
serum half-life, and clearance of glycoproteins from the serum, as
well as physical, chemical, and immunogenic properties of the
glycoprotein. In some embodiments of the present disclosure, sialic
acid associated with alkaline phosphatases, e.g., asfotase alfa,
impacts the half-life of the molecule in physiological conditions.
In some embodiments, precise and predictable control of total
sialic acid content (TSAC) of asfotase alfa serves as a critical
quality attribute for recombinant asoftase alfa. In some
embodiments, the TSAC is 1.2 to 3.0 mol/mol asfotase alfa monomer.
In some embodiments, TSAC is generated in the recombinant protein
production process in the bioreactor. In some embodiments, the
disclosure provides a method of controlling total sialic acid
content (TSAC) in a TSAC-containing recombinant protein through
mammalian cell culture, comprising at least one purification step
and at least one chromatography step. In some embodiments, the
purification and chromatography steps lead to decreased glycosidase
activity, and thus increased total sialic acid content of the
recombinant protein.
[0071] The term "sialylation" refers to a specific type of
glycosylation, i.e., the addition of one or more sialic acid
molecules to biomolecules, particularly, the addition of one or
more sialic acid molecules to proteins. In some embodiments of the
present disclosure, sialylation is performed by a sialyltransferase
enzyme. In some embodiments, sialyltransferases add sialic acid to
nascent oligosaccharides and/or to N- or O-linked sugar chains of
glycoproteins. In some embodiments, sialyltransferases are present
natively in the cells producing recombinant alkaline phosphatase.
In some embodiments, sialyltransferases are present in the cell
culture medium and/or nutrient supplement used in culturing the
cells producing recombinant alkaline phosphatase. In some
embodiments, sialyltransferases are produced recombinantly, using
recombinant protein expression methods known in the art. In some
embodiments, recombinant sialyltransferases produced separately
from the recombinant alkaline phosphatases are added exogenously to
the cell culture, the harvest clarified culture fluid (HCCF),
and/or the filtration pool.
[0072] In some embodiments of the present disclosure, sialic acid
groups are removed from glycoproteins (i.e., "desialylation") by
hydrolysis. In some embodiments, desialylation is performed by a
glycosidase enzyme. As used herein, "glycosidase," also called
"glycoside hydrolase," is an enzyme that catalyzes the hydrolysis
of a bond joining a sugar of a glycoside to an alcohol or another
sugar unit. Examples of glycosidases include amylase, xylanase,
cellulase, and sialidase. In some embodiments, desialylation is
performed by a sialidase enzyme. In some embodiments, sialidases
hydrolyze glycosidic linkages of terminal sialic acid residues in
glycoproteins, glycolipids, oligosaccharides, colominic acid,
and/or synthetic substrates. In some embodiments, sialidases are
present in the cell culture medium producing recombinant alkaline
phosphatase. In some embodiments, sialidase activity is dependent
on and/or correlates with total protein concentration. In some
embodiments, sialidases are essentially inactive until a critically
high protein concentration, at which point the sialidase is
activated. In some embodiments, sialidases are present in the HCCF
or the filtration pool of the cell culture producing recombinant
alkaline phosphatase. In some embodiments, sialidases remove sialic
acid moieties from glycosylation sites on recombinant alkaline
phosphatase, e.g., asfotase alfa, effectively reducing the TSAC of
the recombinant alkaline phosphatase. In some embodiments,
sialidases are selectively removed from the cell culture, the HCCF,
and/or the filtration pool. Sialidases can be selectively removed
by, e.g., one or a combination of sialidase-specific inhibitors,
antibodies, ion exchange and/or affinity chromatography,
immunoprecipitation, and the like. For an overview of how
bioprocess conditions affect the sialic acid content of proteins,
see Gramer et al., Biotechnol. Prog. 9(4):366-373 (1993), the
disclosure of which is hereby incorporated by reference in its
entirety. In some embodiments, the present disclosure provides a
method of controlling glycosidase activity in mammalian cell
culture producing recombinant protein, comprising at least one
purification and at least one chromatography step. In some
embodiments, the purification and chromatography steps lead to
decreased glycosidase activity, and thus increased total sialic
acid content of the recombinant protein.
[0073] The term "harvest clarified culture fluid," abbreviated as
HCCF, refers to a clarified, filtered fluid harvested from a cell
culture, e.g., a cell culture in a bioreactor. The HCCF is
typically free of cells and cellular debris (such as, e.g.,
insoluble biomolecules) which may be present in the cell culture.
In some embodiments of the present disclosure, HCCF is generated
through centrifugation, depth filtration, sterile filtration,
and/or chromatography. In some embodiments, a cell culture fluid
from the bioreactor is first centrifuged and/or filtered, then
subjected to at least one chromatography step in order to generate
the HCCF. In some embodiments, the HCCF is concentrated prior to
and/or after the at least one chromatography step. In some
embodiments, the HCCF is diluted after the at least one
chromatography step. In some embodiments, the HCCF from the cell
culture producing recombinant alkaline phosphatase contains the
recombinant alkaline phosphatase and contaminant proteins. In some
embodiments, the contaminant proteins in the HCCF include sialidase
enzymes.
[0074] The terms "filtration" and "flow filtration" refer to a
pressure driven process that uses membranes to separate components
in a liquid solution or suspension based on their size and charge
differences. Flow filtration may be normal flow filtration or
"tangential flow filtration," also known as TFF or cross-flow
filtration. TFF is typically used for clarifying, concentrating,
and purifying proteins. During a TFF process, fluid is pumped
tangentially along the surface of at least one membrane. An applied
pressure serves to force a portion of the fluid through the
membrane to the downstream side as "filtrate." Particulates and
macromolecules that are too large to pass through the membrane
pores are retained on the upstream side as "retentate." TFF may be
used in various forms, including, for example, microfiltration,
ultrafiltration--which includes virus filtration and high
performance TFF, reverse osmosis, nanofiltration, and
diafiltration. In some embodiments of the present disclosure, one
or more of the TFF forms are used in combination for protein
processing and/or purification. In some embodiments,
ultrafiltration and diafiltration are used in combination for
purifying a recombinant alkaline phosphatase. Ultrafiltration and
diafiltration are described herein.
[0075] "Ultrafiltration," or "UF," is a purification process used
to separate proteins from buffer components for buffer exchange,
desalting, or concentration. Depending on the protein to be
retained, membrane molecular weight limits in the range of about 1
kD to about 1000 kD are used. In some embodiments, UF is a TFF
process.
[0076] "Diafiltration," or "DF," is a purification process that
washes smaller molecules through a membrane and leaves larger
molecules in the retentate without ultimately changing
concentration. Typically, DF is used in combination with another
purification processes to enhance product yield and/or purity.
During DF, solution (e.g., water or buffer) is introduced into the
sample reservoir while filtrate is removed from the unit operation.
In processes where the desired product is in the retentate,
diafiltration washes components out of the product pool into the
filtrate, thereby exchanging buffers and reducing the concentration
of undesirable species. When the product is in the filtrate,
diafiltration washes it through the membrane into a collection
vessel. In some embodiments, DF is a TFF process.
[0077] The term "filtration pool," sometimes also referred to as
the "UFDF pool" or the "UFDF," refers to a total volume of fluid
from a filtration process, typically from a combined
ultrafiltration/diafiltration (UF/DF) process. In the context of
protein purification, the UFDF refers to the retentate from an
ultrafiltration/diafiltration process.
[0078] The present disclosure provides a method of improving the
yield, enzymatic function and consistency of a recombinant protein
which is expressed by cell culture (e.g., mammalian cells including
but not limited to Chinese Hamster Ovary (CHO) cells).
Specifically, a recombinant protein may be produced by a certain
type of cells (e.g., mammalian cells including but not limited to
Chinese Hamster Ovary (CHO) cells) through, for example, a
fermentation process. The total processes of inoculation and growth
of the cells, induction of protein expression, and various
parameter optimizations for protein expression are referred as
upstream processing steps. Correspondingly, the downstream
processing steps may include, e.g., the recovery and purification
of the produced proteins (i.e., separation of the produced proteins
from other impurities and/or contaminants originated from the cells
and the culture medium). Exemplary downstream process steps
include, for example, protein capturing from harvest, removing host
cell debris, host cell proteins (HCPs), and host cell DNAs,
endotoxins, viruses and other containments, buffer-exchanging, and
formulation adjustment, etc.
[0079] The present disclosure provides a method of improving the
yield, enzymatic function and consistency of an alkaline
phosphatase (e.g., asfotase alfa) which is produced by cell
culture.
[0080] The present disclosure provides a method of culturing cells
(e.g., mammalian cells including but not limited to Chinese Hamster
Ovary (CHO) cells) expressing a recombinant protein. The present
disclosure provides manufacturing systems for the production of an
alkaline phosphatase (e.g., asfotase alfa) by cell culture. In
certain embodiments, systems are provided that minimize production
of one or more metabolic products that are detrimental to cell
growth, viability, and/or protein production or quality. In
particular embodiments, the cell culture is a batch culture, a
fed-batch culture, a culture or a continuous culture.
Alkaline Phosphatases (ALPS)
[0081] The present disclosure relates to the manufacturing of an
alkaline phosphatase protein (e.g., asfotase alfa) in recombinant
cell culture. The alkaline phosphatase protein includes any
polypeptides or molecules comprising polypeptides that comprise at
least some alkaline phosphatase activity. In various embodiments,
the alkaline phosphatase disclosed herein includes any polypeptide
having alkaline phosphatase functions, which may include any
functions of alkaline phosphatase known in the art, such as
enzymatic activity toward natural substrates including
phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and
pyridoxal 5'-phosphate (PLP).
[0082] In certain embodiments, such alkaline phosphatase protein,
after being produced and then purified by the methods disclosed
herein, can be used to treat or prevent alkaline
phosphatase-related diseases or disorders. For example, such
alkaline phosphatase protein may be administered to a subject
having decreased and/or malfunctioned endogenous alkaline
phosphatase, or having overexpressed (e.g., above normal level)
alkaline phosphatase substrates. In some embodiments, the alkaline
phosphatase protein in this disclosure is a recombinant protein. In
some embodiments, the alkaline phosphatase protein is a fusion
protein. In some embodiments, the alkaline phosphatase protein in
this disclosure specifically targets a cell type, tissue (e.g.,
connective, muscle, nervous, or epithelial tissues), or organ
(e.g., liver, heart, kidney, muscles, bones, cartilage, ligaments,
tendons, etc.). For example, such alkaline phosphatase protein may
comprise a full-length alkaline phosphatase (ALP) or fragment of at
least one alkaline phosphatase (ALP). In some embodiments, the
alkaline phosphatase protein comprises a soluble ALP (sALP) linked
to a bone-targeting moiety (e.g., a negatively-charged peptide as
described below). In some embodiments, the alkaline phosphatase
protein comprises a soluble ALP (sALP) linked to an immunoglobulin
moiety (full-length or fragment). For example, such immunoglobulin
moiety may comprise a fragment crystallizable region (Fc). In some
embodiments, the alkaline phosphatase protein comprises a soluble
ALP (sALP) linked to both a bone-targeting moiety and an
immunoglobulin moiety (full-length or fragment). For more detailed
description of the alkaline phosphatase protein disclosed herein,
see PCT Publication Nos. WO 2005/103263 and WO 2008/138131, the
teachings of both of which are incorporated by reference herein in
their entirety.
[0083] In some embodiments, the alkaline phosphatase protein
described herein comprises any one of the structures selected from
the group consisting of: sALP--X, X-sALP, sALP--Y, Y-sALP,
sALP--X--Y, sALP--Y--X, X-sALP--Y, X--Y-sALP, Y-sALP-X, and
Y-X-sALP, wherein X comprises a bone-targeting moiety, as described
herein, and Y comprises an immunoglobulin moiety, as described
herein. In one embodiment, the alkaline phosphatase protein
comprises the structure of W-sALP--X-Fc-Y-D.sub.n/E.sub.n-Z,
wherein W is absent or is an amino acid sequence of at least one
amino acid; X is absent or is an amino acid sequence of at least
one amino acid; Y is absent or is an amino acid sequence of at
least one amino acid; Z is absent or is an amino acid sequence of
at least one amino acid; Fc is a fragment crystallizable region;
D.sub.n/E.sub.n is a polyaspartate, polyglutamate, or combination
thereof wherein n=8-20; and sALP is a soluble alkaline phosphatase
(ALP). In some embodiments, D.sub.n/E.sub.n is a polyaspartate
sequence. For example, D.sub.n may be a polyaspartate sequence
wherein n is any number between 8 and 20 (both included) (e.g., n
may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20). In
one embodiment, D.sub.n is D.sub.10 or D.sub.16. In some
embodiments, D.sub.n/E.sub.n is a polyglutamate sequence. For
example, E.sub.n may be a polyglutamate sequence wherein n is any
number between 8 and 20 (both included) (e.g., n may be 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In one embodiment,
E.sub.n is E.sub.10 or E.sub.16.
[0084] For example, such sALPs may be fused to the full-length or
fragment (e.g., the fragment crystallizable region (Fc)) of an
immunoglobulin molecule. In some embodiments, the recombinant
polypeptide comprises a structure of W-sALP--X-Fc-Y-D.sub.n-Z,
wherein W is absent or is an amino acid sequence of at least one
amino acid; X is absent or is an amino acid sequence of at least
one amino acid; Y is absent or is an amino acid sequence of at
least one amino acid; Z is absent or is an amino acid sequence of
at least one amino acid; Fc is a fragment crystallizable region;
D.sub.n is a poly-aspartate, poly-glutamate, or combination
thereof, wherein n=10 or 16; and said sALP is a soluble alkaline
phosphatase. In one embodiment, n=10. In another embodiment, W and
Z are absent from said polypeptide. In some embodiments, said Fc
comprises a CH2 domain, a CH3 domain and a hinge region. In some
embodiments, said Fc is a constant domain of an immunoglobulin
selected from the group consisting of IgG-1, IgG-2, IgG-3, IgG-3
and IgG-4. In one embodiment, said Fc is a constant domain of an
immunoglobulin IgG-1. In one particular embodiment, said Fc
comprises the sequence as set forth in D488-K714 of SEQ ID NO:
1.
