U.S. patent application number 17/419541 was filed with the patent office on 2022-03-17 for methods of producing ustekinumab.
The applicant listed for this patent is Momenta Pharmaceuticals, Inc.. Invention is credited to Ru Zang.
Application Number | 20220081479 17/419541 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-17 |
United States Patent
Application |
20220081479 |
Kind Code |
A1 |
Zang; Ru |
March 17, 2022 |
METHODS OF PRODUCING USTEKINUMAB
Abstract
Methods of manufacturing an ustekinumab product with a target
level of one or more glycans are described.
Inventors: |
Zang; Ru; (Auburndale,
MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Momenta Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
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|
Appl. No.: |
17/419541 |
Filed: |
December 20, 2019 |
PCT Filed: |
December 20, 2019 |
PCT NO: |
PCT/US19/67916 |
371 Date: |
June 29, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62786821 |
Dec 31, 2018 |
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International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1. A method of manufacturing a pharmaceutical composition
comprising ustekinumab having a target level of G0F glycan, the
method comprising: (a) selecting a target level of G0F glycan, (b)
selecting a level of galactose and a time for cell culture to
provide the selected level of G0F glycan, wherein the level of
galactose and the time are inversely related; (c) culturing a
population of cells genetically engineered to express ustekinumab
under conditions comprising the selected level of galactose and
time; (d) harvesting ustekinumab expressed by the population of
cells, thereby producing the preparation of ustekinumab; and (e)
purifying, concentrating, and/or formulating the ustekinumab
preparation to produce pharmaceutical composition comprising
ustekinumab if the preparation meets the target level of G0F
glycan.
2. The method of claim 1, wherein the culturing is performed using
a perfusion culture process.
3. The method of claim 1 or 2, wherein the target level of G0F
glycan is within a range of 20% to 80% G0F relative to total
glycan.
4. The method of claim 1 or 2, wherein the target level of G0F
glycan is within a range of 25% to 65% G0F relative to total
glycan.
5. The method of any one of claims 1-4, wherein the target level of
G0F glycan is within a range of 20% up to 40% G0F relative to total
glycan, wherein the selected level of galactose is 0 mM, and
wherein the time for cell culture is within a range of 7 days to 15
days.
6. The method of any one of claims 1-4, wherein the target level of
G0F glycan is within a range of 40% to 80% G0F relative to total
glycan, wherein the selected level of galactose is within a range
of 15 mM to 30 mM, and wherein the time for cell culture is within
a range of 16 days to 60 days.
7. The method of claim 6, wherein the time for cell culture is
within a range of 25 days to 42 days.
8. The method of any one of claims 1-7, further comprising
measuring a level of G0F glycan.
9. The method of any one of claims 1-8, wherein the population of
cells genetically engineered to express ustekinumab are mammalian
cells.
10. The method of claim 9, wherein the mammalian cells are selected
from CHO cells, HEK 293 cells, fibrosarcoma HT 1080 cells, PER.C6
cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells and SP 2/0
cells.
11. The method of any one of claims 1-10, wherein the selected
level of galactose controlled during culturing from t=0 until
harvest.
12. The method of any one of claims 1-11, wherein the ustekinumab
expressed by the cells is harvested at two or more times within the
range of time of the time for cell culture.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/786,821, filed Dec. 31, 2018, which is hereby
incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 20, 2019, is named M0168PCT_SL.txt and is 6,036 bytes in
size.
BACKGROUND
[0003] Therapeutic antibodies are an important class of therapeutic
biological products. Antibody glycosylation and glycan composition
can impact antibody activity and effector functions. There remains
an ongoing need for improved methods for controlling the glycan
profile of antibody products.
SUMMARY OF THE INVENTION
[0004] The present disclosure provides process levers for producing
(e.g., manufacturing) ustekinumab with a target level of one or
more glycans. The present disclosure provides the insight that a
relationship between galactose concentration in a culture media and
time of culturing can be used as a process lever for controlling a
target level of one or more glycans (e.g., galactosylation) of
ustekinumab in a culture. The present disclosure identifies an
inverse relationship between culture time and galactose
concentration in culture media in controlling a target level of one
or more glycans (e.g., galactosylation).
[0005] In certain aspects, the disclosure provides methods of
manufacturing ustekinumab with a target level of one or more
glycans. Such methods can include providing (e.g., producing,
expressing (e.g., in small scale or large scale cell culture)
and/or manufacturing) or obtaining (e.g., receiving and/or
purchasing from a third party (including a contractually related
third party or a non-contractually-related (e.g., an independent)
third party)) an ustekinumab test protein (e.g., an ustekinumab
drug substance, e.g., a preparation of ustekinumab drug substance,
e.g., a batch of test ustekinumab dug substance).
[0006] In some instances, the disclosure provides methods of
manufacturing a pharmaceutical composition comprising ustekinumab
having a target level of one or more glycans, the method
comprising: selecting a level of galactose and a time for cell
culture, where the level of galactose and the time are inversely
related; culturing a population of cells genetically engineered to
express ustekinumab under conditions comprising the selected level
of galactose and time; harvesting ustekinumab expressed by the
population of cells, thereby producing the preparation of
ustekinumab; and purifying, concentrating, and/or formulating the
ustekinumab preparation to produce pharmaceutical composition
comprising ustekinumab if the preparation meets the target level of
one or more glycans. In some embodiments, the target level of one
or more glycans is a target level of a glycan selected from the
group consisting of G0F, sialylated glycan, and G2F. In some
embodiments, the target level of one or more glycans is a target
level of G0F.
[0007] In some instances, the disclosure provides methods of
manufacturing a pharmaceutical composition comprising ustekinumab
having a target level of G0F glycan, the method comprising:
selecting a target level of G0F glycan; selecting a level of
galactose and a time for cell culture to provide the selected
target level of G0F, where the level of galactose and the time are
inversely related; culturing a population of cells genetically
engineered to express ustekinumab under conditions comprising the
selected level of galactose and time; harvesting ustekinumab
expressed by the population of cells, thereby producing the
preparation of ustekinumab; and purifying, concentrating, and/or
formulating the ustekinumab preparation to produce pharmaceutical
composition comprising ustekinumab if the preparation meets the
target level of G0F glycan.
