U.S. patent application number 16/270494 was filed with the patent office on 2020-06-25 for harvest operations for recombinant proteins.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Rachel L.E. ADAMS, Jane V. GUNSON, Kimberly KALEAS, Michael W. LAIRD, Deepa NADARAJAH, Bradley R. SNEDECOR, Richard ST. JOHN.
Application Number | 20200199639 16/270494 |
Document ID | / |
Family ID | 48040433 |
Filed Date | 2020-06-25 |
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United States Patent
Application |
20200199639 |
Kind Code |
A1 |
LAIRD; Michael W. ; et
al. |
June 25, 2020 |
HARVEST OPERATIONS FOR RECOMBINANT PROTEINS
Abstract
The present invention contemplates a method of producing a
recombinant protein comprising (a) fermenting a prokaryotic host
cell wherein said prokaryotic host cell has been transformed with a
nucleic acid encoding said recombinant protein, and (b) harvesting
said recombinant protein under conditions where dO.sub.2 levels are
greater than 0%, and (c) purifying said recombinant protein to a
filtered bulk, wherein said filtered bulk does not contain
detectable DHNA-recombinant protein adduct, as measured by an IEC
assay at 310 nm. The present invention also contemplates a method
of producing a recombinant protein comprising (a) fermenting a menE
gene-deleted prokaryotic host cell wherein said prokaryotic host
cell has been transformed with a nucleic acid encoding said
recombinant protein, (b) harvesting said recombinant protein; and
(c) purifying said recombinant protein to a filtered bulk, wherein
said filtered bulk does not contain detectable DHNA-recombinant
protein adduct, as measured by an IEC assay at 310 nm, and wherein
the recombinant protein yield is increased by about 20% or
greater.
Inventors: |
LAIRD; Michael W.; (San
Ramon, CA) ; ST. JOHN; Richard; (Millbrae, CA)
; GUNSON; Jane V.; (Redwood City, CA) ; KALEAS;
Kimberly; (San Mateo, CA) ; NADARAJAH; Deepa;
(San Mateo, CA) ; ADAMS; Rachel L.E.; (Guelph,
CA) ; SNEDECOR; Bradley R.; (Portola Valley,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
48040433 |
Appl. No.: |
16/270494 |
Filed: |
February 7, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15908526 |
Feb 28, 2018 |
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16270494 |
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14734848 |
Jun 9, 2015 |
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15908526 |
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13826166 |
Mar 14, 2013 |
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14734848 |
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61616297 |
Mar 27, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/00 20130101;
C12P 21/00 20130101 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07K 16/00 20060101 C07K016/00 |
Claims
1. A method of producing a recombinant protein comprising (a)
fermenting a prokaryotic host cell wherein said prokaryotic host
cell has been transformed with a nucleic acid encoding said
recombinant protein, and (b) harvesting said recombinant protein
under conditions where dissolved oxygen (d0.sub.2) levels are
greater than 0%, and (c) purifying said recombinant protein to a
filtered bulk for storage (FBS), wherein said filtered bulk does
not contain detectable 1,4-dihydroxy-2-naphthoate
(DHNA)-recombinant protein adduct, as measured by an ion exchange
chromatography (IEC) assay at 310 nm.
2. The method of claim 1, wherein said analytical assay is by HPLC,
RP HPLC, HIC HPLC, NMR, mass spectrometry, or UV spectroscopy.
3. The method of claim 1, wherein said dO.sub.2 is maintained at
levels greater than 0% continuously throughout the harvest
operations of step (b).
4. The method of claim 3, wherein the harvest operations comprise a
homogenization stage.
5. The method of claim 4, wherein said dO.sub.2 is maintained at
about 30% to about 75% prior to homogenization.
6. The method of claim 4, wherein said dO.sub.2 is maintained at
levels greater than 75% prior to homogenization.
7. The method of claim 4, wherein said dO.sub.2 is maintained at
about 50% after homogenization.
8. The method of claim 4, wherein said dO.sub.2 is maintained at
levels greater than 50% after homogenization.
9. The method of claim 5, wherein said dO.sub.2 is maintained for a
period of greater than or equal to 1.5 hours.
10. The method of claim 6, wherein said dO.sub.2 is maintained for
a period of greater than or equal to 2 hours.
11. The method of claim 1, wherein said dO.sub.2 is maintained with
overlay or sparged air, with increased back-pressure, or with
agitation rate.
12. The method of claim 11, wherein the overlay air is from about
0.4 vvm to about 0.8 vvm.
13. The method of claim 12, wherein the overlay air is targeted at
0.6 vvm.
14. The method of claim 11, wherein the increased backpressure is
between about 1.0 to 30 psi.
15. The method of claim 14, wherein the increase backpressure is
targeted at 19 psi.
16. The method of claim 11, wherein the agitation rate is from
about 6 Watts/L to about 8 Watts/L.
17. The method of claim 16, wherein the agitation rate is targeted
at about 6 Watts/L.
18. A method of producing a recombinant protein comprising (a)
fermenting a menE gene-deleted prokaryotic host cell wherein said
prokaryotic host cell has been transformed with a nucleic acid
encoding said recombinant protein, (b) harvesting said recombinant
protein; and (c) purifying said recombinant protein to a FBS,
wherein said filtered bulk does not contain detectable
1,4-dihydroxy-2-naphthoate (DHNA)-recombinant protein adduct, as
measured by an ion exchange chromatography (IEC) assay at 310
nm.
19. The method of claim 18, wherein the recombinant protein yield
is increased by about 20% or greater, by about 30% or greater, by
about 40% or greater, by about 50% or greater, by about 60% or
greater, as compared to the yield using a control prokaryotic host
cell.
20. The method of claim 1, wherein the fermentation is
scale-independent.
21. The method of claim 1, wherein said recombinant protein is a
recombinant polypeptide or an isolated antibody.
22. The method of claim 1, wherein said prokaryotic host cell is
Escherichia coli (E. coli), Enterobacter, Azotobacter, Erwinia,
Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia,
Shigella, Rhizobia, Vitreoscilla, and Paracoccus.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U. S. patent
application Ser. No. 15/908,526, filed Feb 28, 2018, which is a
continuation of U.S. patent application Ser. No. 14/734,848, filed
Jun. 9, 2015, which is a continuation of U.S. patent application
Ser. No. 13/826,166, filed Mar. 14, 2013, now abandoned, which
claims benefit under 35 U.S.C. .sctn. 119 to U.S. Patent
Application No. 61/616,297 filed Mar. 27, 2012, the entire contents
of each of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to improved methods for culturing
recombinant proteins in prokaryotic host cells.
BACKGROUND OF THE INVENTION
[0003] The large-scale, economic purification of proteins is
required for viable biotechnology products. Generally, proteins are
produced by cell culture, using either mammalian or bacterial cell
lines engineered to produce the protein of interest by insertion of
a recombinant plasmid containing the gene for that protein. Since
the cell lines used are living organisms, they must be fed with a
complex growth medium which usually contains a mixture of salts,
sugars, amino acids, vitamins, trace elements and peptones.
Separation of the desired protein from the mixture of compounds fed
to the cells and from the by-products of the cells themselves to a
purity sufficient for use as a human therapeutic poses a formidable
challenge.
[0004] Recombinant therapeutic proteins are commonly produced in
several host cell lines including mammalian host cells, such as,
for example, murine myeloma NSO and Chinese Hamster Ovary (CHO)
cells (Anderson, D. C and Krummen, L. (2002) Curr. Opin. Biotech.
13: 117-123; Chu, L. and Robinson, D. K (2001) Curr. Opin.
Biotechnol. 12:180-187) and bacterial host cells including
Escherichia coli (E. coli). Each cell line has advantages and
disadvantages in terms of productivity and the characteristics of
the proteins produced by the cells. Escherichia coli has been most
extensively used for the large-scale production of therapeutic
proteins, which do not require complex glycosylation for
bioactivity. Heterologous proteins expressed by E. coli may
accumulate as soluble product or insoluble aggregates. Generally,
to isolate the proteins, the cells may be subjected to treatments
for periplasmic extraction or be lysed to release intracellular
products that are otherwise inaccessible. Advances in fermentation
and cell culture techniques have greatly increased the titers of
targeted recombinant proteins.
[0005] Choices of commercial production cell lines often balance
the need for high productivity with the ability to deliver the
product quality attributes required of a given product. Under cGMP
fermentation procedures, quality is built into the entire process
ensuring that regulatory agencies requirements are met in terms of
safety, product identity, quality and purity. However, occasionally
issues arise in which a given product does not meet its
specifications. The challenge is to develop a robust process in
which to identify and isolate the issue, then mitigate the issue
such that process controls can be maintained within established
parameter ranges, and make sure the process consistently produces a
product that meets product specifications. There is a need in the
art for mitigating or eliminating the incidence of products that do
not meet specifications.
SUMMARY OF THE INVENTION
[0006] The present invention contemplates a method of producing a
recombinant protein comprising (a) fermenting a prokaryotic host
cell wherein said prokaryotic host cell has been transformed with a
nucleic acid encoding said recombinant protein, and (b) harvesting
said recombinant protein under conditions where dissolved oxygen
(dO2) levels are greater than 0%, and (c) purifying said
recombinant protein to a filtered bulk for storage (FBS), wherein
said filtered bulk does not contain detectable
1,4-dihydroxy-2-naphthoate (DHNA)-recombinant protein adduct, as
measured by an ion exchange chromatography (IEC) assay at 310 nm.