[0085] In some embodiments, the alkaline phosphatase disclosed
herein comprises the structure of W-sALP--X-Fc-Y-D.sub.n-Z, wherein
W is absent or is an amino acid sequence of at least one amino
acid; X is absent or is an amino acid sequence of at least one
amino acid; Y is absent or is an amino acid sequence of at least
one amino acid; Z is absent or is an amino acid sequence of at
least one amino acid; Fc is a fragment crystallizable region;
D.sub.n is a poly-aspartate, poly-glutamate, or combination
thereof, wherein n=10 or 16; and said sALP is a soluble alkaline
phosphatase. Such sALP is capable of catalyzing the cleavage of at
least one of phosphoethanolamine (PEA), inorganic pyrophosphate
(PPi) and pyridoxal 5'-phosphate (PLP). In various embodiments, the
sALP disclosed herein is capable of catalyzing the cleavage of
inorganic pyrophosphate (PPi). Such sALP may comprise all amino
acids of the active anchored form of alkaline phosphatase (ALP)
without C-terminal glycolipid anchor (GPI). Such ALP may be at
least one of tissue-non-specific alkaline phosphatase (TNALP),
placental alkaline phosphatase (PALP), germ cell alkaline
phosphatase (GCALP), and intestinal alkaline phosphatase (IAP), or
their chimeric or fusion forms or variants disclosed herein. In one
particular embodiment, the ALP comprises tissue-non-specific
alkaline phosphatase (TNALP). In another embodiment, the sALP
disclosed herein is encoded by a polynucleotide encoding a
polypeptide comprising the sequence as set forth in L1-S485 of SEQ
ID NO: 1. In yet another embodiment, the sALP disclosed herein
comprises the sequence as set forth in L1-S485 of SEQ ID NO: 1.
[0086] In one embodiment, the alkaline phosphatase protein
comprises the structure of TNALP-Fc-D.sub.10 (SEQ ID NO: 1, as
listed below). Underlined asparagine (N) residues correspond to
potential glycosylation sites (i.e., N 123, 213, 254, 286, 413
& 564). Bold underlined amino acid residues
(L.sub.486-K.sub.487 & D.sub.715-I.sub.716) correspond to
linkers between sALP and Fc, and Fc and D.sub.10 domains,
respectively.
TABLE-US-00001 (SEQ ID NO: 1) 10 20 30 40 LVPEKEKDPK YWRDQAQETL
KYALELQKLN TNVAKNVIMF 50 60 70 80 LGDGMGVSTV TAARILKGQL HHNPGEETRL
EMDKFPFVAL 90 100 110 120 SKTYNTNAQV PDSAGTATAY LCGVKANEGT
VGVSAATERS 130 140 150 160 RCNTTQGNEV TSILRWAKDA GKSVGIVTTT
RVNHATPSAA 170 180 190 200 YAHSADRDWY SDNEMPPEAL SQGCKDIAYQ
LMHNIRDIDV 210 220 230 240 IMGGGRKYMY PKNKTDVEYE SDEKARGTRL
DGLDLVDTWK 250 260 270 280 SFKPRYKHSH FIWNRTELLT LDPHNVDYLL
GLFEPGDMQY 290 300 310 320 ELNRNNVTDP SLSEMVVVAI QILRKNPKGF
FLLVEGGRID 330 340 350 360 HGHHEGKAKQ ALHEAVEMDR AIGQAGSLTS
SEDTLTVVTA 370 380 390 400 DHSHVFTFGG YTPRGNSIFG LAPMLSDTDK
KPFTAILYGN 410 420 430 440 GPGYKVVGGE RENVSMVDYA HNNYQAQSAV
PLRHETHGGE 450 460 470 480 DVAVFSKGPM AHLLHGVHEQ NYVPHVMAYA
ACIGANLGHC 490 500 510 520 APASSLKDKT HTCPPCPAPE LLGGPSVFLF
PPKPKDTLMI 530 540 550 560 SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE
VHNAKTKPRE 570 580 590 600 EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV
SNKALPAPIE 610 620 630 640 KTISKAKGQP REPQVYTLPP SREEMTKNQV
SLTCLVKGFY 650 660 670 680 PSDIAVEWES NGQPENNYKT TPPVLDSDGS
FFLYSKLTVD 690 700 710 720 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL
SPGKDIDDDD DDDDDD
[0087] In this embodiment, the polypeptide is composed of five
portions. The first portion (sALP) containing amino acids L1-S485
is the soluble part of the human tissue non-specific alkaline
phosphatase enzyme, which contains the catalytic function. The
second portion contains amino acids L486-K487 as a linker. The
third portion (Fc) containing amino acids D488-K714 is the Fc part
of the human immunoglobulin gamma 1 (IgG1) containing hinge,
CH.sub.2 and CH.sub.3 domains. The fourth portion contains
D715-I716 as a linker. The fifth portion contains amino acids
D717-D726 (D.sub.10), which is a bone targeting moiety that allows
asfotase alfa to bind to the mineral phase of bone. In addition,
each polypeptide chain contains six potential glycosylation sites
and eleven cysteine (Cys) residues. Cys102 exists as free cysteine.
Each polypeptide chain contains four intra-chain disulfide bonds
between Cys122 and Cys184, Cys472 and Cys480, Cys528 and Cys588,
and Cys634 and Cys692. The two polypeptide chains are connected by
two inter-chain disulfide bonds between Cys493 on both chains and
between Cys496 on both chains. In addition to these covalent
structural features, mammalian alkaline phosphatases are thought to
have four metal-binding sites on each polypeptide chain, including
two sites for zinc, one site for magnesium and one site for
calcium.
[0088] There are four known isozymes of ALP, namely tissue
non-specific alkaline phosphatase (TNALP) further described below,
placental alkaline phosphatase (PALP) (as described e.g., in
GenBank Accession Nos. NP_112603 and NP_001623), germ cell alkaline
phosphatase (GCALP) (as described, e.g., in GenBank Accession No.
P10696) and intestinal alkaline phosphatase (IAP) (as described,
e.g., in GenBank Accession No. NP_001622). These enzymes possess
very similar three-dimensional structures. Each of their catalytic
sites contains four metal-binding domains, for metal ions that are
necessary for enzymatic activity, including two Zn and one Mg.
These enzymes catalyze the hydrolysis of monoesters of phosphoric
acid and also catalyze a transphosphorylation reaction in the
presence of high concentrations of phosphate acceptors. Three known
natural substrates for ALP (e.g., TNALP) include
phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and
pyridoxal 5'-phosphate (PLP) (Whyte et al., 1995 J Clin Invest
95:1440-1445). An alignment between these isozymes is shown in FIG.
30 of WO 2008/138131, the teachings of which are incorporated by
reference herein in their entirety.
[0089] The alkaline phosphatase protein in this disclosure may
comprise a dimer or multimers of any ALP protein, alone or in
combination. Chimeric ALP proteins or fusion proteins may also be
produced, such as the chimeric ALP protein that is described in
Kiffer-Moreira et al. 2014 PLoS One 9:e89374, the entire teachings
of which are incorporated by reference herein in its entirety.
[0090] In one particular embodiment, the alkaline phosphatase
disclosed herein is encoded by a polynucleotide encoding a
polypeptide comprising the sequence as set forth in SEQ ID NO: 1.
In some embodiments, the alkaline phosphatase disclosed herein is
encoded by a polynucleotide encoding a polypeptide comprising a
sequence having 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO: 1. In some embodiments, the
alkaline phosphatase disclosed herein is encoded by a
polynucleotide encoding a polypeptide comprising a sequence having
95% or 99% identity to SEQ ID NO: 1. In another embodiment, the
alkaline phosphatase disclosed herein comprises the sequence as set
forth in SEQ ID NO: 1.
TNALP
[0091] As indicated above, TNALP is a membrane-bound protein
anchored through a glycolipid to its C-terminus (for human TNALP,
see UniProtKB/Swiss-Prot Accession No. P05186). This glycolipid
anchor (GPI) is added post translationally after removal of a
hydrophobic C-terminal end which serves both as a temporary
membrane anchor and as a signal for the addition of the GPI. Hence,
in one embodiment a soluble human TNALP comprises a TNALP wherein
the first amino acid of the hydrophobic C-terminal sequence, namely
alanine, is replaced by a stop codon. The soluble TNALP (herein
called sTNALP) so formed contains all amino acids of the native
anchored form of TNALP that are necessary for the formation of the
catalytic site but lacks the GPI membrane anchor. Known TNALPs
include, e.g., human TNALP [GenBank Accession Nos. NP-000469,
AAI10910, AAH90861, AAH66116, AAH21289, and AAI261661; rhesus TNALP
[GenBank Accession No. XP-001109717]; rat TNALP [GenBank Accession
No. NP_037191]; dog TNALP [GenBank Accession No. AAF64516]; pig
TNALP [GenBank Accession No. AAN64273], mouse TNALP [GenBank
Accession No. NP_031457], bovine TNALP [GenBank Accession Nos.
NP_789828, NP_776412, AAM 8209, and AAC338581, and cat TNALP
[GenBank Accession No. NP_001036028].
[0092] As used herein, the terminology "extracellular domain" is
meant to refer to any functional extracellular portion of the
native protein (e.g., without the peptide signal). Recombinant
sTNALP polypeptide retaining original amino acids 1 to 501 (18 to
501 when secreted), amino acids 1 to 502 (18 to 502 when secreted),
amino acids 1 to 504 (18 to 504 when secreted), or amino acids 1 to
505 (18-505 when secreted) are enzymatically active (see Oda et
al., 1999 J. Biochem 126:694-699). This indicates that amino acid
residues can be removed from the C-terminal end of the native
protein without affecting its enzymatic activity. Furthermore, the
soluble human TNALP may comprise one or more amino acid
substitutions, wherein such substitution(s) does not reduce or at
least does not completely inhibit the enzymatic activity of the
sTNALP. For example, certain mutations that are known to cause
hypophosphatasia (HPP) are listed in PCT Publication No. WO
2008/138131 and should be avoided to maintain a functional
sTNALP.
Negatively-Charged Peptide
[0093] The alkaline phosphatase protein of the present disclosure
may comprise a target moiety which may specifically target the
alkaline phosphatase protein to a pre-determined cell type, tissue,
or organ. In some embodiments, such pre-determined cell type,
tissue, or organ is bone tissues. Such bone-targeting moiety may
include any known polypeptide, polynucleotide, or small molecule
compounds known in the art. For example, negatively-charged
peptides may be used as a bone-targeting moiety. In some
embodiments, such negatively-charged peptides may be a
poly-aspartate, poly-glutamate, or combination thereof (e.g., a
polypeptide comprising at least one aspartate and at least one
glutamate, such as a negatively-charged peptide comprising a
combination of aspartate and glutamate residues). In some
embodiments, such negatively-charged peptides may be D.sub.6,
D.sub.7, D.sub.8, D.sub.9, D.sub.10, D.sub.11, D.sub.12, D.sub.13,
D.sub.14, D.sub.15, D.sub.16, D.sub.17, D.sub.18, D.sub.19,
D.sub.20, or a polyaspartate having more than 20 aspartates. In
some embodiments, such negatively-charged peptides may be E.sub.6,
E.sub.7, E.sub.8, E.sub.9, E.sub.10, E.sub.11, E.sub.12, E.sub.13,
E.sub.14, E.sub.15, E.sub.16, E.sub.17, E.sub.18, E.sub.19,
E.sub.20, or a polyglutamate having more than 20 glutamates. In one
embodiment, such negatively-charged peptides may comprise at least
one selected from the group consisting of D.sub.10 to D.sub.16 or
E.sub.10 to E.sub.16.
Spacer
[0094] In some embodiments, the alkaline phosphatase protein of the
present disclosure comprises a spacer sequence between the ALP
portion and the targeting moiety portion. In one embodiment, such
alkaline phosphatase protein comprises a spacer sequence between
the ALP (e.g., TNALP) portion and the negatively-charged peptide
targeting moiety. Such spacer may be any polypeptide,
polynucleotide, or small molecule compound. In some embodiments,
such spacer may comprise fragment crystallizable region (Fc)
fragments. Useful Fc fragments include Fc fragments of IgG that
comprise the hinge, and the CH2 and CH.sub.3 domains. Such IgG may
be any of IgG-1, IgG-2, IgG-3, IgG-3 and IgG-4, or any combination
thereof.
[0095] Without being limited to this theory, it is believed that
the Fc fragment used in bone-targeted sALP fusion proteins (e.g.,
asfotase alfa) acts as a spacer, which allows the protein to be
more efficiently folded given that the expression of
sTNALP-Fc-D.sub.10 was higher than that of sTNALP-D.sub.10. One
possible explanation is that the introduction of the Fc fragment
alleviates the repulsive forces caused by the presence of the
highly negatively-charged D.sub.10 sequence added at the C-terminus
of the sALP sequence exemplified herein. In some embodiments, the
alkaline phosphatase protein described herein comprises a structure
selected from the group consisting of: sALP-Fc-D.sub.10,
sALP-D.sub.10-Fc, D.sub.10-sALP-Fc, D.sub.10-Fc-sALP,
Fc-sALP-D.sub.10, and Fc-D.sub.10-sALP. In other embodiments, the
D.sub.10 in the above structures is substituted by other
negatively-charged polypeptides (e.g., D.sub.8, D.sub.16, E.sub.10,
E.sub.8, E.sub.16, etc.).
[0096] Useful spacers for the present disclosure include, e.g.,
polypeptides comprising a Fc, and hydrophilic and flexible
polypeptides able to alleviate the repulsive forces caused by the
presence of the highly negatively-charged bone-targeting sequence
(e.g., D.sub.10) added at the C-terminus of the sALP sequence.
Dimers/Tetramers
[0097] In specific embodiments, the bone-targeted sALP fusion
proteins of the present disclosure are associated so as to form
dimers or tetramers.
[0098] In the dimeric configuration, the steric hindrance imposed
by the formation of the interchain disulfide bonds is presumably
preventing the association of sALP domains to associate into the
dimeric minimal catalytically-active protein that is present in
normal cells.