[0008] In some instances, the disclosure provides methods of
producing an ustekinumab drug product having a target level of one
or more glycans, the method comprising: culturing a population of
cells genetically engineered to express ustekinumab under
conditions, where the conditions are characterized by parameters
including a selected level of galactose and a time for cell
culture, and where the level of galactose and the time are
inversely related; harvesting ustekinumab expressed by the cell,
thereby producing an ustekinumab preparation; and purifying,
concentrating, and/or formulating the ustekinumab preparation to
produce an ustekinumab drug product if the ustekinumab preparation
meets the target level of one or more glycans. In some embodiments,
the target level of one or more glycans is a target level of a
glycan selected from the group consisting of G0F, sialylated
glycan, and G2F. In some embodiments, the target level of one or
more glycans is a target level of G0F.
[0009] In some embodiments, provided methods include culturing a
population of mammalian cells that are genetically engineered to
express ustekinumab. In some embodiments, mammalian cells are
selected from: CHO cells, HEK 293 cells, fibrosarcoma HT 1080
cells, PER.C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0
cells and SP 2/0 cells.
[0010] In some embodiments, in the provided methods the culturing
step is performed using continuous culture process. In some
embodiments, provided methods include culturing that is performed
using a perfusion culture process (e.g., an alternating tangential
flow filter (ATF)-based perfusion culture process). In some certain
embodiments, provided methods include culturing mammalian cells
that are genetically engineered to express ustekinumab (e.g., SP
2/0 cells that express ustekinumab) by a perfusion culture
process.
[0011] In some embodiments, provides methods including harvesting
ustekinumab expressed by the cells at two or more times within the
range of time of the time for cell culture.
[0012] In some embodiments, a target level of G0F glycan is within
a range of 20% to 80% G0F relative to total glycan. In some
embodiments, a target level of G0F glycan is within a range of 25%
to 65% G0F relative to total glycan.
[0013] In some embodiments, provided methods are directed to
manufacturing and/or producing ustekinumab having a target level of
G0F glycan within a range of 20% up to 40% G0F relative to total
glycan. In some embodiments, a method for achieving ustekinumab
having a target level of G0F glycan within a range of 20% up to 40%
G0F relative to total glycan includes a selected level of galactose
at 0 mM and a time for cell culturing is within a range of 7 days
to 15 days.
[0014] In some embodiments, provided methods are directed to
manufacturing and/or producing ustekinumab having a target level of
G0F glycan within a range of 40% to 80% G0F relative to total
glycan. In some embodiments, a method for achieving ustekinumab
having a target level of G0F glycan within a range of 40% to 80%
G0F relative to total glycan include a selected level of galactose
within a range of 15 mM to 30 mM and a time for cell culture that
is within a range of 16 days to 60 days. In some certain
embodiments, a time for cell culturing is within a range of 25 days
to 42 days.
[0015] In some embodiments, provided methods include selecting a
target level of G0F glycan. In some embodiments, a target level of
G0F glycan is within a range of 20% to 80% G0F relative to total
glycan. In some certain embodiments, a target level of G0F glycan
within a range of 20% up to 40% G0F relative to total glycan, where
the method includes a selected level of galactose at 0 mM and a
time for cell culture that is within a range of 7 days to 15 days.
In some certain embodiments, a target level of G0F glycan within a
range of 40% to 80% G0F relative to total glycan, where the method
includes a selected level of galactose within a range of 15 mM to
30 mM and a time for cell culture that is within a range of 16 days
to 60 days.
[0016] In some embodiments, provided methods a selected level of
galactose is controlled throughout the culturing step (e.g.,
controlled during culturing from t=0 until harvest).
[0017] In some embodiments, provided methods further include
measuring a level of G0F glycan. In some embodiments, if the
measured level of G0F glycan is within a range of 20% to 80% G0F
relative to total glycan (e.g., within a range of 20% up to 40% G0F
relative to total glycan, e.g., within a range of 40% to 80% G0F
relative to total glycan), then a step of purifying, concentrating,
and/or formulating the ustekinumab preparation is performed.
[0018] These, and other aspects of the invention, are described in
more detail below and in the claims.
BRIEF DESCRIPTION OF THE DRAWING
[0019] The Drawing included herein, which is composed of the
following Figures, is for illustration purposes only and not for
limitation.
[0020] FIG. 1 depicts comparison of glycan profiles of various
preparations and sources of ustekinumab. Abundance of G0F glycan is
provided in the upper left panel; abundance of G1F-A glycan is
provided in the upper right panel; abundance of G1F-B glycan is
provided in the lower left panel; and total sialylation provided in
the lower right panel. Solid circles represent reference protein
products ("RPPs") from the U.S. (black solid circles) and Europe
(shaded solid circles). Symbols in the right-most portion of each
panel represent ustekinumab test preparations cultured at different
volumes. Solid shaded squares depicting samples cultured at 3 L,
open squares depicting samples from satellite culture, solid
triangles depicting samples cultured at 100 L, and solid diamonds
depicting samples cultured at 250 L. The relative abundance of each
glycan depicted in its respective panel, with samples depicted in
each panel sorted into four groups, walking along the x-axis from
left to right: Group 1 RPPs from 90 mg/ml reference samples (left
most), Group 2 RPPs from 90 mg/ml reference samples (second from
left), 5 mg/mL RPPs (third from left) and ustekinumab test products
(right most).
[0021] FIG. 2 depicts G0F glycan profile variation with harvest
duration. Left-most column (white) represents Group 1 RPP G0F
abundance, second column from the left (black) represents Group 2
RPP G0F abundance, third column from the left represents the
average abundance of G0F from samples cultured between 7 days and
42 days. Shaded columns (starting at fourth column from the left
and continuing to the right most column) each represent the G0F
abundance from samples cultured an indicated number of days within
a range of 7 days (D7) up to 42 days (D42, right most column).
[0022] FIG. 3 depicts comparisons of major glycan profiles and
charge variants between various culture conditions. Walking along
the x-axis of each panel from left to right are depicts: Group 1
RPPs (left-most), Group 2 RPPs, ustekinumab cultured at 3 L DS,
ustekinumab cultured at 100 L DS, ustekinumab cultured at 250 L DS,
NCM-2 ustekinumab preparation, and NCM-1 ustekinumab preparation
(right-most).
CERTAIN DEFINITIONS
[0023] In general, terminology used herein is in accordance with
its understood meaning in the art, unless clearly indicated
otherwise. Explicit definitions of certain terms are provided
below; meanings of these and other terms in particular instances
throughout this specification will be clear to those skilled in the
art from context.
[0024] In order that the present invention may be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification.
[0025] As used herein, the terms "about" or "approximately," as
applied to one or more values of interest, refers to a value that
is similar to a stated reference value. In certain embodiments, the
terms "about" or "approximately" refer 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%, 1%, or less of the stated
reference value.