In one embodiment, in the method described above, the analytical
assay is by HPLC, RP HPLC, HIC HPLC, NMR, mass spectrometry, or UV
spectroscopy.
[0007] The present invention contemplates a method of producing a
recombinant protein comprising (a) fermenting a prokaryotic host
cell wherein said prokaryotic host cell has been transformed with a
nucleic acid encoding said recombinant protein, and (b) harvesting
said recombinant protein under conditions where d02 levels are
greater than 0%, and (c) purifying said recombinant protein to a
FBS, wherein said filtered bulk does not contain detectable
DHNA-recombinant protein adduct, as measured by an IEC assay at 310
nm, wherein said recombinant protein is a recombinant polypeptide
or an isolated antibody.
[0008] The present invention contemplates a method of producing a
recombinant protein comprising (a) fermenting a prokaryotic host
cell wherein said prokaryotic host cell has been transformed with a
nucleic acid encoding said recombinant protein, and (b) harvesting
said recombinant protein under conditions where dO.sub.2 levels are
greater than 0%, and (c) purifying said recombinant protein to a
FBS, wherein said filtered bulk does not contain detectable
DINA-recombinant protein adduct, as measured by an IEC assay at 310
nm, wherein the fermentation is scale-independent.
[0009] The present invention contemplates a method of producing a
recombinant protein comprising (a) fermenting a prokaryotic host
cell wherein said prokaryotic host cell has been transformed with a
nucleic acid encoding said recombinant protein, and (b) harvesting
said recombinant protein under conditions where dO.sub.2 levels are
greater than 0%, and (c) purifying said recombinant protein to a
FBS, wherein said filtered bulk does not contain detectable
DHNA-recombinant protein adduct, as measured by an IEC assay at 310
nm, wherein said prokaryotic host cell is Escherichia coli (E.
coli), Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas,
Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia,
Vitreoscilla, and Paracoccus.
[0010] The present invention contemplates a method of producing a
recombinant protein comprising (a) fermenting a prokaryotic host
cell wherein said prokaryotic host cell has been transformed with a
nucleic acid encoding said recombinant protein, and (b) harvesting
said recombinant protein under conditions where dO.sub.2 levels are
greater than 0%, and (c) purifying said recombinant protein to a
FBS, wherein said filtered bulk does not contain detectable
DHNA-recombinant protein adduct, as measured by an IEC assay at 310
mu, wherein said dO.sub.2 is maintained at levels greater than 0%
continuously throughout the harvest operations of step (b). In one
embodiment in the method described above, the harvest operations
comprise a homogenization stage. In another embodiment, the
dO.sub.2 is maintained at about 30% to about 75% prior to
homogenization. In yet another embodiment, the dO.sub.2 is
maintained at levels greater than 75% prior to homogenization. In
still another embodiment, the dO.sub.2 is maintained at about 50%
after homogenization. In another embodiment, the dO.sub.2 is
maintained at levels greater than 50% after homogenization. In one
embodiment, the dO.sub.2 is maintained for a period of greater than
or equal to 1.5 hours. In still another embodiment, the dO.sub.2 is
maintained for a period of greater than or equal to 2 hours.
[0011] The present invention contemplates a method of producing a
recombinant protein comprising (a) fermenting a prokaryotic host
cell wherein said prokaryotic host cell has been transformed with a
nucleic acid encoding said recombinant protein, and (b) harvesting
said recombinant protein under conditions where dO.sub.2 levels are
greater than 0%, and (c) purifying said recombinant protein to a
FBS, wherein said filtered bulk does not contain detectable
DHNA-recombinant protein adduct, as measured by an IEC assay at 310
nm, wherein the dO.sub.2 is maintained with overlay or sparged air,
with increased back-pressure, or with agitation (i.e. stirring). In
one embodiment, the overlay air is from about 0.4 vvm to about 0.8
vvm. In another embodiment, the overlay air is targeted at 0.6 vvm.
In another embodiment, the increased backpressure is between about
1.0 to about 30 psi. In one embodiment, the increased backpressure
is targeted at 19 psi. In still another embodiment, the agitation
rate is from about 6 Watts/L to about 8 Watts/L. In yet another
embodiment, the agitation rate is at least 6 Watts/L. In another
embodiment, the agitation rate is targeted at 6 Watts/L.
[0012] In another aspect of the present invention, a method of
producing a recombinant protein comprising (a) fermenting a menE
gene-deleted prokaryotic host cell wherein said prokaryotic host
cell has been transformed with a nucleic acid encoding said
recombinant protein, (b) harvesting said recombinant protein; and
(c) purifying said recombinant protein to a FBS, wherein said
filtered bulk does not contain detectable DHNA-recombinant protein
adduct, as measured by an IEC assay at 310 nm, is contemplated. As
a further embodiment to the method described above, the recombinant
protein yield is increased by about 20% or greater, by about 30% or
greater, by about 40% or greater, by about 50% or greater, by about
60% or greater, as compared to the yield using a control
prokaryotic host cell.
[0013] In another aspect of the present invention, a method of
producing a recombinant protein comprising (a) fementing a menE
gene-deleted prokaryotic host cell wherein said prokaryotic host
cell has been transformed with a nucleic acid encoding said
recombinant protein, (b) harvesting said recombinant protein; and
(c) purifying said recombinant protein to a FBS, wherein said
filtered bulk does not contain detectable DHNA-recombinant protein
adduct, as measured by an IEC assay at 310 nm, is contemplated,
wherein the recombinant protein yield is increased by about 20% or
greater, by about 30% or greater, by about 40% or greater, by about
50% or greater, by about 60% or greater, as compared to the yield
using a control prokaryotic host cell, wherein the fementation is
scale-independent.
[0014] In another aspect of the present invention, a method of
producing a recombinant protein comprising (a) fermenting a menE
gene-deleted prokaryotic host cell wherein said prokaryotic host
cell has been transformed with a nucleic acid encoding said
recombinant protein, (b) harvesting said recombinant protein; and
(c) purifying said recombinant protein to a FBS, wherein said
filtered bulk does not contain detectable DHNA-recombinant protein
adduct, as measured by an IEC assay at 310 nm, is contemplated,
wherein the recombinant protein yield is increased by about 20% or
greater, by about 30% or greater, by about 40% or greater, by about
50% or greater, by about 60% or greater, as compared to the yield
using a control prokaryotic host cell, wherein said recombinant
protein is a recombinant polypeptide or an isolated antibody.
[0015] In another aspect of the present invention, a method of
producing a recombinant protein comprising (a) fermenting a menE
gene-deleted prokaryotic host cell wherein said prokaryotic host
cell has been transformed with a nucleic acid encoding said
recombinant protein, (b) harvesting said recombinant protein; and
(c) purifying said recombinant protein to a FBS, wherein said
filtered bulk does not contain detectable DHNA-recombinant protein
adduct, as measured by an IEC assay at 310 nm, is contemplated,
wherein the recombinant protein yield is increased by about 20% or
greater, by about 30% or greater, by about 40% or greater, by about
50% or greater, by about 60% or greater, as compared to the yield
using a control prokaryotic host cell, wherein said prokaryotic
host cell is Escherichia coli (E. coli), Enterobacter, Azotobacter,
Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella,
Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows the COC assay results of three manufacturing
runs of a product in which two runs, Run 2 and Run 3, did not meet
the expected results for the COC assay. PW=purified water,
C=development run control, 1=Run 1, 2=Run 2, and 3=Run 3.
[0017] FIG. 2A shows the UV/vis Spectra (10 cm) for Runs 1-3-Near
UV, where Runs 1-3 are represented. New absorbance peaks were
observed approximately at 320 nm and at 460 nm which were not
apparent for Run 1. FIG. 2B shows the UV/vis spectra for Run 3
minus Run 1, in which the difference of the absorbance peaks for
Runs 2 and 3 can be distinguished from Run 1.
[0018] FIG. 3 shows an IEC assay monitored at 310 nm for Runs 1-3.
A slight shoulder peak behind the main peak was observed for Runs 2
and 3, while the profile for Run 1 was comparable to the Reference
Material.
[0019] FIG. 4 shows a 2D LC-MS analysis of intact Runs 1-3,
monitored at 280 nm and 310 nm. An expected mass was observed for
Run 1, while the expected mass and an additional mass at 157 Da
were observed for Runs 2 and 3.
[0020] FIG. 5 shows a 2D-LC MS and mass identification by tryptic
peptide map with MS detection of a collected fraction of the brown
adduct--a minor peak from the IEC assay was collected. From the 2D
LC-MS analysis, in addition to the expected mass, a +156 Da mass
was observed for the fractionated shoulder peak.
[0021] FIG. 6 shows the LC-MS-MS analysis of the novel brown adduct
peak observed at 48.8 minutes at 310 nm was determined to be T20
peptide with Cys182 modified with +154.006 Da. Modified (at
cysteine, +154.006 Da) and free T6 and T16 peptides were also
detected by mass extraction.
[0022] FIG. 7 compares 1H-15N HSQC data of product to a synthetic
peptide (NH2-IVQCR-COOH) and showed a Cys NH correlation was
missing in the product sample.
[0023] FIG. 8 shows the proposed structure confirmed by strong nOe
observed between the CH of Cys and the NH of arginine.
[0024] Based on the NMR data collected, the proposed structure of
the brown adduct is presented in FIG. 9.
[0025] FIG. 10 shows the biosynthesis pathway in prokaryotic cells
to make menaquinones.
[0026] FIG. 11 shows a representative filtered bulk recombinant
product tested for brown adduct formation by ion exchange
chromatography at 310 nm and showed no measurable adduct
formation.