[0099] The bone-targeted sALP may further optionally comprise one
or more additional amino acids 1) downstream from the
negatively-charged peptide (e.g., the bone tag); and/or 2) between
the negatively-charged peptide (e.g., the bone tag) and the Fc
fragment; and/or 3) between the spacer (e.g., an Fc fragment) and
the sALP fragment. This could occur, for example, when the cloning
strategy used to produce the bone-targeting conjugate introduces
exogenous amino acids in these locations. However the exogenous
amino acids should be selected so as not to provide an additional
GPI anchoring signal. The likelihood of a designed sequence being
cleaved by the transamidase of the host cell can be predicted as
described by Ikezawa, 2002 Glycosylphosphatidylinositol
(GPI)-anchored proteins. Biol Pharm Bull. 25:409-17.
[0100] The present disclosure also encompasses a fusion protein
that is post-translationally modified, such as by glycosylation
including those expressly mentioned herein, acetylation, amidation,
blockage, formylation, gamma-carboxyglutamic acid hydroxylation,
methylation, phosphorylation, pyrrolidone carboxylic acid, and
sulfation.
Asfotase Alfa
[0101] Asfotase alfa is a soluble Fc fusion protein consisting of
two TNALP-Fc-D.sub.10 polypeptides each with 726 amino acids as
shown in SEQ ID NO: 1. Each polypeptide or monomer is composed of
five portions. The first portion (sALP) containing amino acids
L1-S485 is the soluble part of the human tissue non-specific
alkaline phosphatase enzyme, which contains the catalytic function.
The second portion contains amino acids L486-K487 as a linker. The
third portion (Fc) containing amino acids D488-K714 is the Fc part
of the human Immunoglobulin gamma 1 (IgG1) containing hinge,
CH.sub.2 and CH.sub.3 domains. The fourth portion contains
D715-I716 as a linker. The fifth portion contains amino acids
D717-D726 (D.sub.10), which is a bone targeting moiety that allows
asfotase alfa to bind to the mineral phase of bone. In addition,
each polypeptide chain contains six potential glycosylation sites
and eleven cysteine (Cys) residues. Cys102 exists as free cysteine.
Each polypeptide chain contains four intra-chain disulfide bonds
between Cys122 and Cys184, Cys472 and Cys480, Cys528 and Cys588,
and Cys634 and Cys692. The two polypeptide chains are connected by
two inter-chain disulfide bonds between Cys493 on both chains and
between Cys496 on both chains. In addition to these covalent
structural features, mammalian alkaline phosphatases are thought to
have four metal-binding sites on each polypeptide chain, including
two sites for zinc, one site for magnesium and one site for
calcium.
[0102] Asfotase alfa can also be characterized as follows. From the
N-terminus to the C terminus, asfotase alfa comprises: (1) the
soluble catalytic domain of human tissue non-specific alkaline
phosphatase (TNSALP) (UniProtKB/Swiss-Prot Accession No. P05186),
(2) the human immunoglobulin G1 Fc domain (UniProtKB/Swiss-Prot
Accession No. P01857) and (3) a deca-aspartate peptide (D.sub.10)
used as a bone-targeting domain (Nishioka et al. 2006 Mol Genet
Metab 88:244-255). The protein associates into a homo-dimer from
two primary protein sequences. This fusion protein contains 6
confirmed complex N-glycosylation sites. Five of these
N-glycosylation sites are located on the sALP domain and one on the
Fc domain. Another important post-translational modification
present on asfotase alfa is the presence of disulfide bridges
stabilizing the enzyme and the Fc-domain structure. A total of 4
intra-molecular disulfide bridges are present per monomer and 2
inter-molecular disulfide bridges are present in the dimer. One
cysteine of the alkaline phosphatase domain is free.
[0103] Asfotase alfa has been used as an enzyme-replacement therapy
for the treatment of hypophosphatasia (HPP). In patients with HPP,
loss-of-function mutation(s) in the gene encoding TNSALP causes a
deficiency in TNSALP enzymatic activity, which leads to elevated
circulating levels of substrates, such as inorganic pyrophosphate
(PPi) and pyridoxal-5'-phosphate (PLP). Administration of asfotase
alfa to patients with HPP cleaves PPi, releasing inorganic
phosphate for combination with calcium, thereby promoting
hydroxyapatite crystal formation and bone mineralization, and
restoring a normal skeletal phenotype. For more details on asfotase
alfa and its uses in treatment, see PCT Publication Nos. WO
2005/103263 and WO 2008/138131
[0104] In some embodiments, the method provides an alkaline
phosphatase (asfotase alfa) having improved enzymatic activity of
the produced alkaline phosphatase (e.g., asfotase alfa) relative to
an alkaline phosphatase produced by conventional means, by
minimizing the concentration of metal ions having potential
negative impact on activity or increasing the concentration of
metal ions having potential positive impact on activity or both as
described herein. Activity may be measured by any known method.
Such methods include, e.g., those in vitro and in vivo assays
measuring the enzymatic activity of the produced alkaline
phosphatase (e.g., asfotase alfa) to substrates of an alkaline
phosphatase, such as phosphoethanolamine (PEA), inorganic
pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP).
[0105] In some embodiments, the alkaline phosphatase disclosed
herein is encoded by a first polynucleotide which hybridizes under
high stringency conditions to a second polynucleotide comparing the
sequence completely complementary to a third polynucleotide
encoding a polypeptide comprising the sequence as set forth in SEQ
ID NO: 1. Such high stringency conditions may comprise:
pre-hybridization and hybridization in 6.times.SSC,
5.times.Denhardt's reagent, 0.5% SDS and 100 mg/ml of denatured
fragmented salmon sperm DNA at 68.degree. C.; and washes in
2.times.SSC and 0.5% SDS at room temperature for 10 minutes; in
2.times.SSC and 0.1% SDS at room temperature for 10 minutes; and in
0.1.times.SSC and 0.5% SDS at 65.degree. C. three times for 5
minutes.
Manufacturing Process
[0106] The alkaline phosphatase protein described herein (e.g.,
asfotase alfa) may be produced by mammalian or other cells,
particularly CHO cells, using methods known in the art. Such cells
may be grown in culture dishes, flask glasses, or bioreactors.
Specific processes for cell culture and producing recombinant
proteins are known in the art, such as described in Nelson and
Geyer, 1991 Bioprocess Technol. 13:112-143 and Rea et al.,
Supplement to BioPharm International March 2008, 20-25. Exemplary
bioreactors include batch, fed-batch, and continuous reactors. In
some embodiments, the alkaline phosphatase protein is produced in a
fed-batch bioreactor.
[0107] Cell culture processes have variability caused by, for
example, variable physicochemical environment, including but not
limited to, changes in pH, temperature, temperature changes, timing
of temperature changes, cell culture media composition, cell
culture nutrient supplements, raw material lot-to-lot variation,
medium filtration material, bioreactor scale difference, gassing
strategy (air, oxygen, and carbon dioxide), etc. As disclosed
herein, the yield, relative activity profile, and glycosylation
profile of manufactured alkaline phosphatase protein may be
affected and may be controlled within particular values by
alterations in one or more of these parameters.
[0108] For recombinant protein production in cell culture, the
recombinant gene with the necessary transcriptional regulatory
elements is first transferred to a host cell by methods known in
the biotechnological arts. Optionally, a second gene is transferred
that confers to recipient cells a selective advantage. In the
presence of the selection agent, which may be applied a few days
after gene transfer, only those cells that express the selector
gene survive. Two exemplary genes for such selection are
dihydrofolate reductase (DHFR), an enzyme involved in nucleotide
metabolism, and glutamine synthetase (GS). In both cases, selection
occurs in the absence of the appropriate metabolite (hypoxanthine
and thymidine, in the case of DHFR, and glutamine in the case of
GS), preventing growth of any nontransformed cells. In general, for
efficient expression of the recombinant protein, it is not
important whether the biopharmaceutical-encoding gene and selector
genes are on the same plasmid or not.
[0109] Following selection, surviving cells may be transferred as
single cells to a second cultivation vessel, and the cultures are
expanded to produce clonal populations. Eventually, individual
clones are evaluated for recombinant protein expression, with the
highest producers being retained for further cultivation and
analysis. From these candidates, one cell line with the appropriate
growth and productivity characteristics is chosen for production of
the recombinant protein. A cultivation process is then developed
that is determined by the production needs and the requirements of
the final product.
Cells
[0110] Any mammalian cell or non-mammalian cell type, which can be
cultured to produce a polypeptide, may be utilized in accordance
with the present disclosure. Non-limiting examples of mammalian
cells that may be used include, e.g., Chinese hamster ovary
cells+/-DHFR (CHO, Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci.
USA, 77:4216); BALB/c mouse myeloma line (NSO/1, ECACC Accession
No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, The
Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7,
ATCC CRL 1651); human embryonic kidney line (293 or 293 cells
subcloned for growth in suspension culture, Graham et al., 1977 J.
Gen Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL 10);
mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251
(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-I 587); human cervical
carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells
(Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2). In a
particular embodiment, culturing and expression of polypeptides and
proteins occurs from a Chinese Hamster Ovary (CHO) cell line.
[0111] Additionally, any number of commercially and
non-commercially available hybridoma cell lines that express
polypeptides or proteins may be utilized in accordance with the
present disclosure. One skilled in the art will appreciate that
hybridoma cell lines might have different nutrition requirements
and/or might require different culture conditions for optimal
growth and polypeptide or protein expression, and will be able to
modify conditions as needed.
[0112] As noted above, in many instances the cells will be selected
or engineered to produce high levels of protein or polypeptide.
Often, cells are genetically engineered to produce high levels of
protein, for example by introduction of a gene encoding the protein
or polypeptide of interest and/or by introduction of control
elements that regulate expression of the gene (whether endogenous
or introduced) encoding the polypeptide of interest.
Seeding Density
[0113] In the present disclosure, Chinese Hamster Ovary (CHO) cells
are inoculated, i.e., seeded, into the culture medium. Various
seeding densities can be used. In some embodiments, a seeding
density of 1.0.times.10.sup.4 cells/mL to 1.0.times.10.sup.7
cells/mL can be used. In some embodiments, a seeding density of
1.0.times.10.sup.5 cells/mL to 1.0.times.10.sup.6 cells/mL can be
used. In some embodiments, a seeding density of 4.0.times.10.sup.5
cells/mL to 8.0.times.10.sup.5 cells/mL can be used. In some
embodiments, increased seeding density can impact fragmentation of
asfotase alfa quality, as measured by SEC. In some embodiments, the
seeding density is controlled when inoculating in order to reduce
the risk of fragment generation.
Temperature
[0114] Prior results indicated that temperature may have an impact
on several parameters including growth rate, aggregation,
fragmentation, and TSAC. In some embodiments, the temperature
remains constant when culturing the CHO cells in the culture
medium. In some embodiments, the temperature is about 30.degree. C.
to about 40.degree. C., or about 35.degree. C. to about 40.degree.
C., or about 37.degree. C. to about 39.degree. C. when culturing
the CHO cells in the culture medium. In some embodiments, the
temperature is about 30.degree. C., about 30.5.degree. C., about
31.degree. C., about 31.5.degree. C., about 32.degree. C., about
32.5.degree. C., about 33.degree. C., about 33.5.degree. C., about
34.degree. C., about 34.5.degree. C., about 35.degree. C., about
35.5.degree. C., about 36.degree. C., about 36.5.degree. C., about
37.degree. C., about 37.5.degree. C., about 38.degree. C., about
38.5.degree. C., about 39.degree. C., about 39.5.degree. C., or
about 40.degree. C. when culturing the CHO cells in the culture
medium. In some embodiments, the temperature is constant for 40 to
200 hours after inoculation. In some embodiments, the temperature
is constant for 50 to 150 hours, or 60 to 140 hours, or 70 to 130
hours, or 80 to 120 hours, or 90 to 110 hours after inoculation. In
some embodiments, the temperature is constant for 80 to 120 hours
after inoculation. In some embodiments, the temperature is constant
for 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours,
102 hours, 104 hours, 106 hours, 108 hours or 110 hours after
inoculation.
Temperature Shifting
[0115] Run times of cell culture processes, especially
non-continuous processes (e.g., fed-batch processes in
bioreactors), are usually limited by the remaining viability of the
cells, which typically declines over the course of the run.
Therefore, extending the length of time for cell viability is
desired for improving recombination protein production. Product
quality concerns also offer a motivation for minimizing decreases
in viable cell density and maintaining high cell viability, as cell
death can release sialidases to the culture supernatant, which may
reduce the sialic acid content of the protein expressed. Protein
purification concerns offer yet another motivation for minimizing
decreases in viable cell density and maintaining high cell
viability. Cell debris and the contents of dead cells in the
culture can negatively impact one's ability to isolate and/or
purify the protein product at the end of the culturing run. Thus,
by keeping cells viable for a longer period of time in culture,
there is a reduction in the contamination of the culture medium by
cellular proteins and enzymes (e.g., cellular proteases and
sialidases) that may cause degradation and ultimate reduction in
the quality of the desired glycoprotein produced by the cells.
[0116] Many methods may be applied to achieve high cell viability
in cell cultures. One involves lowering culture temperature
following initial culturing at a normal temperature. For example,
see Ressler et al., 1996, Enzyme and Microbial Technology
18:423-427). Generally, the mammalian or other types of cells
capable of expressing a protein of interest are first grown under a
normal temperature to increase cell numbers. Such "normal"
temperatures for each cell type are generally around 37.degree. C.