[0026] The terms "control, controlled, controlling," as used herein
in reference to controlling a target level of one or more glycans
(e.g., galactose, e.g., G0F) of ustekinumab means selecting,
maintaining and/or adjusting one or more culture conditions for
production of ustekinumab. Adjusting can including increasing or
decreasing one or more culture conditions for production of
ustekinumab. As used herein, a controlled target level of one or
more glycans relates to production of ustekinumab with a desired
level of one or more glycans that has minimal product drift. In
some embodiments, a controlled target level of G0F will vary no
more than 20%, 15%, 10%, or 5% between samples in the same
production run and/or batch. In some embodiments, controlling a
target level of one or more glycan ensures consistency in the
production of ustekinumab (e.g., batch to batch consistency,
consistency throughout samples from any particular production
process).
[0027] As used herein, a "glycan" refers to a compound comprising
at least one sugar residue (e.g., monosaccharide). Glycans can be
monomers or polymers of sugar residues, and can be linear or
branched. A glycan may include natural sugar residues (e.g.,
glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose,
mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and/or
modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose,
phosphomannose, 6'-sulfo N-acetylglucosamine, etc). The term glycan
includes homopolymers and heteropolymers of sugar residues. The
term "glycan" also encompasses a glycan component of a
glycoconjugate (e.g., a glycoprotein, glycolipid, proteoglycan,
etc.). The term also encompasses free glycans, including glycans
that have been cleaved or otherwise released from a
glycoconjugate.
[0028] As used herein, a "galactosylated glycan" refers to a glycan
that includes at least one galactose sugar residue. In some
embodiments, a galactosylated glycan is a G1, G2, G1F, G2F, A1,
and/or A2 glycan. A non-galactosylated glycan includes G0F or G0.
In some embodiments, a target level of galactosylated glycan may
refer to the presence of galactosylated glycan (e.g., G2F) and/or a
target level of non-galactosylated glycan (e.g., G0F).
[0029] The term "isolated," as used herein, refers to a substance
and/or entity that has been (1) separated from at least some of the
components with which it was associated when initially produced
(whether in nature and/or in an experimental setting), and/or (2)
designed, produced, prepared, and/or manufactured by the hand of
man. Isolated substances and/or entities may be separated from
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
or more than about 99% of the other components with which they were
initially associated. In some embodiments, isolated agents are
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
or more than about 99% pure. As used herein, a substance is "pure"
if it is substantially free of other components. In some
embodiments, as will be understood by those skilled in the art, a
substance may still be considered "isolated" or even "pure", after
having been combined with certain other components such as, for
example, one or more carriers or excipients (e.g., buffer, solvent,
water, etc.); in such embodiments, percent isolation or purity of
the substance is calculated without including such carriers or
excipients. To give but one example, in some embodiments, a
biological polymer such as a polypeptide or polynucleotide that
occurs in nature is considered to be "isolated" when, a) by virtue
of its origin or source of derivation is not associated with some
or all of the components that accompany it in its native state in
nature; b) it is substantially free of other polypeptides or
nucleic acids of the same species from the species that produces it
in nature; c) is expressed by or is otherwise in association with
components from a cell or other expression system that is not of
the species that produces it in nature. Thus, for instance, in some
embodiments, a polypeptide that is chemically synthesized or is
synthesized in a cellular system different from that which produces
it in nature is considered to be an "isolated" polypeptide.
Alternatively or additionally, in some embodiments, a polypeptide
that has been subjected to one or more purification techniques may
be considered to be an "isolated" polypeptide to the extent that it
has been separated from other components a) with which it is
associated in nature; and/or b) with which it was associated when
initially produced.
[0030] As used herein, "process lever" refers to an element of a
culture process (e.g., one or more culture conditions) that can be
controlled in order to increase or decrease the abundance of one or
more glycans on an antibody product. The present disclosure
provides a novel process lever that is a specified relationship
between galactose concentration in the culture media and culture
time. As described herein, an inverse relationship between
galactose concentration in a culture media and the time of culture
in the culture media can be used as a process lever to produce
ustekinumab with a target level of one or more glycans (e.g.,
galactosylation). In some embodiments, a process lever includes a
selected a level of galactose and time for cell culture to control
level of one or more glycans (e.g., galactosylation, e.g., G0F) of
ustekinumab (e.g., level of one or more glycans in a ustekinumab
culture or preparation).
[0031] As used herein, an "N-glycosylation site of an Fc region"
refers to an amino acid residue within an Fc region to which a
glycan is N-linked. In some certain embodiments, a N-glycosylation
site of ustekinumab is located in a heavy chain at position
Asn299.
[0032] In general, a "protein," as used herein, is a polypeptide
(i.e., a string of at least two amino acids linked to one another
by peptide bonds). Proteins may include moieties other than amino
acids (e.g., may be glycoproteins) and/or may be otherwise
processed or modified. Those of ordinary skill in the art will
appreciate that a "protein" can be a complete polypeptide chain as
produced by a cell (with or without a signal sequence), or can be a
functional portion thereof. Those of ordinary skill will further
appreciate that a protein can sometimes include more than one
polypeptide chain, for example linked by one or more disulfide
bonds or associated by other means.
[0033] As used herein "recovering" refers to the process of
rendering an agent or entity substantially free of other
previously-associated components, for example by isolation, e.g.,
using purification techniques known in the art. In some
embodiments, an agent or entity is recovered from a natural source
and/or a source comprising cells.
[0034] As used herein, "sample(s)" refer to separately procured
samples. In some embodiments, evaluation of separate samples
includes evaluation of samples from the same culture run (e.g., at
different time points during preparation) or from different culture
runs (e.g., different rounds of culture).
[0035] "Target value or target level", as used herein, refers to a
predetermined level of one or more particular glycans, such as
galactosylated glycans and/or sialylated glycans. In some
embodiments, a target value is a level of one or more particular
glycans, such as galactosylated glycans (e.g., G0, G1, G2, G0F,
G1F, G2F or combinations) and/or sialylated glycans (e.g.,
monosialylated, disialylated, or combinations), in a reference
ustekinumab product or described in a specification or master batch
record for a pharmaceutical product. In some certain embodiments, a
target value is a level of G0F glycans in an ustekinumab
product.