[0027] FIG. 12 shows an exemplary schematic of the Hi-dO process
enhancements implemented around the harvest operations.
[0028] FIG. 13 shows a schematic that shows the three major stages
of a typical harvest operation: post-fermentation stage, a
homogenization stage, then a post-homogenization stage.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. DEFINITIONS
[0029] Unless stated otherwise, the following terms and phrases as
used herein are intended to have the following meanings:
[0030] The term "agitation rate" is mixing of the culture broth or
of the homogenate, which is typically measured as revolutions per
minute (rpm). In one embodiment, agitation rate can be measured by
a "power per unit volume". For example, at 200 rpm in a 1,000 liter
fermentor, the agitation rate has a value of approximately 6
Watts/L.
[0031] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and
antibody fragments, so long as they exhibit the desired biological
activity. Antibodies may be murine, human, humanized, chimeric, or
derived from other species. The term "antibody," as used herein,
also refers to a full-length immunoglobulin molecule or an
immunologically active portion of a full-length immunoglobulin
molecule, i.e., a molecule that contains an antigen binding site
that immunospecifically binds an antigen of a target of interest or
part thereof, such targets including but not limited to, cancer
cell or cells that produce autoimmune antibodies associated with an
autoimmune disease. The immunoglobulin disclosed herein can be of
any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1,
IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin
molecule. The immunoglobulins can be derived from any species. In
one aspect, however, the immunoglobulin is of human, murine, or
rabbit origin.
[0032] "Antibody fragments" comprise a portion of a full length
antibody, generally the antigen binding or variable region thereof.
Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv
fragments; diabodies; linear antibodies; fragments produced by a
Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR
(complementary determining region), ECD (extracellular domain), and
epitope-binding fragments of any of the above which
immunospecifically bind to cancer cell antigens, viral antigens or
microbial antigens, single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0033] "Clarity, Opalescence and Coloration (COC) Assay" is defined
as using identical test tubes of colourless, transparent, neutral
glass with a flat base and an internal diameter of 15-25 mm,
compare the liquid to be examined with a reference suspension
freshly prepared as described below, the depth of the layer being
40 mm. The Standard color solutions listed in the U.S. Pharmacopeia
2012 (USP Monograph 631, Color and Achromicity) or in the European
Pharmacopoeia 5.0 (EP Method 2.2.2, Degree of Coloration of
Liquids) can be used for confirmation of the appropriate color
assignment.
[0034] The term "1,4-dihydroxy-2-naphthoate (DHNA)" is a chemical
product derived from E. coli cells. Okada Y, Tsuzuki Y, Miyazaki J,
Matsuzaki K, Hokari R, Komoto S, et al. (2006) Gut 55: 681-8. DHNA
is an inteiniediate in the menaquinone (MK), also known as vitamin
K2, biosynthesis pathway of E. coil cells. Neidhardt, F. C. (2010)
Escherichia coli and Salmonella (online version: Module 3.2.2 pgs.
36-37); Inledew, W. J. & R. K. Poole (1984) The respiratory
chains of Escherichia coli. Microbiological reviews. 48: 222-271;
Nowicka, B. & J. Cruk (2010) Occurrence, Biosynthesis and
Function of Isoprenoid Quinones. Biochimica et Biophysica Acta
1797: 1587-1605.
[0035] The term "dissolved oxygen" (dO.sub.2) is a relative measure
of the amount of oxygen that is dissolved or carried in a given
medium. It can be measured with a dissolved oxygen probe such as an
oxygen sensor in liquid media.
[0036] The term "ferment" or "fermenting" as used herein means the
process of culturing prokaryotic host cells that have been
transformed to induce the production of a recombinant protein of
interest.
[0037] The term "filtered bulk" or "filtered bulk substance (FBS)"
means the recombinant protein of interest product after harvest and
purification, wherein the protein has been released from the host
cell, centrifuged and/or filtered to remove any cell debris,
purified over suitable chromatography columns, and subsequently
concentrated by a filtration process.
[0038] The term "harvested cell culture fluid", also denoted as
HCCF, means prokaryotic or eukaryotic cell culture fluid from which
the cells have been removed, by means including centrifugation or
filtration. Cell culture is the process by which either prokaryotic
or eukaryotic cells are grown under controlled conditions. The term
"cell culture" refers to the culturing of cells derived from
multicellular eukaryotes, including animal cells or monocellular
prokaryotes, including bacteria and yeast. Eukaryotic cell cultures
include mammalian cells such as Chinese Hamster Ovary cells,
hybridomas, and insect cells. With an appropriate cell culture
vessel, secreted proteins can be obtained from anchorage dependent
cells or suspension cell lines. Mammalian cell cultures include
Chinese Hamster Ovary (CHO) cells or NSO cells.
[0039] The term "harvest operations" or "harvesting" means, without
limitation, a process comprising the lysing or homogenization, and
then centrifugation and/or filtration of a fermented prokaryotic
host cell culture that has been transformed to produce a
recombinant protein of interest, in order to begin isolating and
purifying said protein of interest.
[0040] The term "Hi-dO" as used herein refers to an enhanced
process as described herein which is the maintenance of a dissolved
oxygen level greater than 0% during harvest operations. To achieve
this, the present invention contemplates a combination of overlay
air, backpressure and agitation rate that can be used to maintain
the dO.sub.2 level at or above a set-point, i.e., above 0%, or at
about 30% to about 75%, or at levels greater than 75%, or at about
50%, or at levels greater than 50%. In another embodiment, those
skilled in the art could also sparge air or pure oxygen into the
broth directly to achieve Hi-dO of dissolved oxygen levels greater
than 0%.
[0041] The term "homogenization" as used herein means a process of
lysing or the mechanical cell lysis of prokaryotic host cells
transformed with a recombinant protein of interest in order to
release said protein from the host cell.
[0042] The term "increased back-pressure" is used to increase the
oxygen transfer rate through the culture broth. Back-pressure is
typically measured either in psi or bar.
[0043] "Menaquinones (MK)" are vitamin K.sub.2 homologs and serve
as electron shuttle molecules in the respiratory chain between
membrane bound protein complexes during micro-aerobic and/or
anaerobic conditions. The term "menE" is a gene in the biosynthesis
pathway to make menaquinones.
[0044] The term "microbial fermentation" means cell culture of
bacteria or yeast which is genetically engineered to produce
proteins and small molecules (e.g. secondary metabolites).
Fermentation is used to propagate recombinant bacteria and yeast as
well as other microorganisms and produce proteins of value. The
cell productivity and growth of these organisms are maximized by
supplying particular growth media and controlling and various
environmental factors (such as pH, temperature, and aeration).
Bacterial fermentation fluid may be derived from E. coil
cultures.
[0045] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different deteiiiiinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al
(1975) Nature 256:495, or may be made by recombinant DNA methods
(U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described for example in Clackson et al (1991) Nature, 352:624-628;
Marks et al (1991) J. Mol. Biol., 222:581-597.
[0046] The term "overlay air" means air blown in from the top of
the fet uentor which contains the culture broth. Typically, oxygen
is supplied to a fermentor by bubbling air through the liquid
culture medium, often accompanied by vigorous agitation to effect a
fine bubble dispersion.
[0047] The term "prokaryotic host cell" as used in the present
invention should encompass those that utilize the menaquinone
biosynthesis pathway. In one embodiment, prokaryotic host cells
encompass, for example, Archaebacteria and Eubacteria, such as
gram-negative or gram-positive organisms. Examples of useful
bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
subtilis), Enterobacteria, Pseudomonas species (e.g., P.
aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In
another embodiment, E. coli cells are used as hosts for the
invention (Bachmann, Cellular and Molecular Biology, vol. 2
(Washington, D.C.: American Society for Microbiology, 1987), pp.
1190-1219; ATCC Deposit No. 27,325) and derivatives thereof,
including strain 33D3 having genotype W3110 .DELTA.fhuA
(.DELTA.tonA) ptr3 lacIq lacL8 .DELTA.ompT .DELTA.(nmpC-fepE)
degP41 kan.sup.R (U.S. Pat. No. 5,639,635). Of course other strains
and derivatives thereof, such as E. coli. 294 (ATCC 31,446), E.
coli. B, E. coli.sub..lamda., 1776 (ATCC 31,537) and E. coli. RV308
(ATCC 31,608) are also suitable. These examples are illustrative
rather than limiting. Methods for constructing derivatives of any
of the above-mentioned bacteria having defined genotypes are known
in the art and described in, for example, Bass et al. (1990)
Proteins, 8: 309-314. It is, of course, necessary to select the
appropriate bacteria taking into consideration replicability of the
replicon in the cells of a bacterium. For example, E. coli,
Serratia, or Salmonella species can be suitably used as the host
when well known plasmids such as pBR322, pBR325, pACYC177, or
pKN410 are used to supply the replicon.
[0048] As used herein, "recombinant protein" refers generally to
peptides and proteins, including antibodies. Such recombinant
proteins are "heterologous," i.e., foreign to the host cell being
utilized, such as a human protein produced by E. coli. The
polypeptide may be produced as an insoluble aggregate or as a
soluble polypeptide in the periplasmic space or cytoplasm.
[0049] The term "scale-independent" means the volume capacity of
the fermentation process of the present invention can be
accomplished using any scale, such as, for example, from about 1
liter or greater, or about 10 liters or greater, or about 100
liters or greater, or about 500 liters or greater, or about 1,000
liters or greater, or about 10, 000 liters or greater, or about
100,000 liters or greater.