(e.g., from about 35.degree. C. to about 39.degree. C., including,
for example, 35.0.degree. C., 35.5.degree. C., 36.0.degree. C.,
36.5.degree. C., 37.0.degree. C., 37.5.degree. C., 38.0.degree. C.,
38.5.degree. C., and/or 39.0.degree. C.). In one particular
embodiment, the temperature for producing asfotase alfa is first
set at about 37.degree. C. When a reasonably high cell density is
reached, the culturing temperature for the whole cell culture can
then be shifted (e.g., decreased) to promote protein production. In
most cases lowering temperature shifts the cells towards the
non-growth G1 portion of the cell cycle, which may increase cell
density and viability, as compared to the previous
higher-temperature environment. In addition, a lower temperature
may also promote recombinant protein production by increasing the
cellular protein production rate, facilitating protein
post-translational modification (e.g., glycosylation), decreasing
fragmentation or aggregation of newly-produced proteins,
facilitating protein folding and formation of 3D structure (thus
maintaining activity), and/or decreasing degradation of newly
produced proteins. In some embodiments, the temperature is
decreased 3.degree. C., 4.degree. C., 5.degree. C., 6.degree. C.,
7.degree. C., 8.degree. C., 9.degree. C., or 10.degree. C. In some
embodiments, the temperature is decreased to about 27.degree. C.,
28.degree. C., 29.degree. C., 30.degree. C., 31.degree. C.,
32.degree. C., 33.degree. C., 34.degree. C., or 35.degree. C. In
some embodiments, the lower temperature is from about 30.degree. C.
to about 35.degree. C. (e.g., 30.0.degree. C., 30.5.degree. C.,
31.0.degree. C., 31.5.degree. C., 32.0.degree. C., 32.5.degree. C.,
33.0.degree. C., 33.5.degree. C., 34.0.degree. C., 34.5.degree. C.,
and/or 35.0.degree. C.). In other embodiments, the temperature for
producing asfotase alfa is first set to from about 35.0.degree. C.
to about 39.0.degree. C. and then shifted to from about
30.0.degree. C. to about 35.0.degree. C. In one embodiment, the
temperature for producing asfotase alfa is first set at about
37.0.degree. C. and then shifted to about 30.degree. C. In another
embodiment, the temperature for producing asfotase alfa is first
set at about 36.5.degree. C. and then shifted to about 33.degree.
C. In yet another embodiment, the temperature for producing
asfotase alfa is first set at about 37.0.degree. C. and then
shifted to about 33.degree. C. In yet a further embodiment, the
temperature for producing asfotase alfa is first set at about
36.5.degree. C. and then shifted to about 30.degree. C. In other
embodiments, multiple (e.g., more than one) steps of temperature
shifting may be applied.
[0117] The time for maintaining the culture at a particular
temperature prior to shifting to a different temperature may be
determined to achieve a sufficient (or desired) cell density while
maintaining cell viability and an ability to produce the protein of
interest. In some embodiments, the cell culture is grown under the
first temperature until the viable cell density reaches about
10.sup.5 cells/mL to about 10.sup.7 cells/mL (e.g.,
1.times.10.sup.5, 1.5.times.10.sup.5, 2.0.times.10.sup.5,
2.5.times.10.sup.5, 3.0.times.10.sup.5, 3.5.times.10.sup.5,
4.0.times.10.sup.5, 4.5.times.10.sup.5, 5.0.times.10.sup.5,
5.5.times.10.sup.5, 6.0.times.10.sup.5, 6.5.times.10.sup.5,
7.0.times.10.sup.5, 7.5.times.10.sup.5, 8.0.times.10.sup.5,
8.5.times.10.sup.5, 9.0.times.10.sup.5, 9.5.times.10.sup.5,
1.0.times.10.sup.6, 1.5.times.10.sup.6, 2.0.times.10.sup.6,
2.5.times.10.sup.6, 3.0.times.10.sup.6, 3.5.times.10.sup.6,
4.0.times.10.sup.6, 4.5.times.10.sup.6, 5.0.times.10.sup.6,
5.5.times.10.sup.6, 6.0.times.10.sup.6, 6.5.times.10.sup.6,
7.0.times.10.sup.6, 7.5.times.10.sup.6, 8.0.times.10.sup.6,
8.5.times.10.sup.6, 9.0.times.10.sup.6, 9.5.times.10.sup.6,
1.times.10.sup.7 cell/mL, or more) before shifting to a different
temperature. In one embodiment, the cell culture is grown under the
first temperature until the viable cell density reaches about 2.5
to about 3.4.times.10.sup.6 cells/mL before shifting to a different
temperature. In another embodiment, the cell culture is grown under
the first temperature until the viable cell density reaches about
2.5 to about 3.2.times.10.sup.6 cells/mL before shifting to a
different temperature. In yet another embodiment, the cell culture
is grown under the first temperature until the viable cell density
reaches about 2.5 to about 2.8.times.10.sup.6 cells/mL before
shifting to a different temperature.
[0118] In some embodiments, the method of the present disclosure
provides the temperature shift occurs 50 to 150 hours, or 60 to 140
hours, or 70 to 130 hours, or 80 to 120 hours, or 90 to 110 hours
after inoculation. In some embodiments, the method of the present
disclosure provides the temperature decreased about 80 hours to 150
hours after inoculation, about 90 hours to 100 hours after
inoculation or about 96 hours after inoculation. In some
embodiments, the temperature shift occurs 80 to 120 hours after
inoculation. In some embodiments, the temperature shift occurs 90
hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102
hours, 104 hours, 106 hours, 108 hours or 110 hours after
inoculation. In some embodiments, the temperature after the
temperature shift is maintained until the CHO cells are
harvested.
[0119] Alteration of the pH of the growth medium in cell culture
may affect cellular proteolytic activity, secretion, and protein
production levels. Most of the cell lines grow well at about pH
7-8. Although optimum pH for cell growth varies relatively little
among different cell strains, some normal fibroblast cell lines
perform best at a pH 7.0-7.7 and transformed cells typically
perform best at a pH of 7.0-7.4 (Eagle, 1973 The effect of
environmental pH on the growth of normal and malignant cells. J
Cell Physiol 82:1-8). In some embodiments, the pH of the culture
medium for producing asfotase alfa is about pH 6.5-7.7 (e.g., 6.50,
6.55, 6.60, 6.65, 6.70, 6.75, 6.80, 6.85, 6.90, 6.95, 7.00, 7.05,
7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.39, 7.40, 7.45, 7.50, 7.55,
7.60, 7.65, or 7.70).
Culture Medium
[0120] In some embodiments, batch culture is used, wherein no
additional culture medium is added after inoculation. In some
embodiments, fed batch is used, wherein one or more boluses of
culture medium are added after inoculation. In some embodiments,
two, three, four, five or six boluses of culture medium are added
after inoculation.
[0121] In various embodiments, alkaline phosphatase (e.g., asfotase
alfa) is produced by a process in which extra boluses of culture
medium are added to the production bioreactor. For example, one,
two, three, four, five, six, or more boluses of culture medium may
be added. In one particular embodiment, three boluses of culture
medium are added. In various embodiments, such extra boluses of
culture medium may be added in various amounts. For example, such
boluses of culture medium may be added in an amount of about 20%,
25%, 30%, 33%, 40%, 45%, 50%, 60%, 67%, 70%, 75%, 80%, 90%, 100%,
110%, 120%, 125%, 130%, 133%, 140%, 150%, 160%, 167%, 170%, 175%,
180%, 190%, 200%, or more, of the original volume of culture medium
in the production bioreactor. In one particular embodiment, such
boluses of culture medium may be added in an amount of about 33%,
67%, 100%, or 133% of the original volume. In various embodiments,
such addition of extra boluses may occur at various times during
the cell growth or protein production period. For example, boluses
may be added at day 1, day 2, day 3, day 4, day 5, day 6, day 7,
day 8, day 9, day 10, day 11, day 12, or later in the process. In
one particular embodiment, such boluses of culture medium may be
added in every other day (e.g., at (1) day 3, day 5, and day 7; (2)
day 4, day 6, and day 8; or (3) day 5, day 7, and day 9. In
practice, the frequency, amount, time point, and other parameters
of bolus supplements of culture medium may be combined freely
according to the above limitation and determined by experimental
practice.
[0122] Various culture mediums are available commercially. In some
embodiments, the culture medium is selected from the group
consisting of EX-CELL.RTM. 302 Serum-Free Medium; CD DG44 Medium;
BD Select.TM. Medium; SFM4CHO Medium, or a combination thereof. In
some embodiments, the culture medium comprises a combination of
commercially available mediums, e.g., SFM4CHO Medium and BD
Select.TM. Medium. In some embodiments, the culture medium
comprises a combination of of commercially available mediums, e.g.,
SFM4CHO Medium and BD Select.TM. Medium, at a ratio selected from
90/10, 80/20, 75/25, 70/30, 60/40, or 50/50.
Nutrient Supplement
[0123] Various nutrient supplements, also referred to as "feed
media," are commercially available and are known to those of skill
in the art. Nutrient supplements include a media (distinct from the
culture media) added to a cell culture after inoculation has
occurred. In some instances, the nutrient supplement can be used to
replace nutrients consumed by the growing cells in the culture. In
some embodiments, the nutrient supplement is added to optimize
production of a desired protein, or to optimize activity of a
desired protein. Numerous nutrient supplements have been developed
and are available commercially. While the expressed purpose of the
nutrient supplements is to increase an aspect of process
development, no universal nutrient supplement exists that works for
all cells and/or all proteins produced. The selection of a scalable
and appropriate cell culture nutrient supplement that can work in
combination with the desired cell line, protein produced and a
given base medium to achieve the desired titer and growth
characteristics is not routine. The typical approach of screening
multiple commercially available nutrient supplements and
identifying the most appropriate supplement with a specific cell
line, specific protein produced and base medium combination may not
be successful due to the myriad of variables present in the cell
culture process. In some embodiments, the nutrient supplement is
selected from the group consisting of Efficient Feed C+ AGT.TM.
Supplement (Thermo Fisher Scientific, Waltham, Mass.), a
combination of Cell Boost.TM. 2+Cell Boost.TM. 4 (GE Healthcare,
Sweden), a combination of Cell Boost.TM. 2+Cell Boost.TM. 5 (GE
Healthcare, Sweden), Cell Boost.TM. 6 (GE Healthcare, Sweden), and
Cell Boost.TM. 7a+Cell Boost.TM. 7b (GE Healthcare, Sweden), or
combinations thereof.
[0124] Cell Boost.TM. 7a can be described as a first animal-derived
component-free (ADCF) nutrient supplement comprising one or more
amino acids, vitamins, salts, trace elements, poloxamer and
glucose, wherein the first ADCF nutrient supplement does not
comprise hypoxanthine, thymidine, insulin, L-glutamine, growth
factors, peptides, proteins, hydrolysates, phenol red and
2-mercaptoethanol. Cell Boost.TM. 7a is a chemically defined
supplement. The phrase "animal-derived component-free" or "ADCF"
refers to a supplement in which no ingredients are derived directly
from an animal source, e.g., are not derived from a bovine source.
In some embodiments, the nutrient supplement is Cell Boost.TM.
7a.
[0125] Cell Boost.TM. 7b can be described as a second ADCF nutrient
supplement comprising one or more amino acids, wherein the second
ADCF nutrient supplement lacks hypoxanthine, thymidine, insulin,
L-glutamine, growth factors, peptides, proteins, hydrolysates,
phenol red, 2-mercaptoethanol and poloxamer. Cell Boost.TM. 7b is a
chemically defined supplement. In some embodiments, the nutrient
supplement is Cell Boost.TM. 7b.
[0126] In some embodiments, combinations of commercially available
nutrient supplements are used. The term "nutrient supplement"
refers to both a single nutrient supplement, as well as
combinations of nutrient supplements. For example, in some
embodiments a combination of nutrient supplements includes a
combination of Cell Boost.TM. 7a and Cell Boost.TM. 7b.
[0127] In various embodiments, alkaline phosphatase (e.g., asfotase
alfa) is produced by a process in which extra additions of nutrient
supplement are added to the production bioreactor. In some
embodiments, the nutrient supplement is added over a period of
time, e.g., over a period of time ranging from 1 minute to 2 hours.
In some embodiments, the nutrient supplement is added in a bolus.
For example, one, two, three, four, five, six, or more boluses of
nutrient supplement may be added. In some embodiments, the nutrient
supplement is added at more than 2 different times, e.g., 2 to 6
different times. In various embodiments, such extra boluses of
nutrient supplement may be added in various amounts. For example,
such boluses of nutrient supplement may be added in an amount of
about 1% to 20%, 1% to 10% or 1% to 5% (w/v) of the original volume
of culture medium in the production bioreactor. In one particular
embodiment, such boluses of nutrient supplement may be added in an
amount of 1% to 20%, 1% to 10% or 1% to 5% (w/v) of the original
volume.
[0128] In some embodiments, a combination of nutrient supplements
is used, and the first nutrient supplement, e.g., Cell Boost.TM.
7a, is added at a concentration of 0.5% to 4% (w/v) of the culture
medium. In some embodiments, a combination of nutrient supplements
is used, and the second nutrient supplement, e.g., Cell Boost.TM.
7b, is added at a concentration of 0.05% to 0.8% (w/v) of the
culture medium. In specific embodiments wherein a combination of
nutrient supplements include Cell Boost.TM. 7a and Cell Boost.TM.
7b, a boluses of nutrient supplement may be added in an amount of
1% to 20%, 1% to 10% or 1% to 5% (w/v) of the original volume.
[0129] In various embodiments, such addition of extra boluses may
occur at various times after inoculation. For example, boluses may
be added at day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8,
day 9, day 10, day 11, day 12, or later after inoculation. In
practice, the frequency, amount, time point, and other parameters
of bolus supplements of nutrient supplement may be combined freely
according to the above limitation and determined by experimental
practice.
[0130] In some embodiments, the method disclosed herein further
comprises adding zinc into said culture medium during production of
the recombinant polypeptide. In some embodiments, zinc may be added
to provide a zinc concentration of from about 1 to about 300 .mu.M
in said culture medium. In one embodiment, zinc may be added to
provide a zinc concentration of from about 10 to about 200 .mu.M
(e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,
or 150 .mu.M) in the culture medium. In some embodiments, zinc is
added to provide a zinc concentration in the culture medium of from
about 25 .mu.M to about 150 .mu.M, or about 60 .mu.M to about 150
.mu.M. In one embodiment, zinc is added to provide a zinc
concentration in the culture medium of from about about 30, 60, or
90 .mu.M of zinc. In some embodiments, the zinc is added into said
culture medium in a bolus, continuously, semi-continuously, or
combinations thereof. In some embodiments, zinc is added one day,
two days, three days, four days, five days, six days, seven days,
eight days, nine days, ten days, eleven days, twelve days, and/or
thirteen days after inoculation.