[0036] In some embodiments, a target value refers to an absolute
level of (e.g., number of moles of) one or more glycans (e.g.,
galactosylated glycans (e.g., one or more species of galactosylated
glycans) and/or sialylated glycans (e.g., one or more species of
sialylated glycans)) in a ustekinumab preparation. In some
embodiments, a target value refers to a level of one or more
glycans (e.g., galactosylated glycans (e.g., one or more species of
galactosylated glycans) and/or sialylated glycans (e.g., one or
more species of sialylated glycans)) in a ustekinumab preparation
relative to total level of glycans in the ustekinumab preparation.
In some embodiments, a target value is expressed as a "percent",
which refers to the number of moles of one or more glycans (e.g.,
Fc glycans) relative to total moles of glycans (e.g., Fc glycans)
in a ustekinumab preparation. In some embodiments, "percent" refers
to the number of moles of one or more PNGase F-released Fc glycans
relative to total moles of PNGase P-released Fc glycans
detected.
[0037] The term "ustekinumab preparation" as used herein refers to
a mixture of ustekinumab proteins obtained according to a
particular production method. Ustekinumab proteins in an
ustekinumab preparation includes multiple copies of ustekinumab
(i.e., having the same or substantially the same amino acid
sequence) but have a mixture of glycans associated with the
protein. In some instances, an ustekinumab preparation is prepared
using a method and/or system as provided herein. Production methods
can include a recombinant preparation step using cultured cells
that have been engineered to express ustekinumab (or to express
ustekinumab at a relevant level or under relevant conditions). In
some embodiments, a production method may include an isolation step
in which ustekinumab is isolated from certain components of the
engineered cells (e.g., by lysing the cells and pelleting the
protein component by centrifugation). In some embodiments,
production method may also include a purification step in which
ustekinumab is separated (e.g., by chromatography) from other
cellular components, e.g., other proteins or organic components
that were used in earlier steps. It will be appreciated that these
steps are non-limiting and that any number of additional
productions steps may be included. Different ustekinumab
preparations may be prepared by the same production method but on
different occasions (e.g., different runs or preparations).
Alternatively, different ustekinumab preparations may be prepared
by different production methods. Two production methods may differ
in any way (e.g., expression vector, engineered cell type, culture
conditions, isolation procedure, purification conditions,
etc.).
[0038] All literature and similar material cited in this
application, including, but not limited to, patents, patent
applications, articles, books, treatises, and web pages, regardless
of the format of such literature and similar materials, are
expressly incorporated by reference in their entirety. In the event
that one or more of the incorporated literature and similar
materials differs from or contradicts this application, including
but not limited to defined terms, term usage, described techniques,
or the like, this application controls. The section headings used
herein are for organizational purposes only and are not to be
construed as limiting the subject matter described in any way.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0039] The present disclosure describes, at least in part, the
discovery of process levers for producing ustekinumab having
targeted levels of one or more glycans (e.g., galactosylation,
e.g., G0F glycans). Controlling glycan composition and levels
during antibody production is a continuing challenge. Glycosylation
of therapeutic antibodies can impact their safety and/or efficacy.
(Zhang et al. (2016) Drug Discovery Today 21(5): 740-765).
Accordingly, it is important to be able to ensure consistency of
glycan composition in the production of ustekinumab (e.g., batch to
batch consistency, consistency throughout samples from any
particular production process). During characterization of
ustekinumab reference protein products ("RPPs"), two distinct
glycan populations of ustekinumab RPPs were observed.
[0040] The present disclosure describes the development of levers
for controlling glycan composition during manufacture of
ustekinumab. Process levers are described herein to produce
ustekinumab with target levels of particular glycans, including
each of the RPPs glycan profiles identified. In the course of
developing levers as described, the present disclosure identified a
relationship between galactose concentration in a culture media and
time of culturing. The present disclosure provides the insight that
a relationship between galactose concentration in a culture media
and time of culturing (e.g., continuously culturing) can be used as
a process lever for controlling a target level of one or more
glycans (e.g., galactosylation) of ustekinumab in a culture.
[0041] The present disclosure further provides the insight that the
for controlling the glycan level (e.g., galactosylation), a
relationship between a level of galactose and time (i.e., duration
of culturing) are inversely related. Culturing methods of the
present disclosure for controlling glycan compositions include
continuous culture methods (e.g., perfusion culture).
Ustekinumab
[0042] The present disclosure provides, in part, methods and
processes for manufacturing, preparing, controlling or otherwise
generating ustekinumab with particular glycan profiles.
[0043] Ustekinumab is an antibody that specifically binds to the
p-40 subunit of both IL-12 and IL-23. Ustekinumab has been studied
in a number of human diseases including psoriasis, psoriatic
arthritis, Crohn's disease and multiple sclerosis.
[0044] In some embodiments, ustekinumab includes HCDR1, HCDR2, and
HCDR3 sequences that differ by no more than 3 amino acid residues
from the HCDR sequences as set forth in SEQ ID NO.: 1 and LCDR1,
LCDR2, and LCDR3 sequences that differ by no more than 3 amino acid
residues from the LCDR sequences as set forth in SEQ ID NO.: 2. In
some embodiments, ustekinumab includes HCDR1, HCDR2, and HCDR3
sequences that differ by no more than 2 amino acid residues or by
no more than 1 amino acid residue from the HCDR sequences as set
forth in SEQ ID NO.: 1 and LCDR1, LCDR2, and LCDR3 sequences that
differ no more than 2 amino acid residues or by no more than 1
amino acid residue from the LCDR sequences as set forth in SEQ ID
NO.: 2. In some embodiments, ustekinumab includes a heavy chain
variable domain that differs by no more than 3 amino acid resides
from the sequence as set forth in SEQ ID NO.: 1 and a light chain
variable domain that differs by no more than 3 amino acid resides
from the sequence as set forth in SEQ ID NO.: 2. In some
embodiments, ustekinumab includes a heavy chain that includes a
sequence that differs by no more than 5 amino acids from the
sequence as set forth in SEQ ID NO.: 1 and a light chain that
includes a sequence that differs by no more than 5 amino acid
resides from the sequence of SEQ ID NO.: 2.
[0045] In some embodiments, ustekinumab includes HCDR1, HCDR2, and
HCDR3 sequences as set forth in SEQ ID NO.: 1 and LCDR1, LCDR2, and
LCDR3 sequences as set forth in SEQ ID NO.: 2. In some embodiments,
ustekinumab includes a heavy chain variable domain as set forth in
SEQ ID NO.: 1 and a light chain variable domain as set forth in SEQ
ID NO.: 2. In some embodiments, ustekinumab includes a heavy chain
comprising a sequence of SEQ ID NO.: 1 and/or a light chain
comprising a sequence of SEQ ID NO.: 2.