II. MODES FOR CARRYING OUT THE INVENTION
[0050] The present invention concerns improved methods of
recombinant production of proteins in a prokaryotic system. The
invention is based on preventing a brown adduct formation
discovered during the manufacturing of a recombinant protein which
caused certain lots of the product to not meet specifications. As
illustrated in the examples provided herein, the problem of the
brown adduct resulted from an inconsistent redox potential during
the harvest operations. It has now been surprisingly discovered
that the brown adduct formation can be prevented by maintaining a
dissolved oxygen environment greater than zero during the harvest
operations or alternatively, by genetically deleting the menE gene
in the prokaryotic host cell genome used to recombinantly produce
the recombinant protein of interest.
Recombinant Production of Recombinant Proteins in Prokaryotic
Cells
[0051] In the first step of the above processes, the heterologous
nucleic acid (e.g., cDNA or genomic DNA) used to produce the
recombinant protein of interest, is suitably inserted into a
replicable vector for expression in the bacterium under the control
of a suitable promoter for bacteria. Many vectors are available for
this purpose, and selection of the appropriate vector will depend
mainly on the size of the nucleic acid to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components depending on its
function (amplification of DNA or expression of DNA) and the
particular host cell with which it is compatible. The vector
components for bacterial transformation may include a signal
sequence for the heterologous polypeptide and will include a signal
sequence and will also include an inducible promoter for the
heterologous polypeptide. They also generally include an origin of
replication and one or more marker genes, described herein.
[0052] If the heterologous polypeptide is to be secreted, the DNA
encoding the heterologous polypeptide of interest herein contains a
signal sequence, such as one at the N-terminus of the mature
heterologous polypeptide. In general, the signal sequence may be a
component of the vector, or it may be a part of the heterologous
polypeptide DNA that is inserted into the vector. The heterologous
signal sequence selected should be one that is recognized and
processed (i.e., cleaved by a signal peptidase) by the host cell.
For bacterial host cells that do not recognize and process the
native heterologous polypeptide signal sequence, the signal
sequence is substituted by any commonly known bacterial signal
sequence.
[0053] Expression vectors contain a nucleic acid sequence that
enables the vector to replicate in one or more selected host cells.
Such sequences are well known for a variety of bacteria. The origin
of replication from the plasmid pBR322 is suitable for most
gram-negative bacteria.
[0054] Expression vectors also generally contain a selection gene,
also termed a selectable marker. This gene encodes a protein
necessary for the survival or growth of transformed host cells
grown in a selective culture medium. Host cells not transformed
with the vector containing the selection gene will not survive in
the culture medium. Typical selection genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media, e.g., the gene encoding D-alanine
racemase for Bacilli. One example of a selection scheme utilizes a
drug to arrest growth of a host cell. Those cells that are
successfully transformed with a heterologous gene produce a protein
conferring drug resistance and thus survive the selection
regimen.
[0055] The expression vector for producing a heterologous
polypeptide also contains an inducible promoter that is recognized
by the host bacterial organism and is operably linked to the
nucleic acid encoding the heterologous polypeptide of interest. It
also contains a separate inducible or low-basal-expression promoter
operably linked to the nucleic acid encoding the lytic enzymes.
Inducible promoters suitable for use with bacterial hosts include
the .beta.-lactamase and lactose promoter systems (Chang et al.,
Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)),
the arabinose promoter system, including the araBAD promoter
(Guzman et al., J. Bacteriol., 174: 7716-7728 (1992); Guzman et
al., J. Bacteriol., 177: 4121-4130 (1995); Siegele and Hu, Proc.
Natl. Acad. Sci. USA, 94: 8168-8172 (1997)), the rhamnose promoter
(Haldimann et al., J. Bacteriol., 180: 1277-1286 (1998)), the
alkaline phosphatase promoter, a tryptophan (tip) promoter system
(Goeddel, Nucleic Acids Res., 8: 4057 (1980) and EP 36,776), the
P.sub.LtetO-1 and P.sub.lac/are-1 promoters (Lutz and Bujard,
Nucleic Acids Res., 25: 1203-1210 (1997)), and hybrid promoters
such as the tac promoter deBoer et al., Proc. Nati. Acad. Sci. USA,
80: 21-25 (1983). However, other known bacterial inducible
promoters and low-basal-expression promoters are suitable. Their
nucleotide sequences have been published, thereby enabling a
skilled worker operably to ligate them to DNA encoding the
heterologous polypeptide of interest or to the nucleic acids
encoding the lytic enzymes (Siebenlist et al., Cell, 20: 269
(1980)) using linkers or adaptors to supply any required
restriction sites. If a strong and highly leaky promoter, such as
the tip promoter, is used, it is generally used only for expression
of the nucleic acid encoding the heterologous polypeptide and not
for lytic-enzyme-encoding nucleic acid. The tac and P.sub.L
promoters could be used for either, but not both. In one
embodiment, the alkaline phosphatase (phoA) promoter is used for
the product and the arabinose (ara) promoter for the lytic
enzymes.
[0056] Promoters for use in bacterial systems also generally
contain a Shine-Dalgarno (SD) sequence operably linked to the DNA
encoding the heterologous polypeptide of interest. The promoter can
be removed from the bacterial source DNA by restriction enzyme
digestion and inserted into the vector containing the desired DNA.
The phoA promoter can be removed from the bacterial-source DNA by
restriction enzyme digestion and inserted into the vector
containing the desired. DNA.
[0057] Construction of suitable vectors containing one or more of
the above-listed components employs standard ligation techniques
commonly known to those of skill in the art. Isolated plasmids or
DNA fragments are cleaved, tailored, and re-ligated in the form
desired to generate the plasmids required.
[0058] Suitable prokaryotic host cells for the claimed invention
include any which utilize the biosynthesis pathway to make
menaquinones, as defined herein. Some non-limiting examples may
include, for example, Escherichia coli (E. coli), Enterobacter,
Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus,
Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and
Paracoccus.
[0059] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0060] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include Luria-Bertani (LB) broth plus necessary nutrient
supplements. In certain embodiments, the media also contains a
selection agent, chosen based on the construction of the expression
vector, to selectively permit growth of prokaryotic cells
containing the expression vector. For example, ampicillin is added
to media for growth of cells expressing ampicillin resistant gene.
Any necessary supplements besides carbon, nitrogen, and inorganic
phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source.
[0061] For accumulation of an expressed gene product, the host cell
is cultured under conditions sufficient for accumulation of the
gene product. Such conditions include, e.g., temperature, nutrient,
and cell-density conditions that permit protein expression and
accumulation by the cell. Moreover, such conditions are those under
which the cell can perform basic cellular functions of
transcription, translation, and passage of proteins from one
cellular compartment to another for the secreted proteins, as are
known to those skilled in the art.
[0062] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, the typical
temperature ranges from about 20.degree. C. to about 39.degree. C.
In one embodiment, the temperature is from about 25.degree. C. to
about 37.degree. C. In another embodiment, the temperature is at
about 30.degree. C.
[0063] The pH of the culture medium may be any pH from about 5-9,
depending mainly on the host organism. For E. coli, the pH is from
about 6.8 to about 7.4, or about 7.0.
[0064] For induction, typically the cells are cultured until a
certain optical density is achieved, e.g., an A.sub.550 of about
80-100, at which point induction is initiated (e.g., by addition of
an inducer, by depletion of a repressor, suppressor, or medium
component, etc.) to induce expression of the gene encoding the
heterologous polypeptide.
[0065] After product accumulation, optionally before product
recovery, the broth lysate is incubated for a period of time
sufficient to release the heterologous polypeptide contained in the
cells. In an alternative embodiment, or subsequent to the
preceding, the cells present in culture may be lysed mechanically,
using any mechanical means known in the art, which may include, for
example, chemical lysis or osmotic shock in order to release said
protein from the host cell.
[0066] Once lysed, the lysate or homogenate may be transferred to a
hold tank where it can await the addition of more batches of
lysate/homogenate and/or where further processing may occur, such
as, for example, dilution with water, addition of buffers or
flocculants, pH adjustment, or altering or maintaining the
temperature of the lysate/homogenate in preparation for subsequent
recovery steps.
[0067] In a subsequent step, the heterologous polypeptide, as a
soluble or insoluble product released from the cellular matrix, is
recovered from the lysate, or homogenate, in a manner that
minimizes co-recovery of cellular debris with the product. The
recovery may be done by any means, but in one embodiment, can
comprise sedimenting refractile particles containing the
heterologous polypeptide or collecting supernatant containing
soluble product. An example of sedimentation is centrifugation. In
this case, the recovery takes place, before expanded bed adsorption
(EBA) or sedimentation, in the presence of an agent that disrupts
the outer cell wall to increase permeability and allows more solids
to be recovered. Examples of such agents include a chelating agent
such as ethylenediaminetetraacetic acid (EDTA) or a zwitterion such
as, for example, a dipolar ionic detergent such as ZWITTERGENT
316.TM. detergent. In one embodiment, the recovery takes place in
the presence of EDTA.
[0068] If centrifugation is used for recovery, the relative
centrifugal force (RCF) is an important factor. The RCF is adjusted
to minimize co-sedimentation of cellular debris with the refractile
particles released from the cell wall at lysis. The specific RCF
used for this purpose will vary with, for example, the type of
product to be recovered, but is at least about 3000.times.g, more
preferably about 3500-6000.times.g, or about 4000-6000.times.g.