Harvest
[0131] Prior studies suggested that delaying harvest timing was
associated with a viability and TSAC decline, so harvest timing can
have a potential impact on other CQAs. In various embodiments,
alkaline phosphatase (e.g., asfotase alfa) is harvested at a time
point of about 200 hr, 210 hr, 220 hr, 230 hr, 240 hr, 250 hr, 260
hr, 264 hr, 270 hr, 280 hr, 288 hr (i.e., 12 days), or more than 12
days.
Downstream Processes
[0132] The term "downstream process(es)" used herein is generally
referred to the whole or part(s) of the processes for recovery and
purification of the alkaline phosphatases (e.g., asfotase alfa)
produced from sources such as culture cells or fermentation
broth.
[0133] Generally, downstream processing brings a product from its
natural state as a component of a tissue, cell or fermentation
broth through progressive improvements in purity and concentration.
For example, the removal of insolubles may be the first step, which
involves the capture of the product as a solute in a
particulate-free liquid (e.g., separating cells, cell debris or
other particulate matter from fermentation broth). Exemplary
operations to achieve this include, e.g., filtration,
centrifugation, sedimentation, precipitation, flocculation,
electro-precipitation, gravity settling, etc. Additional operations
may include, e.g., grinding, homogenization, or leaching, for
recovering products from solid sources, such as plant and animal
tissues. The second step may be a "product-isolation" step, which
removes components whose properties vary markedly from that of the
desired product. For most products, water is the chief impurity and
isolation steps are designed to remove most of it, reducing the
volume of material to be handled and concentrating the product.
Solvent extraction, adsorption, ultrafiltration, and precipitation
may be used alone or in combinations for this step. The next step
is about product purification, which separates contaminants that
resemble the product very closely in physical and chemical
properties. Possible purification methods include, e.g., affinity,
ion-exchange chromatography, hydrophobic interaction
chromatography, mixed-mode chromatography, size exclusion, reversed
phase chromatography, ultrafiltration-diafiltration,
crystallization and fractional precipitation. In some embodiments,
the downstream processes comprise at least one of harvest
clarification, ultrafiltration, diafiltration, viral inactivation,
affinity capture, and combinations thereof. Downstream processes
are described herein.
Determination of Total Sialic Acid Content
[0134] Commercial methods of carbohydrate quantification are
available, e.g., from ThermoFisher. Generally, TSAC is released
from a glycoprotein, e.g., asfotase alfa, using acid hydrolysis,
and released sugars/TSAC are detected via electrochemical detection
using column chromatography such as High-Performance Anion-Exchange
Chromatography with Pulsed Amperometric Detection technique
(HPAE-PAD). The resulting levels are quantified per mole against an
internal standard and expressed as a function of the total mole
protein.
[0135] In some embodiments, the methods described herein further
comprise measuring the total sialic acid content (TSAC) of the
recombinant alkaline phosphatase. As described herein, TSAC impacts
the half-life of the recombinant alkaline phosphatase in
physiological conditions, and thus serves as a critical quality
attribute for recombinantly-produced alkaline phosphatases such as,
e.g., asfotase alfa. Tight control of the TSAC range is important
for reproducibility and cGMP. In some embodiments, the TSAC is
about 0.8 mol/mol to about 4.0 mol/mol recombinant alkaline
phosphatase. In some embodiments, the TSAC is about 0.9 mol/mol to
about 3.0 mol/mol recombinant alkaline phosphatase. In some
embodiments, the TSAC is about 1.0 mol/mol to about 2.8 mol/mol
recombinant alkaline phosphatase. In some embodiments, the TSAC is
about 1.2 mol/mol to about 3.0 mol/mol recombinant alkaline
phosphatase. In some embodiments, the TSAC is about 1.2 mol/mol to
about 2.4 mol/mol recombinant alkaline phosphatase. In some
embodiments, the TSAC is about 0.9 mol/mol, about 1.0 mol/mol,
about 1.1 mol/mol, about 1.2 mol/mol, about 1.3 mol/mol, about 1.4
mol/mol, about 1.5 mol/mol, about 1.6 mol/mol, about 1.7 mol/mol,
about 1.8 mol/mol, about 1.9 mol/mol, about 2.0 mol/mol, about 2.1
mol/mol, about 2.2 mol/mol, about 2.3 mol/mol, about 2.4 mol/mol,
about 2.5 mol/mol, about 2.6 mol/mol, about 2.7 mol/mol, about 2.8
mol/mol, about 2.9 mol/mol, or about 3.0 mol/mol recombinant
alkaline phosphatase.
[0136] In some embodiments, the TSAC of recombinant alkaline
phosphatase decreases during downstream processing. In some
embodiments, the TSAC of recombinant alkaline phosphatases
decreases as a result of sialidase enzymes present in the solution
containing recombinant alkaline phosphatase, e.g., the cell
culture, the HCCF, and/or the UFDF filtration pool. In some
embodiments, sialidases are selectively removed from the cell
culture, the HCCF, and/or the UFDF filtration pool to achieve a
TSAC of about 0.9 mol/mol to about 3.0 mol/mol recombinant alkaline
phosphatase. Sialidases can be selectively removed by, e.g., one or
a combination of sialidase-specific inhibitors, antibodies, ion
exchange and/or affinity chromatography, immunoprecipitation, and
the like.
[0137] In some embodiments, sialic acid moieties are added to
recombinant alkaline phosphatase by sialyltransferase enzymes
present in the solution containing recombinant alkaline
phosphatase, e.g., the cell culture, the HCCF, and/or the UFDF
filtration pool. In some embodiments, recombinant
sialyltransferases are added exogenously to the cell culture, the
HCCF, and/or the UFDF filtration pool to achieve a TSAC of about
0.9 to about 3.0 mol/mol recombinant alkaline phosphatase.
Determination of Recombinant Alkaline Phosphatase Activity
[0138] In some embodiments, the methods described herein further
comprise measuring recombinant alkaline phosphatase activity. In
some embodiments, the activity is selected from a method selected
from at least one of a pNPP-based alkaline phosphatase enzymatic
assay and an inorganic pyrophosphate (PPi) hydrolysis assay. In
some embodiments, at least one of the recombinant alkaline
phosphatase Kcat and Km values increases in an inorganic
pyrophosphate (PPi) hydrolysis assay. In some embodiments, the
method comprises determining an integral of viable cell
concentration (IVCC).
[0139] The last step may be used for product polishing, the
processes which culminate with packaging of the product in a form
that is stable, easily transportable and convenient. Storage at
2-8.degree. C., freezing at -20.degree. C. to -80.degree. C.,
crystallization, desiccation, lyophilization, freeze-drying and
spray drying are exemplary methods in this final step. Depending on
the product and its intended use, product polishing may also
sterilize the product and remove or deactivate trace contaminants
(e.g., viruses, endotoxins, metabolic waste products, and
pyrogens), which may compromise product safety.
[0140] Product recovery methods may combine two or more steps
discussed herein. For example, expanded bed adsorption (EBA)
accomplishes removal of insolubles and product isolation in a
single step. For a review of EBA, see Kennedy, Curr Protoc Protein
Sci. 2005 June; Chapter 8: Unit 8.8. In addition, affinity
chromatography often isolates and purifies in a single step.
[0141] For a review of downstream processes for purifying a
recombinant protein produced in culture cells, see Rea, 2008
Solutions for Purification of Fc-fusion Proteins. BioPharm Int.
Supplements March 2:20-25. The downstream processes for alkaline
phosphatases disclosed herein may include at least one, or any
combination, of exemplary step described herein.
Harvest Clarification Process
[0142] In some embodiments of the method, the recombinant alkaline
phosphatase is isolated from the cell culture by at least one
purification step to form harvest clarified culture fluid (HCCF),
i.e., a "harvesting" step or harvest clarification step.
"Harvesting" the cell culture typically refers to the process of
collecting the cell culture from the culture container, e.g., a
bioreactor. In some embodiments, the at least one purification step
comprises at least one of filtration, centrifugation, and
combinations thereof. In some embodiments, the harvest
clarification step comprises centrifuging and/or filtering the
harvested cell culture in order to remove cells and cellular debris
(e.g., insoluble biomaterials) to recover the product, i.e., the
recombinant alkaline phosphatase. In some embodiments, the cells
and cellular debris are removed in order to yield a clarified,
filtered fluid suitable for chromatography. In some embodiments,
the clarified, filtered fluid is known as harvest clarified culture
fluid, or HCCF. In some embodiments, the cell culture is subjected
to a combination of centrifugation and depth filtration to generate
the HCCF. Possible used solutions in this step may include a
recovery buffer (e.g., 50 mM Sodium Phosphate, 100 mM NaCl, pH
7.50). The composition of suitable recovery buffers may be selected
by the skilled artisan.
[0143] In some embodiments, the HCCF has a total sialic acid
content (TSAC) of from about 2.1 mol/mol to about 4.3 mol/mol. In
some embodiments, the HCCF has a TSAC of from about 2.2 mol/mol to
about 3.6 mol/mol. In some embodiments, the HCCF has a TSAC of from
about 2.2 mol/mol to about 3.4 mol/mol. In some embodiments, the
HCCF has a TSAC of about 2.0, about 2.1, about 2.2, about 2.3,
about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9,
about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5,
about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1,
about 4.2, about 4.3, about 4.4, or about 4.5 mol/mol.
Post-Harvest Ultrafiltration and/or Diafiltration
[0144] In some embodiments of the method, an additional
purification step is performed after the at least one purification
step to form a filtration pool, also known as an "UFDF pool" or
"UFDF." In some embodiments, the at least one purification step is
for concentration and buffer dilution. In some embodiments, the at
least one purification step comprises at least one of harvest
clarification, filtration, ultrafiltration, diafiltration, viral
inactivation, affinity capture, and combinations thereof. In some
embodiments, the at least one purification step comprises
ultrafiltration (UF) and/or diafiltration (DF). Exemplary steps for
the UF process include, e.g., pre-use cleaning/storage of the
filter membrane, post-clean/post-storage flush, equilibration
(e.g., with a buffer containing 50 mM sodium phosphate, 100 mM
NaCl, pH 7.50), loading, concentration, dilution/flush/recovery
(e.g., with a buffer containing 50 mM sodium phosphate, 100 mM
NaCl, pH 7.50), and post-use flush/clean/storage of the filter
membrane.
[0145] In some embodiments, after UF/DF, the UFDF is diluted to a
protein concentration of about 1.7 g/L to about 5.3 g/L, then
maintained at about 13.degree. C. to about 27.degree. C. for about
1 to about 60 hours, prior to storage and/or further purification.
"Holding" or "maintaining" the UFDF, as used herein, refers to the
UFDF being kept at the same temperature (within .+-.about 1.degree.
C.) for a target length of time, i.e., the "hold time" (within
.+-.about 2 hours). In some embodiments, the UFDF is held in order
to serve as a control point in the recombinant alkaline phosphatase
production process. In some embodiments, the UFDF is held in order
to ensure uniform product quality. In some embodiments, the UFDF is
held in order to facilitate downstream processing.
[0146] In some embodiments, the TSAC of the recombinant alkaline
phosphatase decreases during the UFDF hold time. In some
embodiments, the TSAC decline is correlated with the protein
concentration, length of time, and/or temperature during the UFDF
hold time.
[0147] In some embodiments, the start of the UFDF hold time is
immediately after the end of the chromatography step. In some
embodiments, the start of the UFDF hold time is immediately after
the end of the UF/DF. In some embodiments, the start of the UFDF
hold time is immediately after the completion of a recirculation at
the end of the UF/DF step. In some embodiments, the start of the
UFDF hold time is immediately after the UF/DF product filtration
and transfer is completed.
[0148] In some embodiments, the UFDF is diluted to achieve a
desired protein concentration. In some embodiments, the UFDF has a
protein concentration of about 1.0 g/L to about 6.0 g/L. In some
embodiments, the UFDF has a protein concentration of about 1.7 g/L
to about 5.3 g/L. In some embodiments, the UFDF has a protein
concentration of about 2.0 g/L to about 5.0 g/L. In some
embodiments, the UFDF has a protein concentration of about 2.3 g/L
to about 4.3 g/L. In some embodiments, the UFDF has a protein
concentration of about 3.0 g/L to about 4.5 g/L. In some
embodiments, the UFDF has a protein concentration of about 3.3 g/L
to about 4.1 g/L. In some embodiments, the UFDF has a protein
concentration of about 2.0 g/L, about 2.1 g/L, about 2.2 g/L, about
2.3 g/L, about 2.4 g/L, about 2.5 g/L, about 2.6 g/l, about 2.7
g/L, about 2.8 g/L, about 2.9 g/L, about 3.0 g/L, about 3.1 g/L,
about 3.2 g/L, about 3.3 g/L, about 3.4 g/L, about 3.5 g/L, about
3.6 g/L, about 3.7 g/L, about 3.8 g/L, about 3.9 g/L, about 4.0
g/L, about 4.1 g/L, about 4.2 g/L, about 4.3 g/L, about 4.4 g/L, or
about 4.5 g/L.
[0149] In some embodiments, the UFDF comprises a combination of
recombinant alkaline phosphatase and other proteins. In some
embodiments, the UFDF has an alkaline phosphatase concentration of
about 2.0 g/L to about 6.0 g/L. In some embodiments, the UFDF has
an alkaline phosphatase concentration of about 2.5 g/L to about 5.0
g/L. In some embodiments, the UFDF has an alkaline phosphatase
concentration of about 3.0 g/L to about 4.5 g/L. In some
embodiments, the UFDF has an alkaline phosphatase concentration of
about 3.3 g/L to about 4.1 g/L. In some embodiments, the UFDF has
an alkaline phosphatase concentration of about 3.0 g/L, about 3.1
g/L, about 3.2 g/L, about 3.3 g/L, about 3.4 g/L, about 3.5 g/L,
about 3.6 g/L, about 3.7 g/L, about 3.8 g/L, about 3.9 g/L, about
4.0 g/L, about 4.1 g/L, about 4.2 g/L, about 4.3 g/L, about 4.4
g/L, or about 4.5 g/L.