TABLE-US-00001 ustekinumab heavy chain sequence (bold indicates
variable domain sequence with CDR sequences underlined) SEQ ID NO.:
1 EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIM
SPVDSDIRYSPSFQGQVTMSVDKSITTAYLQWNSLKASDTAMYYCARRRPG
QGYFDFWGQGTLVTVSSSSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ustekinumab light chain
sequence (bold indicates variable domain sequence with CDR
sequences underlined) SEQ ID NO.: 2
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAA
SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPYTFGQGT
KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC
Culture Methods
[0046] In some instances, the disclosure provides methods of
manufacturing a pharmaceutical composition comprising ustekinumab
having a target level of one or more glycans, the method
comprising: selecting a level of galactose and a time for cell
culture, where the level of galactose and the time are inversely
related; culturing a population of cells genetically engineered to
express ustekinumab under conditions comprising the selected level
of galactose and time; harvesting ustekinumab expressed by the
population of cells, thereby producing the preparation of
ustekinumab; and purifying, concentrating, and/or formulating the
ustekinumab preparation to produce pharmaceutical composition
comprising ustekinumab if the preparation meets the target level of
one or more glycans. In some embodiments, the target level of one
or more glycans is a target level of a glycan selected from the
group consisting of G0F, sialylated glycan, and G2F. In some
embodiments, the target level of one or more glycans is a target
level of G0F.
[0047] In some instances, the disclosure provides methods of
producing an ustekinumab drug product having a target level of one
or more glycans, the method comprising: culturing a population of
cells genetically engineered to express ustekinumab under
conditions, where the conditions are characterized by parameters
including a selected level of galactose and a time for cell
culture, and where the level of galactose and the time are
inversely related; harvesting ustekinumab expressed by the cell,
thereby producing an ustekinumab preparation; and purifying,
concentrating, and/or formulating the ustekinumab preparation to
produce an ustekinumab drug product if the ustekinumab preparation
meets the target level of one or more glycans. In some embodiments,
the target level of one or more glycans is a target level of a
glycan selected from the group consisting of G0F, sialylated
glycan, and G2F. In some embodiments, the target level of one or
more glycans is a target level of G0F.
[0048] In some embodiments, a target level of G0F glycan is within
a range of 20% to 80% G0F relative to total glycan. In some
embodiments, a target level of G0F glycan is within a range of 25%
to 65% G0F relative to total glycan.
[0049] In some embodiments, provided methods are directed to
manufacturing and/or producing ustekinumab having a target level of
G0F glycan within a range of 20% up to 40% G0F relative to total
glycan. In some embodiments, a method for achieving ustekinumab
having a target level of G0F glycan within a range of 20% up to 40%
G0F relative to total glycan includes a selected level of galactose
at 0 mM and a time for cell culture that is within a range of 7
days to 15 days.
[0050] In some embodiments, provided methods are directed to
manufacturing and/or producing ustekinumab having a target level of
G0F glycan within a range of 40% to 80% G0F relative to total
glycan. In some embodiments, a method for achieving ustekinumab
having a target level of G0F glycan within a range of 40% to 80%
G0F relative to total glycan include a selected level of galactose
within a range of 15 mM to 30 mM and a time for cell culture that
is within a range of 16 days to 60 days. In some certain
embodiments, a time for cell cultures is within a range of 25 days
to 42 days.
[0051] In some embodiments, provided methods further include
selecting a target level of G0F glycan. In some embodiments, a
target level of G0F glycan is within a range of 20% to 80% G0F
relative to total glycan. In some certain embodiments, a target
level of G0F glycan within a range of 20% up to 40% G0F relative to
total glycan, where the method includes a selected level of
galactose at 0 mM and a time for cell culture that is within a
range of 7 days to 15 days. In some certain embodiments, a target
level of G0F glycan within a range of 40% to 80% G0F relative to
total glycan, where the method includes a selected level of
galactose within a range of 15 mM to 30 mM and a time for cell
culture that is within a range of 16 days to 60 days.
[0052] In some embodiments, provided methods further include
measuring a level of G0F glycan. In some embodiments, if the
measured level of G0F glycan is within a range of 20% to 80% G0F
relative to total glycan (e.g., within a range of 20% up to 40% G0F
relative to total glycan, e.g., within a range of 40% to 80% G0F
relative to total glycan), then a step of purifying, concentrating,
and/or formulating the ustekinumab preparation is performed.
[0053] In some embodiments, provided methods for manufacturing
and/or producing ustekinumab having a target glycan level include a
target level of sialylated glycan and/or G2F glycan. Provided in
Table 1 below are conditions for ustekinumab with target levels of
sialylated glycan and/or G2F glycan.
TABLE-US-00002 TABLE 1 Examplary Conditions for Production of
Ustekinumab Preparations with Target G2F and Sialylated Glycan
Levels Total Glycan Range G2F Sialylation NCM-1 >5%-10%
>15%-30% (0 mM galactose, culture time 7-15 days) NCM-2 1%-5%
5%-15% (15-30 mM galactose, culture time 25-60 days)
[0054] In some embodiments, provided methods are directed to
manufacturing and/or producing ustekinumab having a target level of
sialylated glycan within a range of 15% to 30% sialylated glycan
relative to total glycan. In some embodiments, a method for
achieving ustekinumab having a target level of sialylated glycan
within a range of 15% to 30% sialylated glycan relative to total
glycan includes a selected level of galactose at 0 mM and a time
for cell culturing is within a range of 7 days to 15 days.
[0055] In some embodiments, provided methods are directed to
manufacturing and/or producing ustekinumab having a target level of
sialylated glycan within a range of 5% up to 15% sialylated glycan
relative to total glycan. In some embodiments, a method for
achieving ustekinumab having a target level of G0F glycan within a
range of 5% up to 15% sialylated glycan relative to total glycan
include a selected level of galactose within a range of 15 mM to 30
mM and a time for cell culture that is within a range of 16 days to
60 days. In some certain embodiments, a time for cell culturing is
within a range of 25 days to 42 days.
[0056] In some embodiments, provided methods are directed to
manufacturing and/or producing ustekinumab having a target level of
G2F glycan within a range of 5% to 10% G2F relative to total
glycan. In some embodiments, a method for achieving ustekinumab
having a target level of G2F glycan within a range of 5% to 10% G2F
relative to total glycan includes a selected level of galactose at
0 mM and a time for cell culture that is within a range of 7 days
to 15 days.