[0069] The duration of centrifugation will depend on several
factors. The sedimentation rate will depend upon, e.g., the size,
shape, and density of the retractile particle and the density and
viscosity of the fluid. The sedimentation time for solids will
depend, e.g., on the sedimentation distance and rate. It is
reasonable to expect that the continuous disc-stack centrifuges
would work well for the recovery of the released heterologous
polypeptide aggregates or for the removal of cellular debris at
large scale, since these centrifuges can process at high fluid
velocities because of their relatively large centrifugal force and
the relatively small sedimentation distance.
[0070] The heterologous polypeptide captured in the initial
recovery step may then be further purified from the contaminating
protein. In one embodiment, the aggregated heterologous polypeptide
is isolated, followed by a simultaneous solubilization and
refolding of the polypeptide, as disclosed in U.S. Pat. No.
5,288,931. Alternatively, the soluble product is recovered by
standard techniques as described below.
[0071] General chromatographic methods and their use are known to a
person skilled in the art. See for example, Chromatography, 5th
edition, Part A: Fundamentals and Techniques, Heftmann, E. (ed),
Elsevier Science Publishing Company, New York, (1992); Advanced
Chromatographic and Electromigration Methods in Biosciences, Deyl,
Z. (ed.), Elsevier Science BV, Amsterdam, The Netherlands, (1998);
Chromatography Today, Poole, C. F., and Poole, S. K., Elsevier
Science Publishing Company, New York, (1991); Scopes, Protein
Purification Principles and Practice (1982); Sambrook, J., et al.
(ed), Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; or
Current Protocols in Molecular Biology, Ausubel, F. M., et al.
(eds), John Wiley & Sons, Inc., New York. The following
procedures are exemplary of suitable purification procedures for
the soluble heterologous polypeptide released from the periplasm or
the cytoplasm, and are well known in the art: fractionation on
immunoaffinity or ion-exchange columns; ethanol precipitation;
reversed-phase HPLC; chromatography on silica or on a
cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; and gel filtration using, for
example, SEPHADEX.TM. G-75.
[0072] In one aspect of the invention, the antibody production is
conducted in large quantity by a fermentation process. Various
large-scale fed-batch fermentation procedures are available for
production of recombinant proteins. Large-scale fermentations have
at least 1000 liters of capacity, preferably about 1,000 to 100,000
liters of capacity. These fermentors use agitator impellers to
distribute oxygen and nutrients, especially glucose (the preferred
carbon/energy source). Small-scale fermentation refers generally to
fetmentation in a fermentor that is no more than approximately 20
liters in volumetric capacity.
[0073] As discussed herein, the claimed invention can be used to
produce recombinant proteins, including, for example, peptides and
proteins, including antibodies.
[0074] Examples of recombinant peptides and proteins that can be
produced by the method of the invention include, but are not
limited to, molecules such as, e.g., renin, a growth hormone,
including human growth hormone; bovine growth hormone; growth
hormone releasing factor; parathyroid hormone; thyroid stimulating
hotinone; lipoproteins; al-antitrypsin; insulin A-chain; insulin
B-chain; proinsulin; thrombopoietin; follicle stimulating hormone;
calcitonin; luteinizing hormone; glucagon; clotting factors such as
factor VIIIC, factor IX, tissue factor, and von Willebrands factor;
anti-clotting factors such as Protein C; atrial naturietic factor;
lung surfactant; a plasminogen activator, such as urokinase or
human urine or tissue-type plasminogen activator (t-PA); bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha
and- beta; enkephalinase; a serum albumin such as human serum
albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin
B-chain; prorelaxin; mouse gonadotropin-associated peptide; a
microbial protein, such as beta-lactamase; DNase; inhibin; activin;
vascular endothelial growth factor (VEGF); receptors for hormones
or growth factors; integrin; protein A or D; rheumatoid factors; a
neurotrophic factor such as brain-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-.beta.; cardiotrophins
(cardiac hypertrophy factor) such as cardiotrophin-1 (CT-1);
platelet-derived growth factor (PDGF); fibroblast growth factor
such as aFGF and bFGF; epidermal growth factor (EGF); transforming
growth factor (TGF) such as TGF-alpha and TGF-beta, including
TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or TGF-.beta.5;
insulin-like growth factor-I and -II (IGF-I and IGF-II);
des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding
proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;
erythropoietin; osteoinductive factors; immunotoxins; a bone
morphogenetic protein (BMP); an interferon such as
interferon-alpha, -beta, and -gamma; colony stimulating factors
(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g.,
IL-1 to IL-13; anti-HER-2 antibody; superoxide dismutase; T-cell
receptors; surface membrane proteins; decay accelerating factor;
viral antigen such as, for example, a portion of the AIDS envelope;
transport proteins; homing receptors; addressins; regulatory
proteins.
[0075] Antibodies produced by the claimed invention may be
monoclonal antibodies that are homogeneous populations of
antibodies to a particular antigenic determinant (e.g., a cancer
cell antigen, a viral antigen, a microbial antigen, a protein, a
peptide, a carbohydrate, a chemical, nucleic acid, or fragments
thereof). A monoclonal antibody (MAb) to a target-of-interest can
be prepared by using any technique known in the art which provides
for the production of antibody molecules by continuous cell lines
in culture. These include, but are not limited to, the hybridoma
technique originally described by Kohler and Milstein (1975) Nature
256:495-497), the human B cell hybridoma technique (Kozbor et al
(1983) Immunology Today 4:72), and the EBV-hybridoma technique
(Cole et al (1985) in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any
immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any
subclass thereof. The hybridoma producing the MAbs of use in this
invention may be cultivated in vitro or in vivo.
[0076] Useful monoclonal antibodies include, but are not limited
to, human monoclonal antibodies, humanized monoclonal antibodies,
antibody fragments, or chimeric human-mouse (or other species)
monoclonal antibodies. Human monoclonal antibodies may be made by
any of numerous techniques known in the art (Teng et al (1983)
Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al (1983)
Immunology Today 4:72-79; and Olsson et al (1982) Methods in
Enzymology 92:3-16).
[0077] The antibody can also be a bispecific antibody. Bispecific
antibodies may have a hybrid immunoglobulin heavy chain with a
first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. This asymmetric structure
facilitates the separation of the desired bispecific compound from
unwanted immunoglobulin chain combinations, as the presence of an
immunoglobulin light chain in only one half of the bispecific
molecule provides for a facile way of separation (WO 94/04690;
Suresh et al (1986) Methods in Enzymology, 121:210; Rodrigues et al
(1993) J. of immunology 151:6954-6961; Carter et al (1992)
Bio/Technology 10:163-167; Carter et al (1995) J. of Hematotherapy
4:463-470; Merchant et al (1998) Nature Biotechnology 16:677-681.
Methods for making bispecific antibodies are known in the art
(Milstein et al (1983) Nature 305:537-539; WO 93/08829; Traunecker
et al (1991) EMBO J. 10:3655-3659. Using such techniques,
bispecific antibodies can be prepared for conjugation as an
antibody drug conjugate (ADC) in the treatment or prevention of
disease as defined herein.
[0078] The antibody, as defined, can be a functionally active
fragment, derivative or analog of an antibody that
immunospecifically binds to cancer cell antigens, viral antigens,
or microbial antigens or other antibodies bound to tumor cells or
matrix. In this regard, "functionally active" means that the
fragment, derivative or analog is able to elicit anti-anti-idiotype
antibodies that recognize the same antigen that the antibody from
which the fragment, derivative or analog is derived recognized.
Specifically, in an exemplary embodiment the antigenicity of the
idiotype of the immunoglobulin molecule can be enhanced by deletion
of framework and CDR sequences that are C-terminal to the CDR
sequence that specifically recognizes the antigen. To determine
which CDR sequences bind the antigen, synthetic peptides containing
the CDR sequences can be used in binding assays with the antigen by
any binding assay method known in the art, e.g. the BIA core assay
(Kabat et al, (1991) in Sequences of Proteins of Immunological
Interest, Fifth Edition, National Institute of Health, Bethesda,
Md.; Kabat et al (1980) J. of Immunology 125(3):961-969).
[0079] Other useful antibodies include fragments of antibodies such
as, but not limited to, F(ab')2 fragments, which contain the
variable region, the light chain constant region and the CH1 domain
of the heavy chain can be produced by pepsin digestion of the
antibody molecule, and Fab fragments, which can be generated by
reducing the disulfide bridges of the F(ab')2 fragments. Other
useful antibodies are heavy chain and light chain dimers of
antibodies, or any minimal fragment thereof such as Fvs or single
chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.
4,946,778; Bird (1988) Science 242:423-42; Huston et al., (1988)
Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883; and Ward et al (1989)
Nature 334:544-54), or any other molecule with the same specificity
as the antibody.
[0080] The antibody may be a fusion protein of an antibody, or a
functionally active fragment thereof, for example in which the
antibody is fused via a covalent bond (e.g., a peptide bond), at
either the N-terminus or the C-terminus to an amino acid sequence
of another protein (or portion thereof, such as at least 10, 20 or
50 amino acid portion of the protein) that is not the antibody. The
antibody or fragment thereof may be covalently linked to the other
protein at the N-terminus of the constant domain.
[0081] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl.
Acad. Sci. 81:6851-6855). A chimeric antibody is a molecule in
which different portions are derived from different animal species,
such as those having a variable region derived from murine
monoclonal and human immunoglobulin constant regions (U.S. Pat.
Nos. 4,816,567; 4,816,397). Chimeric antibodies include
"primatized" antibodies comprising variable domain antigen-binding
sequences derived from a non-human primate (e.g., Old World Monkey,
Ape etc) and human constant region sequences.