[0150] In some embodiments, the UFDF is held for about 1 hour to
about 60 hours. In some embodiments, the UFDF is held for about 10
hours to about 50 hours. In some embodiments, the UFDF is held for
about 12 hours to about 48 hours. In some embodiments, the UFDF is
held for about 14 hours to about 42 hours. In some embodiments, the
UFDF is held for about 17 hours to about 34 hours. In some
embodiments, the UFDF is held for about 19 hours to about 33 hours.
In some embodiments, the UFDF is held for about 25 to about 38
hours. In some embodiments, the UFDF is held for about 29 to about
35 hours. In some embodiments, the UFDF is held for about 12 hours,
about 13 hours, about 14 hours, about 15 hours, about 16 hours,
about 17 hours, about 18 hours, about 19 hours, or about 20 hours.
In some embodiments, the UFDF is held for about 29 hours, about 30
hours, about 31 hours, about 32 hours, about 33 hours, about 34
hours, or about 35 hours. In some embodiments, the UFDF is held for
about 42 hours, about 43 hours, about 44 hours, about 45 hours,
about 46 hours, about 47 hours, or about 48 hours.
[0151] In some embodiments, the UFDF is held at a temperature of
about 10.degree. C. to about 30.degree. C. In some embodiments, the
UFDF is held at a temperature of about 13.degree. C. to about
27.degree. C. In some embodiments, the UFDF is held at a
temperature of about 14.degree. C. to about 26.degree. C. In some
embodiments, the UFDF is held at a temperature of about 15.degree.
C. to about 26.degree. C. In some embodiments, the UFDF is held at
a temperature of about 15.degree. C. to about 25.degree. C. In some
embodiments, the UFDF is held at a temperature of about 19.degree.
C. to about 25.degree. C. In some embodiments, the UFDF is stored
at the end of the hold time until further downstream processing
steps are performed. In some embodiments, the UFDF is stored at
-80.degree. C. after flash freezing.
[0152] In some embodiments, the at least one additional
purification step further comprises a viral inactivation step. In
some embodiments, the viral inactivation step comprises a
solvent/detergent viral inactivation process to chemically
inactivate viral particles. Exemplary solvent/detergent may
comprise 10% Polysorbate 80, 3% TNBP, 50 mM Sodium Phosphate, and
100 mM NaCl.
Chromatography
[0153] In some embodiments of the method, the UFDF is subjected to
at least one chromatography step to obtain partially purified
recombinant alkaline phosphatase. In some embodiments, the UFDF is
subjected to at least one chromatography step to obtain partially
purified recombinant alkaline phosphatase, wherein the recombinant
alkaline phosphatase has a total sialic acid content (TSAC) of
about 0.9 mol/mol to about 3.0 mol/mol. In some embodiments, the at
least one chromatography step is performed to further purify the
product and/or separate the impurities/contaminants. In some
embodiments, the at least one chromatography step is protein
chromatography. In some embodiments, the protein chromatography is
gel filtration chromatography, ion exchange chromatography,
reversed-phase chromatography (RP), affinity chromatography,
expanded bed adsorption (EBA), mixed-mode chromatography, and/or
hydrophobic interaction chromatography (HIC). In some embodiments,
the protein chromatography is affinity chromatography. In some
embodiments, the protein chromatography is Protein A
chromatography. In some embodiments, the Protein A chromatography
captures the product (i.e., the alkaline phosphatase, such as
asfotase alfa). For example, a process of GE Healthcare Mab Select
SuRe Protein A chromatography may be used. Exemplary buffers and
solutions used in Protein A chromatography include, e.g.,
equilibration/wash buffer (e.g., 50 mM Sodium Phosphate, 100 mM
NaCl, pH 7.50), elution buffer (e.g., 50 mM Tris, pH 11.0), strip
buffer (e.g., 100 mM Sodium Citrate, 300 mM NaCl, pH 3.2), flushing
buffer, cleaning solution (e.g., 0.1 M NaOH), etc.
[0154] In some embodiments, the at least one chromatography step
comprises an additional chromatography and/or purification step. In
some embodiments, the at least one additional chromatography step
comprises column chromatography. In some embodiments, the column
chromatography is gel filtration chromatography, ion exchange
chromatography, reversed-phase chromatography (RP), affinity
chromatography, expanded bed adsorption (EBA), mixed-mode
chromatography, and/or hydrophobic interaction chromatography
(HIC). In some embodiments, the column chromatography comprises
hydrophobic interaction chromatography (HIC). In some embodiments,
the HIC uses Butyl Sepharose or CAPTO.RTM. Butyl agarose columns.
Exemplary buffers and solutions used in a CAPTO.RTM. Butyl agarose
HIC process include, e.g., loading dilution
buffer/pre-equilibration buffer (e.g., 50 mM sodium phosphate, 1.4
M sodium sulfate, pH 7.50), equilibration buffer/wash
buffer/elution buffer (e.g., all containing sodium phosphate and
sodium sulfate), strip buffer (e.g., containing sodium phosphate),
etc. Exemplary buffers and solutions used in a Butyl HIC process
include, e.g., loading dilution buffer/pre-equilibration buffer
(e.g., 10 mM HEPES, 2.0 M ammonium sulfate, pH 7.50), equilibration
buffer/wash buffer(s)/elution buffer (e.g., all containing sodium
phosphate or HEPES and ammonium sulfate), and strip buffer (e.g.,
containing sodium phosphate).
[0155] In some embodiments, the at least one additional
purification step comprises an additional diafiltration. In some
embodiments, the at least one additional chromatography and/or
purification step comprises hydrophobic interaction chromatography
and/or at least an additional diafiltration step. In some
embodiments, the additional diafiltration step is performed after a
hydrophobic interaction chromatography step. In some embodiments,
the additional diafiltration step is performed for product
concentration and/or buffer exchange. Exemplary buffers and
solutions used in this process include, e.g., equilibration buffer
(e.g., 20 mM Sodium Phosphate, 100 mM NaCl, pH 6.75), diafiltration
buffer (20 mM Sodium Phosphate, 100 mM NaCl, pH 6.75), etc.
[0156] In some embodiments, the at least one additional
chromatography and/or purification step is performed to obtain
recombinant alkaline phosphatase with a TSAC of about 0.5 mol/mol
to about 4.0 mol/mol. In some embodiments, the at least one
additional chromatography and/or purification step is performed to
obtain recombinant alkaline phosphatase with a TSAC of about 0.9
mol/mol to about 3.9 mol/mol. In some embodiments, the at least one
additional chromatography and/or purification step is performed to
obtain recombinant alkaline phosphatase with a TSAC of about 1.1
mol/mol to about 3.2 mol/mol. In some embodiments, the at least one
additional chromatography and/or purification step is performed to
obtain recombinant alkaline phosphatase with a TSAC of about 1.4
mol/mol to about 2.6 mol/mol. In some embodiments, the at least one
additional chromatography and/or purification step is performed to
obtain recombinant alkaline phosphatase with a TSAC of about 1.2
mol/mol to about 3.0 mol/mol. In some embodiments, the at least one
additional chromatography step is performed to obtain recombinant
alkaline phosphatase with a TSAC of about 0.8 mol/mol, about 0.9
mol/mol, 1.0 mol/mol, about 1.1 mol/mol, about 1.2 mol/mol, about
1.3 mol/mol, about 1.4 mol/mol, about 1.5 mol/mol, about 1.6
mol/mol, about 1.7 mol/mol, about 1.8 mol/mol, about 1.9 mol/mol,
about 2.0 mol/mol, about 2.1 mol/mol, about 2.2 mol/mol, about 2.3
mol/mol, about 2.4 mol/mol, about 2.5 mol/mol, about 2.6 mol/mol,
about 2.7 mol/mol, about 2.8 mol/mol, about 2.9 mol/mol, about 3.0
mol/mol, about 3.1 mol/mol, about 3.2 mol/mol, about 3.3 mol/mol,
about 3.4 mol/mol, about 3.5 mol/mol, about 3.6 mol/mol, about 3.7
mol/mol, about 3.8 mol/mol, about 3.9 mol/mol, or about 4.0
mol/mol.
Additional Downstream Processes
[0157] In some embodiments, additional downstream processes are
performed in addition to the at least one purification step, the
additional purification step, the at least one chromatography step,
and/or the additional chromatography step. In some embodiments, the
additional downstream processes further purify the product, i.e.,
the recombinant alkaline phosphatase.
[0158] In some embodiments, the additional downstream processes
include a viral reduction filtration process to further remove any
viral particles. In some embodiments, the viral reduction
filtration process is nanofiltration.
[0159] In some embodiments, the additional downstream processes
include at least one further chromatography step. In some
embodiments, the at least one further chromatography step is
protein chromatography. In some embodiments, the protein
chromatography is gel filtration chromatography, ion exchange
chromatography, reversed-phase chromatography (RP), affinity
chromatography, expanded bed adsorption (EBA), mixed-mode
chromatography, and/or hydrophobic interaction chromatography
(HIC). In some embodiments, the third chromatography step is
mixed-mode chromatography, such as CAPTO.RTM. Adhere agarose
chromatography. Commercially available mixed-mode materials
include, e.g., resins containing hydrocarbyl amine ligands (e.g.,
PPA Hypercel and HEA Hypercel from Pall Corporation, Port
Washington, N.Y.), which allow binding at neutral or slightly basic
pH, by a combination of hydrophobic and electrostatic forces, and
elution by electrostatic charge repulsion at low pH (see Brenac et
al., 2008 J Chromatogr A. 1177:226-233); resins containing
4-mercapto-ethyl-pyridine ligand (MEP Hypercel, Pall Corporation),
which achieves hydrophobic interaction by an aromatic residue and
the sulfur atom facilitates binding of the target protein by
thiophilic interaction (Lees et al., 2009 Bioprocess Int. 7:42-48);
resins such as CAPTO.RTM. MMC mixed-mode chromatography and
CAPTO.RTM. adhere agarose chromatography (GE Healthcare, Amersham,
UK) containing ligands with hydrogen bonding groups and aromatic
residues in the proximity of ionic groups, which leads to the
salt-tolerant adsorption of proteins at different conductivities
(Chen et al., 2010 J Chromatogr A. 1217:216-224); and other known
chromatography materials, such as affinity resins with dye ligands,
hydroxyapatite, and some ion-exchange resins (including, but not
limited to, Amberlite CG 50 (Rohm & Haas, Philadelphia, Pa.) or
Lewatit CNP 105 (Lanxess, Cologne, Del.). For an exemplary agarose
HIC chromatography step, exemplary buffers and solutions used in
this process include, e.g., pre-equilibration buffer (e.g., 0.5 M
Sodium Phosphate, pH 6.00), equilibration/wash buffer (e.g., 20 mM
Sodium Phosphate, 440 mM NaCl, pH 6.50), load titration buffer
(e.g., 20 mM Sodium Phosphate, 3.2 M NaCl, pH 5.75), pool dilution
buffer (e.g., 25 mM Sodium Phosphate, 150 mM NaCl, pH 7.40), and
strip buffer (0.1 M Sodium Citrate, pH 3.20.
[0160] In some embodiments, the additional downstream processes
comprise a virus filtration step for viral clearance. In some
embodiments, the viral filtration step is performed by size
exclusion chromatography. Exemplary buffers and solutions used in
this process include, e.g., pre-use and post-product flush buffer
(e.g., 20 mM Sodium Phosphate, 100 mM NaCl, pH 6.75).
[0161] In some embodiments, the additional downstream processes
comprise a formulation process. In some embodiments, the
formulation process comprises at least one further ultrafiltration
and/or diafiltration for further concentration and/or buffer
exchange. Exemplary buffers and solutions used in this process
include, e.g., filter flush/equilibration/diafiltration/recovery
buffer (e.g., 25 mM Sodium Phosphate, 150 mM NaCl, pH 7.40).
[0162] In some embodiments, the additional downstream processes
comprise a bulk fill process. In some embodiments, the bulk fill
process comprises sterile filtration. Exemplary filters for sterile
filtration are Millipak 60 or Equivalent sized PVDF filters (EMD
Millipore, Billerica, Mass.
[0163] In some embodiments, the steps used for producing,
purifying, and/or separating the alkaline phosphatase from the
culture cells, as disclosed herein, further comprise at least one
of steps selected from the group consisting of: a harvest
clarification process (or a similar process to remove the intact
cells and cell debris from the cell culture), an ultrafiltration
(UF) process (or a similar process to concentrate the produced
alkaline phosphatase), a diafiltration (DF) process (or a similar
process to change or dilute the buffer comprising the produced
alkaline phosphatase from previous processes), a viral inactivation
process (or a similar process to inactivate or remove viral
particles), an affinity capture process (or any one of
chromatography methods to capture the produced alkaline phosphatase
and separate it from the rest of the buffer/solution components), a
formulation process and a bulk fill process. In one embodiment, the
steps for producing, purifying, and/or separating the alkaline
phosphatase from the culture cells, as disclosed herein, comprise
at least a harvest clarification process (or a similar process to
remove the intact cells and cell debris from the cell culture), a
post-harvest ultrafiltration (UF) process (or a similar process to
concentrate the produced alkaline phosphatase), a post-harvest
diafiltration (DF) process (or a similar process to change or
dilute the buffer comprising the produced alkaline phosphatase from
previous processes), a solvent/detergent viral inactivation process
(or a similar process to chemically inactivate viral particles an
intermediate purification process (such as hydrophobic interaction
chromatography (HIC) or any one of chromatography methods to
capture the produced alkaline phosphatase and separate it from the
rest of the buffer/solution components), a post-HIC UF/DF process
(or a similar process to concentrate and/or buffer exchange for the
produced alkaline phosphatase), a viral reduction filtration
process (or a similar process to further remove any viral particles
or other impurities or contaminants); a mixed-mode chromatography
(such as CAPTO.RTM. Adhere agarose chromatography, or a similar
process to further purify and/or concentrate the produced alkaline
phosphatase), a formulation process and a bulk fill process. In one
embodiment, the separating step of the method provided herein
further comprises at least one of harvest clarification,
ultrafiltration, diafiltration, viral inactivation, affinity
capture, HIC chromatography, mixed-mode chromatography and
combinations thereof. FIG. 1 is an exemplary illustration of an
embodiment of the production process of a recombinant alkaline
phosphatase, asfotase alfa.