[0057] In some embodiments, provided methods are directed to
manufacturing and/or producing ustekinumab having a target level of
G2F glycan within a range of 1% up to 5% G0F relative to total
glycan. In some embodiments, a method for achieving ustekinumab
having a target level of G2F glycan within a range of 1% up to 5%
G2F relative to total glycan include a selected level of galactose
within a range of 15 mM to 30 mM and a time for cell culture that
is within a range of 16 days to 60 days. In some certain
embodiments, a time for cell culturing is within a range of 25 days
to 42 days.
[0058] In some embodiments, provided methods a selected level of
galactose is controlled throughout the culturing step (e.g.,
controlled during culturing from t=0 until harvest).
[0059] In some embodiments, provided methods include culturing a
population of mammalian cells that are genetically engineered to
express ustekinumab. In some embodiments, mammalian cells are
selected from: CHO cells, HEK 293 cells, fibrosarcoma HT 1080
cells, PER.C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0
cells and SP 2/0 cells.
[0060] In some embodiments, in the provided methods the culturing
step is performed using continuous culture process. In some
embodiments, provided methods include culturing that is performed
using a perfusion culture process (e.g., an alternating tangential
flow filter (ATF)-based perfusion culture process). In some certain
embodiments, provided methods include culturing mammalian cells
that are genetically engineered to express ustekinumab (e.g., SP
2/0 cells that express ustekinumab) by a perfusion culture
process.
[0061] In some embodiments, provides methods including harvesting
ustekinumab expressed by the cells at two or more times within the
range of time of the time for cell culture.
[0062] In some embodiments, the target value is a predetermined
pharmaceutical product specification or a quality control criterion
for a pharmaceutical preparation, e.g., a Certificate of Analysis
(CofA), a Certificate of Testing (CofT), or a Master Batch Record.
In some embodiments, the product specification is a product
description in an FDA label, a Physician's Insert, a USP monograph,
or an EP monograph.
[0063] Generally, cell culture methods of the present disclosure
including culturing at a temperature within a range of 25.degree.
C. to 40.degree. C. and with gravity as it is encountered on earth.
In some embodiments, provided methods of culturing a population of
cells is sufficient for expression of an ustekinumab product. Cell
culture media generally comprise an appropriate source of energy
and compounds which regulate the cell cycle. Generally, culture
media includes, for example amino acids, vitamins, inorganic salts,
and glucose, which is known to those of skill in the art. In some
embodiments, a cell culture media has a pH of 6 to 8. Media for
animal cell culture are well established in the art, and are
routinely optimized by the skilled artisan for the particular
purpose and/or cell type.
[0064] In some embodiments, methods of the present disclosure
include culturing a population of cells via a continuous cell
culture system (e.g., perfusion cell culture system). In some
embodiments, a continuous cell culture system includes bioreactor
tank and a cell retention device. In some embodiments, a continuous
cell culture system includes a bioreactor tank, a cell retention
device, a media supply, and a bleed waste collection. In some
embodiments, a continuous cell culture system includes a population
of cells (e.g., a population of cells engineered to express
ustekinumab, e.g., a population of cells consisting of cells
engineered to express ustekinumab) and cell culture media.
[0065] In some embodiments, a cell retention device is or includes
a continuous centrifuge, an alternating tangential flow filter
(ATF), a tangential flow membrane filter (TFF), a dynamic filter, a
spin-filter, an ultrasonic and dielectrophoretic separator, or a
gravity settler. In some certain embodiments, a cell retention
device is or includes an ATF.
[0066] In some embodiments, a bioreactor system includes a
stirred-tank bioreactor, a cell retention device, a media supply,
and a bleed waste collection.
[0067] In some embodiments, a bioreactor system (e.g., a perfusion
bioreactor system) includes a sparger. In some embodiments, a
bioreactor system includes a drilled hole sparger. In some
embodiments, a bioreactor system includes an open pipe sparger. In
some embodiments, a bioreactor system includes a sintered
sparger.
[0068] In some embodiments, methods of the present disclosure
include culturing a population of cells (e.g., a population of
cells engineered to express ustekinumab, e.g., a population of
cells consisting of cells engineered to express ustekinumab) at a
volume within a range of 1 L to 3000 L. In some embodiments,
provided methods include culturing mammalian cells that are
genetically engineered to express ustekinumab at a volume of at
least 25 L, 50 L, 100 L, 200 L, 250 L, 400 L, 500 L, 600 L, 800 L,
1000 L, or 2000 L. In some embodiments, provided methods include
culturing mammalian cells that are genetically engineered to
express ustekinumab at a volume of about 25 L to about 250 L.
Glycan Evaluation
[0069] In some embodiments, glycans of ustekinumab are analyzed
(e.g., measured) by any available suitable method. In some
instances, glycan structure and composition as described herein are
analyzed, for example, by one or more, enzymatic, chromatographic,
mass spectrometry (MS), chromatographic followed by MS,
electrophoretic methods, electrophoretic methods followed by MS,
nuclear magnetic resonance (NMR) methods, and combinations thereof.
Exemplary enzymatic methods include contacting a ustekinumab
preparation with one or more enzymes under conditions and for a
time sufficient to release one or more glycan(s) (e.g., one or more
exposed glycan(s)). In some instances, the one or more enzymes
include(s) PNGase F. Exemplary chromatographic methods include, but
are not limited to, Strong Anion Exchange chromatography using
Pulsed Amperometric Detection (SAX-PAD), liquid chromatography
(LC), high performance liquid chromatography (HPLC),
ultra-performance liquid chromatography (UPLC), thin layer
chromatography (TLC), amide column chromatography, and combinations
thereof. Exemplary mass spectrometry (MS) include, but are not
limited to, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser
desorption ionization mass spectrometry (MALDI-MS), Fourier
transform mass spectrometry (FTMS), ion mobility separation with
mass spectrometry (IMS-MS), electron transfer dissociation
(ETD-MS), and combinations thereof. Exemplary electrophoretic
methods include, but are not limited to, capillary electrophoresis
(CE), CE-MS, gel electrophoresis, agarose gel electrophoresis,
acrylamide gel electrophoresis, SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) followed by Western blotting using
antibodies that recognize specific glycan structures, and
combinations thereof. Exemplary nuclear magnetic resonance (NMR)
include, but are not limited to, one-dimensional NMR (1D-NMR),
two-dimensional NMR (2D-NMR), correlation spectroscopy
magnetic-angle spinning NMR (COSY-NMR), total correlated
spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum
coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence
(HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR
(ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR),
and combinations thereof.