[0082] Chimeric and humanized monoclonal antibodies, comprising
both human and non-human portions, can be made using standard
recombinant DNA techniques (WO 87/02671; EP 184,187; EP 171496; EP
173494; WO 86/01533; U.S. Pat. No. 4,816,567; EP 12023; Berter et
al (1988) Science 240: 1041-1043; Liu et al (1987) Proc. Nati.
Acad. Sci. U.S.A. 84: 3439-3443; Liu et al (1987) J. Immunol. 139:
3521-3526; Sun et al (1987) Proc. Natl. Acad. Sci. U.S.A. 84:
214-218; Nishimura et al (1987) Cancer. Res. 47: 999-1005; Wood et
al (1985) Nature 314: 446-449; and Shaw et al (1988) J. Natl.
Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207;
Oi et al (1986) BioTechniques 4: 214; U.S. Pat. No. 5,225,539;
Jones et al (1986) Nature 321:552-525; Verhoeyan et al (1988)
Science 239: 1534; and Beidler et al (1988) J. Immunol. 141:
4053-4060; each of which is incorporated herein by reference in its
entirety.
[0083] Therapeutic monoclonal antibodies that may be produced by
the methods of the invention include, for are not limited to,
trastuzumab (HERCEPTIN.RTM., Genentech, Inc., Carter et al (1992)
Proc. Natl. Acad. Sci. U.S.A., 89:4285-4289; U.S. Pat. No.
5,725,856); anti-CD20 antibodies such as chimeric anti-CD20 "C2B8"
(U.S. Pat. No. 5,736,137); rituximab (RITUXAN.RTM.), ocrelizumab, a
chimeric or humanized variant of the 2H7 antibody (U.S. Pat. No.
5,721,108; WO 04/056312) or tositumomab (BEXXAR.RTM.); anti-IL-8
(St John et al (1993) Chest, 103:932, and WO 95/23865); antibodies
targeting other interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-9, IL-10, IL-12, IL-13; anti-VEGF antibodies
including humanized and/or affinity matured anti-VEGF antibodies
such as the humanized anti-VEGF antibody huA4.6.1 bevacizumab
(AVASTIN.RTM., Genentech, Inc., Kim et al (1992) Growth Factors 7:
53-64, WO 96/30046, WO 98/45331); anti-PSCA antibodies (WO
01/40309); anti-CD40 antibodies, including S2C6 and humanized
variants thereof (WO 00/75348); anti-CD11a (U.S. Pat. No.
5,622,700; WO 98/23761; Steppe et al (1991) Transplant Intl. 4:3-7;
Hourmant et al (1994) Transplantation 58:377-380); anti-IgE (Presta
et al (1993) J. Immunol. 151:2623-2632; WO 95/19181); anti-CD18
(U.S. Pat. No. 5,622,700; WO 97/26912); anti-IgE, including E25,
E26 and E27 (U.S. Pat. Nos. 5,714,338; 5,091,313; WO 93/04173; U.S.
Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO 98/51793);
anti-TNF-alpha antibodies including cA2 (REMICADE.RTM.), CDP571 and
MAK-195 (U.S. Pat. No. 5,672,347; Lorenz et al (1996) J. Immunol.
156(4): 1646-1653; Dhainaut et al (1995) Crit. Care Med.
23(9):1461-1469); anti-Tissue Factor (TF) (EP 0 420 937 B1);
anti-human alpha 4 beta 7 integrin (WO 98/06248); anti-EGFR,
chimerized or humanized 225 antibody (WO 96/40210); anti-CD3
antibodies such as OKT3 (U.S. Pat. No. 4,515,893); anti-CD25 or
anti-tac antibodies such as CHI-621 SIMULECT.RTM. and ZENAPAX.RTM.
(U.S. Pat. No. 5,693,762); anti-CD4 antibodies such as the cM-7412
antibody (Choy et al (1996) Arthritis Rheum 39(1): 52-56);
anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al (1988)
Nature 332: 323-337); anti-Fc receptor antibodies such as the M22
antibody directed against Fc gamma RI as in Graziano et al (1995)
J. Immunol. 155(10): 4996-5002; anti-carcinoembryonic antigen (CEA)
antibodies such as hMN-14 (Sharkey et al (1995) Cancer Res. 55(23
Suppl): 5935s-5945s; antibodies directed against breast epithelial
cells including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al (1995)
Cancer Res. 55(23): 5852s-5856s; and Richman et al (1995) Cancer
Res. 55(23 Supp): 5916s-5920s); antibodies that bind to colon
carcinoma cells such as C242 (Litton et al (1996) Eur J. Immunol.
26(1):1-9); anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al (1995)
J. Immunol. 155(2): 925-937); anti-CD33 antibodies such as Hu M195
(Jurcic et al (1995) Cancer Res 55(23 Suppl): 5908s-5910s and
CMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide
(Juweid et al (1995) Cancer Res 55(23 Suppl): 5899s-5907s);
anti-EpCAM antibodies such as 17-1A (PANOREX.RTM.); anti-GpIIb/IIIa
antibodies such as abciximab or c7E3 Fab (REOPRO.RTM.); anti-RSV
antibodies such as MEDI-493 (SYNAGIS.RTM.); anti-CMV antibodies
such as PROTOVIR.RTM.); anti-HIV antibodies such as PR0542;
anti-hepatitis antibodies such as the anti-Hep B antibody
OSTAVIRO); anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitope
antibody BEC2; anti-human renal cell carcinoma antibody such as
ch-G250; ING-1; anti-human 17-1A antibody (3622W94); anti-human
colorectal tumor antibody (A33); anti-human melanoma antibody R24
directed against GD3 ganglioside; anti-human squamous-cell
carcinoma (SF-25); and anti-human leukocyte antigen (HLA)
antibodies such as Smart ID10 and the anti-HLA DR antibody Oncolyrn
(Lym-1).
II. METHODS AND ASSAYS
Analytical Methods/Assays
Clarity, Opalescence and Coloration (COC) Assay
[0084] The degree of opalescence may also be determined by
instrumental measurement of the light absorbed or scattered on
account of submicroscopic optical density in homogeneities of
opalescent solutions and suspensions. Such techniques are
nephelometry and turbidimetry. For turbidity measurement of
coloured samples, ratio turbidimetry and nephelometry with ratio
selection are used. The light scattering effect of suspended
particles can be measured by observation of either the transmitted
light (turbidimetry) or the scattered light (nephelometry). Ratio
turbidimetry combines the principles of both nephelometry and
turbidimetry. Turbidimetry and nephelometry are useful for the
measurement of slightly opalescent suspensions. Reference
suspensions produced under well-defined conditions must be used.
Standard color solutions listed in the U.S. Pharmacopeia 2012 (USP
Monograph 631, Color and Achromicity) or in the European
Pharmacopoeia 5.0 (EP Method 2.2.2, Degree of Coloration of
Liquids) for confirmation of the appropriate color assignment. For
quantitative measurements the construction of calibration curves is
essential, since the relationship between the optical properties of
the suspension and the concentration of the dispersed phase is at
best semi-empirical. The determination of opalescence of coloured
liquids is done with ratio turbidimeters or nephelometers with
ratio selection since colour provides a negative interference,
attenuating both incident and scattered light and lowering the
turbidity value. The effect is so great for even moderately
coloured samples that conventional nephelometers cannot be used.
The instrumental assessment of clarity and opalescence provides a
more discriminatory test that does not depend on the visual acuity
of the analyst. Numerical results are more useful for quality
monitoring and process control, especially in stability studies.
For example, previous numerical data on stability can be projected
to determine whether a given batch of dosage formulation or active
pharmaceutical ingredient will exceed shelf-life limits prior to
the expiry date.
HPLC Assay
[0085] High Perfoimance Liquid Chromatography, also known as High
Pressure Liquid Chromatography, abbreviated as HPLC, is a special
form of liquid chromatography and nowadays used frequently in
biochemistry and analytical chemistry. The analyte is forced
through a column of the stationary phase in a liquid (mobile phase)
at high pressure, which decreases the time the separated components
remain on the stationary phase and thus the time they have to
diffuse within the column. This leads to narrower peaks in the
resulting chromatogram and thence to better resolution and
sensitivity as compared to LC. The mobile phase is chosen to ensure
solubility of the sample solutes. For the stationary phase,
preferably microparticulate silica (bare or chemically modified) is
used, because its high surface area accentuates the differences in
solute-stationary phase interactions. The use of a stationary phase
that interacts strongly with solutes relative to solute
mobile-phase interactions will result in very long retention times,
a situation which is not analytically useful. Hence the stationary
phase must be selected so as to provide weak to moderate solute
interactions relative to those in the mobile phase. As a
consequence, the nature of the solute governs the type of LC
selected. The stronger interactions should occur in the mobile
phase to ensure sample solubility and ready elution, while the
stationary phase should be responsive to more subtle differences
among the solutes. For example, polar neutral compounds are usually
better analyzed using a polar mobile phase together with a nonpolar
stationary phase that distinguishes subtle differences in the
dispersive character of the solutes. One of the powerful aspects of
HPLC is that the mobile phase can be varied to alter the retention
mechanism. Modifiers can be added to the mobile phase to control
retention. For example, pH is an important variable in aqueous
mobile phases.
[0086] Reversed-phase chromatography (RP-HPLC) calls for the use of
a non-polar stationary phase and a polar mobile phase (composed of
one or more of the polar solvents, e.g. water, methanol,
acetonitrile, and tetrahydrofuran).