[0164] In some embodiments, the disclosure provides a method for
controlling total sialic acid content (TSAC) in a TSAC-containing
recombinant protein through mammalian cell culture, comprising at
least one purification step and at least one chromatography step.
In some embodiments, the disclosure provides a method for
controlling glycosidase activity in mammalian cell culture
producing recombinant protein, comprising at least one purification
step and at least one chromatography step. In some embodiments, the
at least one purification step comprises at least one of
filtration, centrifugation, harvest clarification, filtration,
ultrafiltration, diafiltration, viral inactivation, affinity
capture, and combinations thereof. In some embodiments, the at
least one chromatography step comprises protein chromatography. In
some embodiments, the protein chromatography is gel filtration
chromatography, ion exchange chromatography, reversed-phase
chromatography (RP), affinity chromatography, expanded bed
adsorption (EBA), mixed-mode chromatography, and/or hydrophobic
interaction chromatography (HIC). In some embodiments, the
purification step and chromatography step are
ultrafiltration/diafiltration and protein A chromatography.
[0165] All references cited herein are incorporated by reference in
their entirety. Although the foregoing disclosure has been
described in some detail by way of illustration and example for
purposes of clarity of understanding, it is apparent to those
skilled in the art that certain minor changes and modifications
will be practiced. Therefore, the description and examples should
not be construed as limiting the scope of the disclosure.
EXAMPLES
Example 1: Production of Asfotase Alfa and TSAC Measurement
[0166] Asfotase alfa (or other glycoprotein of interest) is
manufactured by known methods. In particular, inoculum expansion is
performed from the appropriate recombinant cell line (i.e.,
transfected CHO cells carrying GS resistance marker, selected in
the presence of MSX), which is sample aliquoted into the
appropriate growth media, then further grown in a bioreactor at
controlled temperature, pH, DO.sub.2, and agitation. Typical
production methods for asfotase alfa are fed-batch bioreactor
methods with CHO cells, though other methods are also acceptable.
When a desired cell density and/or cell viability has been reached,
the primary recovery from the raw Cell Culture Fluid (CCF) is
performed by centrifugation and depth filtration which results in
the Harvest Cell Culture Fluid (HCCF).
[0167] The HCCF is further purified by ultrafiltration and
diafiltration to form the UFDF. Further chromatographic
purification steps are performed, such as viral inactivation,
protein A chromatography (resulting in a protein A pool),
multimodal chromatography purification, and additional
ultrafiltration and diafiltration steps. The final bulk drug
substance, or purified recombinant asfotase alfa, is tested for
release specifications. The bulk drug substance proceeds to final
fill finish steps, which result in final packaging suitable for
administration.
[0168] During manufacturing, the TSAC content is an important value
and is monitored closely. However, the specific impact of hold time
and temperature on TSAC was not fully understood. Therefore,
identification and quantitation of sialic acid in asfotase alfa
samples at various stages of manufacture was performed by high
performance anion exchange chromatography with pulsed amperometric
detection (HPAE-PAD). (Commercial systems for carbohydrate
detection and quantitation are available, i.e., ThermoScientific
and others.) Samples were first spiked with internal standard and
dried in a rotary evaporator and sialic acids were released via
acid hydrolysis (0.1M HCl). Samples were dried again to remove
residual acid then reconstituted in water to be injected into a
capillary ion chromatography system (Dionex IC-5000). After
injection, samples undergo anion exchange chromatography in an
alkaline environment using an acetate gradient for separation of
sialic acid species, from 5% acetate buffer to 30% acetate buffer
over 30 minutes with a 5 minute equilibration at 5% acetate buffer
at the end of each injection. Separation occurs in an HPLC column
designed for carbohydrate separation (i.e., CarboPac PA100 column,
where a stationary phase of nonporous beads coated with latex in
conjunction with alkaline mobile phases separates based on charge).
Post separation, samples were detected via pulsed amperometric
detection. A repeating waveform oxidized samples at the surface of
a gold electrode, where the difference of electrical potential
between oxidized sample and the gold electrode is measured. Results
are plotted as electrical potential (nC) vs time and peaks are
integrated. Percent of internal standard (% ISTD) is calculated via
NeuSAc or Neu5Gc peak area divided by the internal standard,
3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN,
Sigma-Aldrich), area multiplied by 100, and quantitated by
comparison to a standard curve of standard % ISTD vs standard
concentration. Sample concentration is determined by interpolating
sample % ISTD from the standard curve. A blend of Neu5Ac and Neu5Gc
sialic acids was used as an assay standard, allowing the method to
quantitate both species. Results are reported in nmol Neu5Ac per
nmol monomer. Neu5Gc is not expected to be present in amounts above
the LOQ of the assay; however samples exhibiting quantifiable
Neu5Gc are noted in routine experimentation.
[0169] The mole ratio of sialic acid to protein for each sample is
calculated by dividing the nmol amount of sialic acid recovered by
the nmol amount of protein (i.e., asfotase alfa) hydrolyzed by
using the concentration (as determined by absorbance of the sample
at 280 nm and adjusted by the molar extinction coefficient and the
molecular weight).
[0170] Additional details on the some embodiments of the methods of
the production of asfotase alfa can be found in International
Publications WO 2017/031114 and WO 2017/214130, the disclosures of
which are hereby incorporated by reference in their entireties.
Example 2: Evaluation of UFDF Hold Time, Temperature, and Protein
Concentration on TSAC
[0171] During large-scale production of asfotase alfa (as described
generally in Example 1), a drop in TSAC from harvested cell culture
fluid (HCCF) to Protein A pool was observed. This difference was
expected to be largely due to the post-harvest
concentration/diafiltration (UF/DF1) hold step. The average
decrease in TSAC observed from HCCF to protein A pool was
approximately 1.1 mol/mol with a standard deviation of 0.2 mol/mol,
with a range of 0.9 mol/mol to 1.3 mol/mol.
[0172] FIG. 2 illustrates the relative protein sialyation of cells
grown with the addition of various nutrient supplements for 14
days, and with and without a temperature shift of about 37.degree.
C. to about 30.degree. C. when the culture reaches a cell density
of at least about 2.5.times.10.sup.6 viable cells. Brx-1=Control
Process with temperature shift; Brx-2=Control Process with
temperature shift; Brx-3=Cell Boost 2+5 with temperature shift;
Brx-4=Cell Boost 2+5 with temperature shift; Brx 5=Cell Boost 6
with temperature shift; Brx-6=Cell Boost 6 without temperature
shift; Brx-7=Cell Boost 7a+7b with temperature shift; Brx-8=Cell
Boost 7a+7b without temperature shift.
[0173] FIG. 3 illustrates the correlation between TSAC decline and
protein concentration (Panel A) and hold temperature (Panel B).
[0174] A small-scale characterization study was executed to assess
and characterize the impact of UF/DF1 hold time, temperature, and
protein concentration on TSAC during the UF/DF1 hold.
Example 2.1: UF/DF1 Operation and Hold
[0175] FIG. 4 outlines the small-scale UF/DF1 operation and hold
time performed for each of three 10 L clarified harvest lots. Table
1 and Table 2 list the target process conditions and process
parameters for the post-harvest UF/DF1 step.
TABLE-US-00002 TABLE 1 UF/DF1 Process Conditions Process Parameter
Target TFF membrane used Millipore Biomax C screen Membrane cutoff
(Da) 50,000 TMP 15 PSI (10-20 PSI target range; 22 PSI maximum)
Cross flow rate 6-8 L/m.sup.2/min Filter area (m.sup.2) 0.1 Filter
load (L/m.sup.2) .ltoreq.250
TABLE-US-00003 TABLE 2 Post Harvest Concentration/Diafiltration
(UF/DF1) Cross Flow Step Buffer Volume Rate WFI Rinse WFI 5
L/m.sup.2 8 L/m.sup.2/min Pre-use Clean 0.5M NaOH 5 L/m.sup.2 8
L/m.sup.2/min WFI Rinse WFI 5 L/m.sup.2 8 L/m.sup.2/min NWP 0.1M
NaOH N/A 5 L/m.sup.2/min Equilibration 50 mM Sodium 5 L/m.sup.2 8
L/m.sup.2/min Phosphate 100 mM Sodium Chloride pH 7.5 Concentration
Harvest 25X maximum 6 L/m.sup.2/min Diafiltration 50 mM Sodium 7X 6
L/m.sup.2/min Phosphate 100 mM Sodium Chloride pH 7.5 Product 50 mM
Sodium Chase/concentrate as 4 L/m.sup.2/min Recovery Phosphate 100
needed to target mM Sodium 4.9 mg/mL and Chloride pH 7.5 dilute
aliquots immediately to target final concentrations..sup.1 WFI
Rinse WFI 20 L/m.sup.2 5 L/m.sup.2/min Clean 0.5M NaOH, 5 L/m.sup.2
8 L/m.sup.2/min 400 ppm bleach WFI Rinse WFI 5 L/m.sup.2 8
L/m.sup.2/min Store 0.2M NaOH 5 L/m.sup.2 8 L/m.sup.2/min
.sup.1Generating samples at high concentration required a final
concentration factor above the transferred range (.ltoreq.25X) and
was governed by harvest titer. To achieve the higher concentration,
a final concentration step (UF1') was performed prior to product
recovery.
[0176] In-process samples at the end of UF1 and end of DF were
pulled for purification and TSAC analysis. Generating samples at
high concentration required a final concentration step (UF1') that
resulted in a concentration factor above the transferred range
(<25 X) and was governed by the estimated harvest titer of each
individual lot. The lower concentrations were achieved by dilution
with DF buffer Immediately following completion of the UF/DF1, the
pool was diluted according to the study design (FIG. 4) and divided
into aliquots and used for the hold study. The UF/DF1 pool was then
split into 15 mL conical tubes and held at the specific target
temperatures.+-.1.degree. C. (15, 19, or 25.degree. C.) in
controlled water baths for 0, 12, 24, 36, 48, and 60 hours before
further processing. All holds were executed .+-.2 hours of hold
target and once timepoints were reached, aliquots were frozen
(-80.degree. C.) until Protein A purification.
[0177] T=0 in this study was defined as the time at which a 10
minute recirculation was completed at the end of the diafiltration
(DF) step. During manufacturing operations, the start of the hold
time is currently defined as the end of bag fill following UF/DF1
product filtration and transfer. The transfer takes approximately 5
hours on average from the end of diafiltration, which is in
addition to the hold time for the UF/DF1 pool.
Example 2.2: High Throughput MabSelect SuRe Protein A
Chromatography
[0178] UF/DF1 hold samples were processed through MabSelect SuRe
pre packed RoboColumns without the solvent/detergent viral
inactivation (S/D VI) step. Removal of the S/D VI step does not
affect TSAC results and improves processing time for the high
throughput RoboColumn method (DVL 16 0128). Table 3 presents the
MabSelect SuRe RoboColumn affinity chromatography step. The
operation was performed using pre-packed MabSelect SuRe RoboColumns
and the Tecan Freedom Evo Liquid Handling system.
[0179] Samples were purified in batches with up to 8 samples per
batch. Prior to purification of each sample batch, samples were
thawed in a water bath (18-25.degree. C.) for .ltoreq.1 hour.
[0180] The elution from each RoboColumn purification was collected
across four 96-well plates (approximately 200 .mu.l per well) and
the absorbance (A280) measured for each well. For each column,
elution wells containing product were pooled via micropipetting on
the basis of the collection criteria defined in Table 3 and pools
were sterile filtered (0.22 .mu.m). The ProA pools were not pH
adjusted or diluted.
TABLE-US-00004 TABLE 3 MabSelect SuRe RoboColumn Chromatography
Flow Rate Step Buffer CV (cm/hr) Clean 0.1M Sodium Hydroxide 5 150
Equilibration 50 mM Sodium 8 150 Phosphate 100 mM Sodium Chloride
pH 7.5 Load UF/DF1 Retentate 100 Post Load 50 mM Sodium 3.5 150
Wash 1 Phosphate 100 mM Sodium Chloride pH 7.5 Elution 50 mM Tris
pH 11.0 16 100 Strip 100 mM Sodium Citrate 5.5 150 300 mM Sodium
Chloride pH 3.2 WFI Flush WFI 3 150 Clean 0.1M Sodium Hydroxide 5
150 Equilibration 50 mM Sodium 3 150 Phosphate 100 mM Sodium
Chloride pH 7.5 Store 18% Ethanol 3 100
Example 2.3: Results--TSAC at HCCF/in-Process Samples
[0181] Table 4 lists the UF/DF1 in-process TSAC data generated
across all three harvest lots. There is a drop in TSAC expected
from HCCF to the start of the UF/DF1 hold (T=0) due to the higher
concentrations of both product and sialidase that are achieved in
the UF1 step and then maintained throughout the remainder of the
UF/DF1 operation. However, this TSAC decrease is only expected to
be a small fraction of the total TSAC drop from HCCF to Protein A
given the processing time is minimal relative to the total UF/DF1
pool hold time. To assess this decrease, TSAC results from all T=0
samples for each harvest UF/DF1 batch were averaged and then
subtracted from the starting TSAC at HCCF of the batch. Given the
duration from end of UF/DF1 operation and T=0 sampling (diluted and
frozen within 2 hours of hold start time), minimal variability
(within .+-.10% assay variability) is expected across the T=0
samples despite the varying protein concentrations. Therefore,
averaging the results from the T=0 samples provides more confidence
in the T=0 result and minimizes the impact of assay
variability.