Cells
[0070] Any host cell that can be used to express ustekinumab can be
used in the methods described herein. In some embodiments, a cell
engineered to express ustekinumab includes one or more nucleic
acids that encode an ustekinumab product that includes a heavy
chain variable domain as set forth in SEQ ID NO.: 1 and/or a light
chain variable domain as set forth in SEQ ID NO.: 2. In some
embodiments, a cell engineered to express ustekinumab includes one
or more nucleic acids that encode an ustekinumab product that
includes a heavy chain comprising a sequence of SEQ ID NO.: 1
and/or a light chain comprising a sequence of SEQ ID NO.: 2. In
some embodiments, a cell engineered to express ustekinumab includes
one or more nucleic acids that encode an ustekinumab product that
includes HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID
NO.: 1 and LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID
NO.: 2.
[0071] In some embodiments, a cell engineered to express
ustekinumab includes one or more nucleic acids that encode an
ustekinumab product that has the same primary amino acid sequence
as a protein that has been approved, e.g., under a secondary
approval process, for therapeutic or diagnostic use in humans or
animals. In some embodiments, a cell engineered to express
ustekinumab includes one or more nucleic acids that encode an
ustekinumab product that differs by no more than 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, or 20 residues with an approved therapeutic or
diagnostic protein. In some embodiments, a cell engineered to
express ustekinumab includes one or more nucleic acids that encode
a protein that has at least 90, 95, 98, 99% or 100% sequence
identity with that of an approved therapeutic or diagnostic
protein. The terms the "same primary amino acid sequence", "a
primary amino acid sequence that differs by no more than 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, or 20 residues", "sequences that have at
least 98% or more sequence identity", or similar terms, relate to
level of identity between primary amino acid sequences. In some
embodiments, a protein preparation or product includes amino acid
variants, e.g., species that differ at terminal residues, e.g., at
one or two terminal residues. In some embodiments of such cases,
sequence identity compared is the identity between the primary
amino acid sequence of the most abundant (e.g., most abundant
active) species in each of the products being compared. In some
embodiments, sequence identity refers to the amino acid sequence
encoded by a nucleic acid that can be used to make the ustekinumab
product.
[0072] In some embodiments, cells engineered to express ustekinumab
are mammalian cells.
[0073] In some embodiments, cells engineered to express ustekinumab
are murine cells. In some embodiments, a cell engineered to express
ustekinumab is from a mouse cell line. Murine (e.g., mouse) cell
lines include, for example, mouse myeloma cell lines, e.g., NS0
cells and SP 2/0 cells.
[0074] In some embodiments, cells engineered to express ustekinumab
are human cells. In some embodiments, a cell engineered to express
ustekinumab is from a human cell line. Human cell lines include,
for example, HEK 293: human embryonic kidney 293; HT-1080: from a
fibrosarcoma with an epithelial-like phenotype; PER.C6: from human
embryonic retinal cells immortalized via transfection with the
adenovirus E1 gene; CAP: from human amniocytes immortalized through
an adenovirus type 5 E1 gene; HKB-11: created through polyethylene
glycol fusion of HEK293-S and a human B-cell line; and HuH-7: from
a human hepatocellular carcinoma. In some certain embodiments,
shear-sensitive cells are selected from: HEK 293 cells,
fibrosarcoma HT 1080 cells, PER.C6 cells, CAP cells, HKB-11 cells
and HuH-7 cells.
[0075] In some embodiments, a population of cells that express an
ustekinumab product as described herein is produced using
recombinant methods. Recombinant expression of a gene, such as a
gene encoding a polypeptide, such as an antibody agent described
herein, can include construction of an expression vector containing
a polynucleotide that encodes the polypeptide. Once a
polynucleotide has been obtained, a vector for the production of
the polypeptide can be produced by recombinant DNA technology using
techniques known in the art. Known methods can be used to construct
expression vectors containing polypeptide coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination.
[0076] Once an ustekinumab product described herein has been
produced by recombinant expression, it can be purified by any
method known in the art for purification, for example, by
chromatography (e.g., ion exchange, affinity, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. For
example, ustekinumab can be isolated and purified by appropriately
selecting and combining affinity columns such as Protein A column
with chromatography columns, filtration, ultra-filtration,
salting-out and dialysis procedures (see Antibodies: A Laboratory
Manual, Ed Harlow, David Lane, Cold Spring Harbor Laboratory,
1988). Further, as described herein, an ustekinumab product can be
fused to heterologous polypeptide sequences to facilitate
purification.
Pharmaceutical Compositions
[0077] A ustekinumab product produced or manufactured using any of
the methods, systems and/or processes described herein can be
incorporated into a pharmaceutical composition. Such a
pharmaceutical composition may be useful in the prevention and/or
treatment of diseases. Pharmaceutical compositions comprising
ustekinumab can be formulated by methods known to those skilled in
the art (see, e.g., Remington's Pharmaceutical Sciences, 20th Ed.,
Lippincott Williams & Wilkins, 2000). The pharmaceutical
composition can be administered parenterally in the form of an
injectable formulation comprising a sterile solution or suspension
in water or another pharmaceutically acceptable liquid. For
example, the pharmaceutical composition can be formulated by
suitably combining the cell product (e.g., a recombinant protein,
e.g., a glycoprotein, e.g., an antibody agent) with
pharmaceutically acceptable vehicles or media, such as sterile
water and physiological saline, vegetable oil, emulsifier,
suspension agent, surfactant, stabilizer, flavoring excipient,
diluent, vehicle, preservative, binder, followed by mixing in a
unit dose form required for generally accepted pharmaceutical
practices. The amount of active ingredient included in a
pharmaceutical preparation is such that a suitable dose within the
designated range is provided.
[0078] In some embodiments, a preparation of ustekinumab includes
sucrose as a stabilizer/tonicifier. In some embodiments, a
preparation of ustekinumab includes histidine (e.g., L-histidine)
as a buffer. In some embodiments, a preparation of ustekinumab
includes polisorbate 80 as a surfactant. In some certain
embodiments, a preparation of ustekinumab includes histidine (e.g.,
L-histidine), sucrose, and polisorbate 80.
[0079] In some embodiments, a preparation of ustekinumab is
formulated for parenteral administration, e.g., intravenous
injection, intramuscular injection, intraperitoneal injection,
subcutaneous injection. In some embodiments, a preparation of
ustekinumab is formulated for subcutaneous administration.