[0087] Hydrophobic interaction chromatography (HIC) HPLC: This
chromatographic method is good for analyzing proteins or
antibody/protein bioconjugates based on their hydrophobicity. The
theory behind hydrophobic interaction chromatography is that
proteins are bound to the resin by employing an aqueous high salt
mobile phase. The salt conditions contribute to a lyotropic effect
which allows the proteins to bind to the lower surface coverage of
a hydrophobic ligand. Proteins are eluted by the simple technique
of decreasing the salt concentration. Most therapeutic targets are
eluted in a low salt or a no salt buffer. Thus, the compound can be
eluted in a more polar and less denaturing environment. For
example, HIC has been used extensively to analyze drug loading in
antibody-drug or protein-drug conjugates.
NMR Assay
[0088] Nuclear magnetic resonance (NMR) detection is based on the
fact that certain nuclei with odd-numbered masses, including H and
13C, spin about an axis in, a random fashion. However, when placed
between poles of a strong magnet, the spins are aligned either
parallel or anti-parallel to the magnetic field, with the parallel
orientation favored since it is slightly lower in energy. The
nuclei are then irradiated with electromagnetic radiation which is
absorbed and places the parallel nuclei into a higher energy state;
consequently, they are now in "resonance" with the radiation. Each
H or C will produce different spectra depending on their location
and adjacent molecules, or elements in the compound, because all
nuclei in molecules are surrounded by electron clouds which change
the encompassing magnetic field and thereby alter the absorption
frequency.
Mass Spectrometry
[0089] Mass spectrometry is an analytical technique used to measure
the mass-to-charge ratio (m/z or m/q) of ions. It is most generally
used to analyze the composition of a physical sample by generating
a mass spectrum representing the masses of sample components. The
technique has several applications including identifying unknown
compounds by the mass of the compound and/or fragments thereof
determining the isotopic composition of one or more elements in a
compound, determining the structure of compounds by observing the
fragmentation of the compound, quantitating the amount of a
compound in a sample using carefully designed methods (mass
spectrometry is not inherently quantitative), studying the
fundamentals of gas phase ion chemistry (the chemistry of ions and
neutrals in vacuum), and determining other physical, chemical or
even biological properties of compounds with a variety of other
approaches.
[0090] A mass spectrometer is a device used for mass spectrometry,
and it produces a mass spectrum of a sample to analyze its
composition. This is normally achieved by ionizing the sample and
separating ions of differing masses and recording their relative
abundance by measuring intensities of ion flux. A typical mass
spectrometer comprises three parts: an ion source, a mass analyzer,
and a detector.
[0091] The kind of ion source is a contributing factor that
strongly influences-what types of samples can be analyzed by mass
spectrometry. Electron ionization and chemical ionization are used
for gases and vapors. In chemical ionization sources, the analyte
is ionized by chemical ion-molecule reactions during collisions in
the source. Two techniques often used with liquid and solid
biological samples include electrospray ionization (ESI) and
matrix-assisted laser desorption/ionization (MALDI). Other
techniques include fast atom bombardment (FAB), thermospray,
atmospheric pressure chemical ionization (APCI), secondary ion mass
spectrometry (SIMS), and thermal ionisation.
UV Spectroscopy
[0092] Ultraviolet--visible spectroscopy or ultraviolet-visible
spectrophotometry (UV-Vis or UV/Vis) refers to absorption
spectroscopy or reflectance spectroscopy in the ultraviolet-visible
spectral region. This means it uses light in the visible and
adjacent (near-UV and near-infrared (NIR)) ranges. The absorption
or reflectance in the visible range directly affects the perceived
color of the chemicals involved. In this region of the
electromagnetic spectrum, molecules undergo electronic transitions.
This technique is complementary to fluorescence spectroscopy, in
that fluorescence deals with transitions from the excited state to
the ground state, while absorption measures transitions from the
ground state to the excited state. A UV spectrometer is an
instrument that uses a beam of light from a visible and/or UV light
source (colored red) is separated into its component wavelengths by
a prism or diffraction grating. Each monochromatic (single
wavelength) beam in turn is split into two equal intensity beams by
a half-mirrored device. One beam, the sample beam (colored
magenta), passes through a small transparent container (cuvette)
containing a solution of the compound being studied in a
transparent solvent. The other beam, the reference (colored blue),
passes through an identical cuvette containing only the solvent.
The intensities of these light beams are then measured by
electronic detectors and compared. The intensity of the reference
beam, which should have suffered little or no light absorption, is
defined as I0. The intensity of the sample beam is defined as I.
Over a short period of time, the spectrometer automatically scans
all the component wavelengths in the manner described. The
ultraviolet (UV) region scanned is normally from 200 to 400 nm, and
the visible portion is from 400 to 800 nm.
III. Examples
[0093] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Example 1
Adduct Detection
[0094] During a manufacture for a particular recombinant protein,
seven filtered bulks for storage (FBS) were produced where typical
results against product appearance criteria were obtained for five
of the seven bulks. Per manufacturing specification, the product
specific test instructions require the use of Yellow (Y) color
series for the evaluation of the product samples by the COC assay,
a method for the determination of clarity/degree of opalescence,
degree of coloration, and appearance. However, two bulks (Runs 2
and 3) appeared brown in color and_did not meet the expected Yellow
series color criterion of .gtoreq.Y7 for the COC assay. A
comparison of the COC results for Runs 1-3 is shown in FIG. 1. To
investigate the discrepancy further, the seven FBS samples were
concentrated to increase the intensity of the color. The
concentrated samples were compared against all the Standard color
solutions listed in the U.S. Pharmacopeia 2012 (USP Monograph 631,
Color and Achromicity) or in the European Pharmacopoeia 5.0 (EP
Method 2.2.2, Degree of Coloration of Liquids) for confirmation of
the appropriate color assignment. The samples were compared in
diffused daylight 5 min after preparation of the reference sample,
viewing vertically against a black background. The diffusion of
light must be such that reference sample I can readily be
distinguished from water and that reference suspension. II can
readily be distinguished from reference suspension I. A liquid was
considered clear if its clarity was the same as that of water R or
of the solvent used when examined under the conditions described
above, or if its opalescence was not more pronounced than that of
the reference sample I.
[0095] Since the cause of the coloration was unknown for Runs 2 and
3, multiple investigational studies were completed to determine the
source and cause of the atypical brown color. Samples from Runs 1-3
were analyzed for metals, trace elements (other than metals), and
chromophores. These studies suggested that the coloration observed
in Runs 2 and 3 were not due to metals or other trace elements
(data not shown).
[0096] To determine whether chromophores were associated with the
unexpected color observed in the FBS, Runs 1-3 were analyzed using
ultraviolet and visible (UV/vis) spectroscopy with a 1 cm path
length cuvette. The UV spectra (200-600 nm) did not display any
significant differences in the observance profile for the samples
analyzed.
[0097] To increase the sensitivity of the UV spectrophotometer, the
experiment was repeated using a 10 cm path length cuvette. The 10
cm cuvette offers increased sensitivity to the 1 cm cuvette due to
the absorbance of a sample is proportional to the number of
absorbing molecules in the spectrophotometer meter light beam. The
samples were scanned between 200-700 nm to determine the absorption
spectrum of Runs 1-3. The shape of the spectra for Runs 2 and 3 was
different than Run 1: new absorbance peaks were observed
approximately at 320 nm and at 460 nm which were not apparent for
Run 1 (FIG. 2A). This difference can be observed more clearly when
the spectrum of Run 1 is subtracted from the spectrum of Run 3
(FIG. 2B). The peak observed at 460 nm for Runs 2 and 3 is
consistent with a flavin (e.g., vitamin) fingerprint.
[0098] Based on the 10 cm UV/vis results, full spectrum analysis
for RP-HPLC and IEC with options for MS detection were performed on
FBS from Runs 1-3.
[0099] Using full spectrum detection for RP-HPLC, no
chromatographic differences were observed for Run 1-3 (data not
shown). However, for IEC at 310 nm, minor differences were
observed. As shown in FIG. 3, a slight peak behind the Main Peak is
observed for Runs 2 and 3 while the profile for Run 1 is comparable
to the Reference Material.
[0100] Intact samples were submitted for 2D LC-MS and monitored at
both 280 and 310 nm. The 2D LC-MS analysis consists of two
parts--first dimension is separation by RP-HPLC with the second
dimension as fractionated peaks for mass spectrometry analysis.
From this experiment, the expected mass was observed for Run 1
while the expected mass and an additional mass of approximately
+157 Da were observed for Runs 2 and 3 (FIG. 4).
Example 2
Elucidation of the Adduct
[0101] To better elucidate the adduct, Run 3 was selected for
fractionation (the minor peak from the IEC assay (FIG. 3) was
collected) and further analyzed by 2D-LC MS and mass identification
by tryptic peptide map with MS detection.
[0102] From the 2D LC-MS analysis (FIG. 5), in addition to the
expected mass, an approximate +156 Da mass increase was again
observed for the fractionated shoulder peak. Upon on-line reduction
(with DTT) of the sample, the expected reduced mass was observed.
The four additional Daltons observed between the reduced and native
analyses are due to the breakage of the disulfide bonds and the
addition of four hydrogens. The additional mass was again observed,
suggesting the modification was non-reversible or covalent.
[0103] From the tryptic peptide map, the sample was collected at
both 214 nm and 310 nm. As shown in FIG. 6, novel peaks are
enhanced in the 45-55 minute region. LC-MS-MS analysis of the novel
peak observed at 48.8 minutes at 310 nm was determined to be T20
peptide with the cysteine residue modified with +154.006 Da.