[0182] As shown in Table 4, the reported TSAC at HCCF for harvest
batch #B is 3.6 mol/mol and the average TSAC result for the T=0
samples is 2.5 mol/mol. A 0.9 mol/mol drop in TSAC is therefore
calculated during the UF/DF1 operation for this lot. This extent of
TSAC drop is more typically seen after the UF/DF1 hold (14-48 hrs)
has been completed, suggesting one of the TSAC results for this lot
may have been an outlier. The additional small scale in process
results for batch #B provided evidence that the TSAC value for HCCF
was the outlier assay result since the averaged TSAC at T=0 aligned
well with the UF/DF1 in process results (End UF1, End DF) shown
below. Additionally, the TSAC value measured for CCF of this
manufacturing batch is 2.7 mol/mol and the difference between the
TSAC results at CCF and HCCF for this batch was inconsistent with
process expectations.
TABLE-US-00005 TABLE 4 HCCF and UF/DF1 In-process sample TSAC Data
summary A B C Harvest Lot # TSAC (mol/mol) HCCF 3.4 3.6 2.9 End UF1
3.3 2.4 2.7 End DF 3.1 2.2 2.7 End of UF1' (T = 0, average) 3.0
2.7.sup.1 2.4 Drop from HCCF to end of UF/DF1 process.sup.2 0.4 0.9
0.5 .sup.1Average of only two TSAC values (excluded low
concentration sample as it will not be included in final analysis)
.sup.2Calculation: TSAC at HCCF - TSAC at End of UF1' (T = 0)
[0183] An accurate TSAC at HCCF result is required to analyze the
TSAC drop from HCCF to the Protein A pool (TSAC drop=TSAC at
HCCF-TSAC at ProA Pool). To minimize the impact of a potentially
aberrant result for TSAC at HCCF for harvest batch #B on subsequent
analyses, an average TSAC value was calculated using in process
results from HCCF through T=0. Included in the average is the
original TSAC at HCCF result of 3.6 mol/mol, the TSAC at end of UF1
(2.4 mol/mol), the TSAC at end of DF (2.2 mol/mol), and the TSAC
values at T=0. In this case, only two of the three T=0 values were
used in this calculation, since the result from the lowest hold
temperature and lowest UF/DF1 protein concentration was ultimately
excluded from the data set (see FIG. 5). As shown in Table 5, an
averaged TSAC at HCCF value of 2.7 mol/mol will be used in the
small scale characterization data analysis for harvest batch #B.
The assumption that 2.7 mol/mol is an appropriate estimate of TSAC
at HCCF for this lot will be assessed as part of the statistical
analysis and validation of the predictive model.
TABLE-US-00006 TABLE 5 Revised HCCF and UF/DF1 In-process sample
TSAC Data summary Harvest Lot # A B C TSAC HCCF 3.4 2.7.sup.1 2.9
(mol/mol) End UF1 3.3 2.4 2.7 End DF 3.1 2.2 2.7 End of UF1' (T =
0, average) 3.0 2.7.sup.2 2.4 Drop from HCCF at end 0.4 0.0 0.5 of
UF/DF1 process.sup.3 .sup.1"Harvest" value = average of QC result,
end UF, end DF, and both T = 0 samples .sup.2Average of only two
TSAC values (excluded low concentration sample as it will not be
included in final analysis) .sup.3Calculation: TSAC at HCCF - TSAC
at End of UF1' (T = 0)
Example 2.4: TSAC at Protein A
[0184] FIG. 5 through FIG. 7 plot the data to show the TSAC decline
during the UF/DF1 hold at the various protein concentrations and
hold temperatures, starting at the T=12 timepoint samples. Overall,
the combined dataset confirms the expected linear drop during the
UF/DF1 hold time. There is also a consistent increase in the slope
of the TSAC decline with increasing protein concentration of the
UF/DF1 pool. However, there is no clear trend between TSAC drop and
the UF/DF1 hold temperature. Similar trends in TSAC drop are
observed across all three datasets indicating consistent TSAC
behavior for the three harvest lots used in this study.
Example 3: TSAC Measurement after Protein A Chromatography and Bulk
Fill
[0185] Total sialic acid content (TSAC) was measured at two steps
of the manufacturing process for asfotase alfa: after Protein A
Chromatography (also referred to as the Protein A or ProA pool) and
Bulk Fill (also referred to as the bulk drug substance or BDS
release) (see FIG. 1). The TSAC data from the ProA pool and the BDS
release were reviewed for 51 manufacturing batches, as shown in
FIG. 8. The BDS TSAC results range from 1.3 to 2.6 mol/mol with a
mean 1.9 mol/mol and standard deviation of 0.3 mol/mol (RSD=17.5%).
As shown in FIG. 8, the BDS TSAC data and the ProA TSAC data trend
closely for each batch, except for batch #524759. The data from
this example indicate that unit operations downstream of the
Protein A pool step do not significantly contribute to TSAC
variability.
Example 4: TSAC Measurement Before and After Harvest
[0186] Total sialic acid content (TSAC) was measured in the cell
culture fluid (CCF) and harvest clarified culture fluid (HCCF) (see
FIG. 1). The TSAC data from the CCF was measured for 39
manufacturing batches, as shown in FIG. 9. The CCF samples were
taken at the end of the Production Bioreactor run and were filtered
to remove cells and cellular debris, and then stored at appropriate
conditions prior to small scale Protein A purification and TSAC
analysis. The measured results of TSAC at the CCF range from 1.9 to
2.9 mol/mol with a mean 2.4 mol/mol and standard deviation of 0.3
mol/mol (RSD=10.4%). TSAC data at harvest (HCCF) was measured for
35 of the 39 batches. The HCCF TSAC results ranged from 2.2 to 3.4
mol/mol with a mean of 2.7 mol/mol and standard deviation of 0.3
mol/mol (RSD=10.7%). As shown in FIG. 9, for batches where data are
available at both the CCF and HCCF, the HCCF TSAC trends similarly
with the CCF TSAC, with approximately a 0.2 mol/mol increase on
average (with the exception of batch #B). The ProA and BDS TSAC
results for this batch align with the CCF TSAC result, and the HCCF
result is considered an outlier. The data from this example
indicate that the harvest process does not add variability to
TSAC.
[0187] Additional multivariate analysis performed on cell culture
fluid did not identify a statistically significant impact from cell
generation number (32 to 51 generations) at the inoculation of
production bioreactor on CCF TSAC (data not shown). A subsequent
analysis confirmed no statistically significant correlation between
generation number with the tested ranged and TSAC at CCF or
HCCF.
Example 5: TSAC Measurement Before and After Harvest
[0188] A review of TSAC measurement collected at various points in
the manufacturing process shows that the decrease in TSAC between
the HCCF and Protein A pool occurs over the hold after UF/DF1 (also
known as the UF1 hold; see FIG. 1). As shown in FIG. 10, TSAC
decreases significantly during the UF1 hold for all batches except
for batch #583389, which specific data point was excluded as an
outlier. The corresponding BDS result for this batch confirms that
a decrease in TSAC result similar to the other batches was observed
during UF1 hold for this batch. The average decrease in TSAC result
over the duration of the UF1 hold was approximately 1.0 mol/mol
with a standard deviation of 0.3 mol/mol (RSD=26.3%), with a range
of 0.5 mol/mol to 1.4 mol/mol.
[0189] Previous analysis identified a correlation between UFDF1
hold time and TSAC variability at BDS. Since the impact of the
UFDF1 hold time, temperature, and protein concentration on TSAC
decrease were not fully understood, additional temperature and
protein concentration dependent data were collected at small scale
to understand the impact on TSAC decrease during the UF1 hold. A
multivariate evaluation of the three factors during UFDF1 hold was
accomplished using a full factorial experimental study design
evaluating a range for each factor (Table 6). This small scale
characterization study design was executed using harvest from three
batches (A, B, and C). Following small scale UFDF 1 operations for
each harvest, UFDF1 pools were held at various hold times,
temperatures, and protein concentrations (Table 6). Samples were
purified using small scale Protein A column for TSAC analysis. The
TSAC at Protein A pool was compared to the TSAC value at HCCF for
the respective harvest and the total decrease in TSAC from HCCF
(TSAC drop) was calculated for each UF1 hold condition.
TABLE-US-00007 TABLE 6 Parameter/Attribute Studied Characterization
Range Hold Temperature (.degree. C.) 15-25 Hold Time (hours) 0-60
Protein Concentration (g/L) 1.8-5.3
[0190] A JMP model was created by performing fit model analysis on
the TSAC drop during UFDF1 hold as per the experimental results.
Each factor (hold time, temperature, and protein concentration) was
included in a factorial analysis using the stepwise regression.
Model outputs included the actual by predicted plot (FIG. 11),
sorted parameter estimates, prediction expression, and prediction
profiler (FIG. 12). The prediction expression was derived from the
parameter estimates and was plotted in the prediction profiler.
[0191] To ensure that the small scale UFDF1 model as well as the
JMP model created from these data are representative of large scale
process, the predictive model was verified for its ability to
predict BDS TSAC for 35 manufactured batches where HCCF TSAC and
UFDF1 hold data (average hold time, protein concentration, and
average hold temperature) are both available. For each manufactured
batch, the predicted TSAC drop was subtracted from the measured
HCCF TSAC to yield predicted ProA TSAC. Based on an average 0.2
mol/mol TSAC increase from ProA to BDS, the predicted BDS TSAC was
calculated. The BDS TSAC was predictable within assay variability
(.+-.20%) for all manufacturing batches with the exception of batch
#B.
[0192] Characterization studies demonstrated that UFDF 1 protein
concentration has a statistically significant (p-value<0.0001)
impact on TSAC decrease during UFDF1 hold. The TSAC decrease during
UF1 hold is believed to occur due to presence of sialidase enzyme
which is known to be present in mammalian cell culture harvest
(Gramer and Goochee, Biotechnol. Prog. 9:366-373 (1993),
incorporated specifically herein by reference). The protein
concentration of asfotase alfa is considered a surrogate for
concentration of sialidase enzyme during UFDF1 hold. However, this
attribute was not defined for TSAC control in the drug substance
manufacturing process.
[0193] In conclusion, the data suggests reducing the range of the
hold time of the UFDF from 14-48 hours to 14-42 hours.
Additionally, the data suggested adding a new performance attribute
for protein concentration at the UFDF hold step with the range
2.0-4.3 g/L.
[0194] All publications, patents, and patent applications mentioned
in the above specification are hereby incorporated by reference to
the same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety. Various modifications
and variations of the described methods, pharmaceutical
compositions, and kits of the instant disclosure will be apparent
to those skilled in the art without departing from the scope and
spirit of the claimed invention. Although the disclosure has been
described in connection with specific embodiments, it will be
understood that it is capable of further modifications and that the
invention as claimed should not be unduly limited to such specific
embodiments.
Sequence CWU 1
1
11726PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 1Leu Val Pro Glu Lys Glu Lys Asp
Pro Lys Tyr Trp Arg Asp Gln Ala1 5 10 15Gln Glu Thr Leu Lys Tyr Ala
Leu Glu Leu Gln Lys Leu Asn Thr Asn 20 25 30Val Ala Lys Asn Val Ile
Met Phe Leu Gly Asp Gly Met Gly Val Ser 35 40 45Thr Val Thr Ala Ala
Arg Ile Leu Lys Gly Gln Leu His His Asn Pro 50 55 60Gly Glu Glu Thr
Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala Leu65 70 75 80Ser Lys
Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly Thr 85 90 95Ala
Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val Gly 100 105
110Val Ser Ala Ala Thr Glu Arg Ser Arg Cys Asn Thr Thr Gln Gly Asn
115 120 125Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys
Ser Val 130 135 140Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr
Pro Ser Ala Ala145 150 155 160Tyr Ala His Ser Ala Asp Arg Asp Trp
Tyr Ser Asp Asn Glu Met Pro 165 170 175Pro Glu Ala Leu Ser Gln Gly
Cys Lys Asp Ile Ala Tyr Gln Leu Met 180 185 190His Asn Ile Arg Asp
Ile Asp Val Ile Met Gly Gly Gly Arg Lys Tyr 195 200 205Met Tyr Pro
Lys Asn Lys Thr Asp Val Glu Tyr Glu Ser Asp Glu Lys 210 215 220Ala
Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Val Asp Thr Trp Lys225 230
235 240Ser Phe Lys Pro Arg Tyr Lys His Ser His Phe Ile Trp Asn Arg
Thr 245 250 255Glu Leu Leu Thr Leu Asp Pro His Asn Val Asp Tyr Leu
Leu Gly Leu 260 265 270Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn
Arg Asn Asn Val Thr 275 280 285Asp Pro Ser Leu Ser Glu Met Val Val
Val Ala Ile Gln Ile Leu Arg 290 295 300Lys Asn Pro Lys Gly Phe Phe
Leu Leu Val Glu Gly Gly Arg Ile Asp305 310 315 320His Gly His His
Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala Val 325 330 335Glu Met
Asp Arg Ala Ile Gly Gln Ala Gly Ser Leu Thr Ser Ser Glu 340 345
350Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr Phe
355 360 365Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala
Pro Met 370 375 380Leu Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile
Leu Tyr Gly Asn385 390 395 400Gly Pro Gly Tyr Lys Val Val Gly Gly
Glu Arg Glu Asn Val Ser Met 405 410 415Val Asp Tyr Ala His Asn Asn
Tyr Gln Ala Gln Ser Ala Val Pro Leu 420 425 430Arg His Glu Thr His
Gly Gly Glu Asp Val Ala Val Phe Ser Lys Gly 435 440 445Pro Met Ala
His Leu Leu His Gly Val His Glu Gln Asn Tyr Val Pro 450 455 460His
Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Gly His Cys465 470
475 480Ala Pro Ala Ser Ser Leu Lys Asp Lys Thr His Thr Cys Pro Pro
Cys 485 490 495Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 500 505 510Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 515 520 525Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 530 535 540Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu545 550 555 560Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 565 570 575His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 580 585
590Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
595 600 605Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu 610 615 620Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr625 630 635 640Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 645 650 655Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 660 665 670Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 675 680 685Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 690 695 700Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys Asp Ile Asp Asp Asp Asp705 710
715 720Asp Asp Asp Asp Asp Asp 725
* * * * *