[0080] The disclosure is further illustrated by the following
examples. The examples are provided for illustrative purposes only.
They are not to be construed as limiting the scope or content of
the disclosure in any way.
EXAMPLES
Example 1: Identification of Shift in Glycan Composition of
Ustekinumab Products
[0081] This example identifies a significant shift in glycan
composition among lots of commercially available ustekinumab
reference protein products ("RPPs"). Specifically, the present
example demonstrates that the variability in the glycan composition
among lots of commercially available ustekinumab that segregates
into two distinct glycan profiles. These two groups of glycan
variants are referred to herein as "Group 1," which are
characterized by a relatively high level of G0F glycan, and "Group
2," which characterized by a relatively low level of G0F
glycan.
[0082] Thirty (30) lots of ustekinumab RPPs were analyzed. Among
these 30 lots, 28 lots were from 90 mg/mL preparations and 2 lots
were 5 mg/mL preparations. Of the 28 lots of 90 mg/mL ustekinumab
RPPs, eight (8) of these lots were identified as having a Group 1
glycan profile and twenty (20) lots were identified as having a
Group 2 glycan profile. See, FIG. 1.
[0083] A summary of the average abundance of each of the major
glycan species for Group 1 and Group 2 samples is summarized in
Table 2 below.
TABLE-US-00003 TABLE 2 Abundance of Major Glycans in Group 1 and
Group 2 Ustekinumab Samples Glycan (%) Group 1 Group 2 4,3,1,0,0
(G0F) 27.7 62.1 4,4,1,0,0 A (G1F-A) 28.1 14.1 4,4,1,0,0 B (G1F-B)
6.6 3.5 4,4,1,0,1 A 5.4 4.1 4,4,1,0,1 B 3.2 2.9 4,5,1,0,0 (G2F) 8.4
2.3 4,6,1,0,0 1.4 0.2 4,5,1,0,1 6.7 2.1 4,5,1,0,2 2.0 0.9 4,6,1,0,1
B 3.4 0.7 Sum of above 92.9 92.9
[0084] Ustekinumab test products generated by culturing (e.g., by
an ATF-based perfusion culture process) with 5 mM galactose for a
duration of 42 days at different volumes within a range of 3 L to
250 L were also analyzed. The glycan abundance of these ustekinumab
test products is depicted in the right most grouping of each panel
of FIG. 1. The glycan profiles of the ustekinumab test products
were within the range of those of Group 1 and Group 2 RPPs. Thus,
the present example identified two distinct groups of glycan
profiles for commercially available ustekinumab RPPs and
demonstrated that contemporaneous culture conditions yield
ustekinumab test products with intermediate glycan abundances
within the ranges of these two groups.
Example 2: Culture Time Affects Ustekinumab Glycan Composition
[0085] The present example demonstrates that harvest collection
timing is an element for controlling ustekinumab glycan
composition. As depicted in FIG. 2 the abundance of G0F glycan
relative to total glycan generally decreased with culture duration.
For example, ustekinumab preparations that were harvested at 13
days of culturing had an almost 50% abundance of G0F glycans, while
samples with harvested after 34 or more days of culturing had less
than 30% G0F glycans relative to total glycan composition.
[0086] Interesting, the present disclosure provides the recognition
that a decrease in the culture duration (i.e. time till harvest) is
coupled with an increase in G0F level; while an increase in the
culture duration (i.e. time till harvest) is coupled with a
reduction in G0F level.
[0087] Thus, this example demonstrates that increasing culture time
can decrease abundance of G0F glycans relative to total glycan
composition and further describes that culture time can be varied
to control ustekinumab glycan composition.
Example 3: Generation of Ustekinumab Products with Group 1 and
Group 2 Glycan Profiles
[0088] The present example identifies an inverse relationship
between culture time and galactose concentration in culture media
that can serve as a process lever for controlling ustekinumab
glycan levels (e.g., galactosylation). The present example
demonstrates production of non-conforming materials (i.e.,
non-conforming ustekinumab preparations) with engineered glycan
profiles. Specifically, the present example demonstrate production
of two non-conforming ustekinumab preparations, referred to herein
as NCM-1 and NCM-2, that have been engineered to have glycan
profiles that match that of Group 1 RPP and Group 2 RPP,
respectively, as described above in Example 1.
[0089] Specifically, NCM-1 preparations were generated by culturing
(e.g., by an ATF-based perfusion culture process) with 15 mM
galactose and harvesting ustekinumab before day 16 of culturing
(e.g., harvesting at time points within a range of day 7 to day
15). In contrast, NCM-2 preparations were generated by culturing
(e.g., by an ATF-based perfusion culture process) in a medium
lacking galactose (0 mM galactose) and harvesting ustekinumab after
day 25 of culturing (e.g., harvesting at time points within a range
of day 34 to day 45). As depicted in FIG. 3, NCM-1 glycan profiles
correlated well with those of Group 1 RPP and NCM-2 glycan profiles
correlated well with those of Group 2 RPP for each of the glycan
species analyzed, including G0F, G1FA, G1FB, G2F, total sialyation,
main, acidic and basic glycans. Table 3 provides exemplary target
ranges for each glycan as engineered by controlling the process
levers of galactose concentration and culture time.
TABLE-US-00004 TABLE 3 Exemplary Target Glycan Ranges of NCM-1 and
NCM-2 Ustekinumab Preparations Glycan Total Range G0F G1F-A G1F-B
G2F Sialylation NCM-1 20%- >25%- >5.5%- >5%- >15%- (0
mM 40% 35% 10% 10% 30% galactose, culture time 7-15 days) NCM-2
>40%- 10%- 2%- 1%- 5%- (15-30 80% 25% 5.5% 5% 15% mM galactose,
culture time 25- 60 days)
[0090] Thus, the present example demonstrated that inversely
related elements of galactose concentration and culture time can be
used as process levers to control glycan composition and can be
used to produce non-conforming ustekinumab preparations with target
glycan levels.
EQUIVALENTS
[0091] It is to be understood that while the disclosure has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
21449PRTHomo sapiens 1Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly
Tyr Ser Phe Thr Thr Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro
Gly Lys Gly Leu Asp Trp Ile 35 40 45Gly Ile Met Ser Pro Val Asp Ser
Asp Ile Arg Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Met Ser
Val Asp Lys Ser Ile Thr Thr Ala Tyr65 70 75 80Leu Gln Trp Asn Ser
Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Arg
Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ser Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Lys2214PRTHomo
sapiens 2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ile Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210
* * * * *