Modified (at cysteine, +154.006 Da) and free T6 and T16 peptides
were also detected by mass extraction. Reduced T21 or modified T21
were not detected but this may have been due to the low levels
present. The other two peaks observed eluting between 50 to 56
minutes at 310 nm did not contain any unique species when compared
to the reference.
[0104] 1D and 2D 1H NMR analysis was collected to determine the
adduct structure. Additional data was acquired using TOSCY (Total
Correlation Spectroscopy), HSQC (Heteronuclear Single Quantum
Coherence), HMBC (Heteronuclear Multiple Bond Correlation), and
ROESY (Rotating-frame Overhauser Effect Spectroscopy (nOe)).
[0105] TOCSY creates correlations between all protons that are
coupled to each other as well as all other protons within a given
spin system. HSQC experiment correlates chemical shifts of directly
bound nuclei (i.e. two types of chemical nuclei) while HMBC
experiment correlates chemical shifts of two types of nuclei
separated from each other with two or more chemical bonds. ROESY
utilizes nOe which uses space, not through chemical bonds to
confirm a precise molecular conformation (i.e., three dimensional
structure of a molecule). The collected peptide observed long range
1H-13C coupling between aromatic (quinone) protons and C=O at 182
ppm. The 1H-13C HSQC chemical shifts for the collected peptide in
the aromatic region are a close match to those observed for the
synthetic model compound bound to naphthalene-1,4-dione. TOCSY data
assigns the Q, V, and R resonances in the product. Comparing 1H-15N
HSQC data of product to a synthetic peptide (NH.sub.2-IVQCR-COOH)
showed a Cys NH correlation was missing in the product sample as
shown in FIG. 7. The proposed structure is confirmed by strong nOe
observed between the CH of Cys and the NH of Arg (FIG. 8). Based on
the NMR data collected, the proposed structure is presented in FIG.
9.
[0106] The identification of the colored species as
1,4-dihydroxy-2-naphthoate (DHNA) which formed the recombinant
protein-brown adduct was based upon MS, NMR and genetic data. NMR
data confirmed that DHNA was attached to the recombinant protein
via cysteine residues. DHNA is a product derived from the
menaquinone biosynthesis pathway of E. coli cells (FIG. 10).
Menaquinone is present in E. coli but production of it is increased
when the culture is in an anaerobic and/or micro-aerobic condition.
Menaquinone is used for electron transport in limited oxygen
environments and used for returning the disulfide bond forming
protein DsbB to the active oxidized state in anaerobic
(micro-aerobic) conditions.
Example 3
Hi-dO Process to Mitigate Formation of DHNA-Product Adduct
[0107] A control strategy was developed to prevent the generation
of a product's free thiols and the subsequent formation of the
DHNA-product adduct. The cause of the color formation was
determined to be the result of a low redox environment during the
harvest operations because Runs 2 and 3 exhibited the highest
titers and cell densities, both were subjected to longer hold times
for their diluted homogenates, endured longer durations for the
homogenates to achieve less than the 15.degree. C. target
temperature and had suboptimal homogenate mixing times and rates
(data not shown). These factors contributed to generating a low
oxygen environment which promoted the reduction of the product
disulfide bonds and permitted the opportunity for DHNA to attach to
the free thiols of the protein product.
[0108] Since the DHNA-protein adduct was formed during the low
redox environment during the harvest operations which led to
reduced disulfide bonds (i.e. free thiols), an approach was
developed to prevent the generation of free thiols and the
formation of the DHNA-product adduct. This enhanced process
control, called Hi-dO, maintains the dissolved oxygen levels in the
harvest operations at greater than zero (>0%) to eliminate the
reducing environment (i.e. no free thiol generation).
[0109] The formation of the DHNA-product adduct is a complex
biological reaction that requires the combination of multiple
events across the fermentation and harvest operations. The output
of the fermentation process is the production of considerable
levels and/or availability of DHNA. The schematic of the three
major stages of a typical harvest operation is shown on FIG.
13.
[0110] Several process steps were tested
post-fermentation/pre-homogenization and tested
post-homogenization, to determine if such actions would mitigate
the reducing environment or free thiol generation. Such process
enhancements tested are shown in Table 1 and FIG. 12.
TABLE-US-00001 TABLE 1 Process Enhancements (Hi-dO)
Post-Fermentation/Pre-Homogenization In HMG Hold
Tank/Post-Homogenization Initiate WCB Hi-dO process control: Dilute
homogenate with 2x water prior to homogenate transfer 1. Target
dO.sub.2 >75% 1. Temperature control to 10.degree. C. 2.
Increase agitation rate (6.3 Watts/L) 2. Target dO.sub.2 >50% by
increasing agitation and/or air sparging 3. Apply overlay air (0.6
vvm) 4. Back-pressure added to about 18.85 psi (1.3 bar) 5. Process
time = 1.5 hours Transfer homogenate in water for immediate
dilution Initiate Hi-dO.sub.2 homogenate process control: 6.
Maintain Target dO.sub.2 >50% 7. Increase agitation (1-6
Watts/L) 8. Apply overlay or sparged air (if required) 9. Process
time = 2 hours
[0111] The results of the process enhancements outlined in Table 1
and FIG. 12 are shown in Table 2.
TABLE-US-00002 TABLE 2 Product Quality Analyses of Development Runs
Performed with the Hi-dO Enhanced Process Controls IEC % Anomalous
IEC Fermentation Peak @ 280/ % Main RP-HPLC SEC Native SEC Run 310
nm Peak @ 280 % Peak A % Monomer % Monomer Small-scale 0.00/0.00
99.48 98.82 100.00 99.99 (10 L) #1 Small-scale 0.00/0.00 99.69
99.00 100.00 99.97 (10 L) #2 Manufacturing- 0.00/0.00 99.58 99.03
100.00 99.99 scale (1,000 L) Release Spec Not defined, .gtoreq.97%
Main .gtoreq.97% Peak .gtoreq.98% .gtoreq.98% FBS CofA but should
Peak A monomer monomer not be detectable
[0112] A root cause analysis was carried out to understand the
origins of the brown coloration. This analysis resulted in the
identification of the colored species (DHNA), its attachment to a
recombinant protein product, the adduct (DHNA-protein) structure,
its origination and the proposed mechanism of how and when DHNA
became attached to the product during the production process. As
Table 1 summarizes, a mitigation strategy was implemented to
prevent formation of the brown adduct, by maintaining the dissolved
oxygen level greater than zero (>0%) throughout the harvest
operations to eliminate the reducing environment and prevent the
formation of product free thiols. As a result, as shown by IEC
analyses as the % anomalous peak demonstrated 0%, the brown adduct
foimation was not detected in the FBS (Table 2).
Example 4
Generating menE Gene-Deleted E. coil Host Cells
[0113] In addition to the Hi-dO harvest process of the invention,
another approach was undertaken to mitigate the brown adduct
formation. This involved genetically engineering the prokaryotic
host cell such that the inenE gene was deleted from the genome,
thereby preventing the production of any DHNA intemiediate from the
menaquinone biosynthesis pathway that could be attached to the
recombinant product.
[0114] The menE gene deleted host cells were generated as an
in-frame, single-gene knockout mutant following the methods
described in Baba et al., Construction of E. coli K-12 in-frame,
single-gene knockout mutants: the Keio collection, Molecular
Systems Biology, vol. 21, p.1-10 (2006) which is hereby
incorporated by reference. The menE gene was targeted for
mutagenesis with PCR products containing a resistance cassette
(such as kanamycin) flanked by FLP recognition target sites and a
50 base pair homologies to the adjacent chromosomal sequences.
[0115] The mutagenesis yielded approximately 10-1000 kanamycin
resistance colonies when the host cells were incubated aerobically
at 37.degree. C. on Luria-Bertani broth (LB) agar containing 30
.mu.g/mL kanamycin.
Example 5
Production of Recombinant Proteins using menE Gene-Deleted E. Coli
Host Cells
[0116] The ability of the menE gene-deleted E. coli host cells to
produce recombinant protein that did not exhibit DHNA-associated
protein adduct was tested. Briefly, the menE gene-deleted E. coli
cells were transformed with plasmid constructs that encoded for two
recombinant proteins, PROT 1 and PROT 2, and two recombinant
antibodies, AB 1 and AB2, per standard techniques well-known to
those of skill in the art (see for example, Simmons et al.,
Expression of full-length immunoglobulins in E. coli: rapid and
efficient production of aglycosylated antibodies, J of Immunol
Methods 263 p. 133-147 (2002)). Fermentation of the four
recombinant proteins/antibodies proceeded as described herein (see
also U.S. Pat. No. 6,979,556 which is hereby incorporated by
reference).
[0117] The filtered bulk recombinant product for all four
recombinant protein/antibodies were tested for DHNA-protein adduct
formation by IEC assay at 310 nm and showed no detectable
DHNA-protein adduct formation (see FIG. 11 for exemplary results
for PROT 1).
[0118] Surprisingly, it was found that the yield of recombinant
product as a result of using the menE deleted E. coli cells
increased appreciably by about 20% to 50% as compared to the yield
using E. coli host cells with an intact, wild-type menE gene. Table
3 shows these results.
TABLE-US-00003 TABLE 3 Recombinant Protein Yields using menE
gene-deleted host cells Yield using Yield using menE Recombinant
wild-type gene-deleted % Protein E. coli host E. coli host change
PROT 1 1.9 g/L 2.5 g/L 30% PROT 2 5.5 g/L 6.5 g/L 20% AB1 0.7 g/L
1.0 g/L 40% AB2 0.46 g/L 0.72 g/L 50%
* * * * *