U.S. patent application number 12/705773 was filed with the patent office on 2010-06-03 for production of recombinant il-18 binding protein.
This patent application is currently assigned to ARES TRADING S.A.. Invention is credited to URS WEBER, Thierry Ziegler.
Application Number | 20100137195 12/705773 |
Document ID | / |
Family ID | 44352415 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100137195 |
Kind Code |
A1 |
WEBER; URS ; et al. |
June 3, 2010 |
Production of Recombinant IL-18 Binding Protein
Abstract
The invention relates to a process for the production of IL-18
binding protein (IL-18BP), and to a composition comprising IL-18BP
characterized by a specific glycosylation pattern.
Inventors: |
WEBER; URS; (La
Tour-de-Peilz, CH) ; Ziegler; Thierry; (Leognan,
FR) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO Box 142950
GAINESVILLE
FL
32614
US
|
Assignee: |
ARES TRADING S.A.
Aubonne
FR
|
Family ID: |
44352415 |
Appl. No.: |
12/705773 |
Filed: |
February 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11915453 |
Nov 26, 2007 |
7691611 |
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PCT/EP2006/062851 |
Jun 1, 2006 |
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12705773 |
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60687631 |
Jun 3, 2005 |
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Current U.S.
Class: |
514/1.4 |
Current CPC
Class: |
A61P 1/04 20180101; C07K
14/47 20130101; A61P 1/16 20180101; A61P 17/06 20180101; A61K 38/00
20130101; A61P 29/00 20180101; A61P 31/04 20180101; A61P 19/02
20180101; A61P 1/00 20180101 |
Class at
Publication: |
514/8 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 37/06 20060101 A61P037/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 2005 |
EP |
05104878.3 |
Jul 13, 2005 |
EP |
05106429.3 |
Claims
1. An IL-18BP composition comprising a pharmaceutically acceptable
surfactant, excipient, carrier, diluent or vehicle and IL-18BP
having a glycoform profile comprising about 0% to about 15% of
basic glycoforms, about 15% to about 30% of less acidic glycoforms,
about 45% to about 65% of acidic glycoforms, and about 10% to about
25% of highly acidic glycoforms.
2. The IL-18BP composition according to claim 1, wherein said
composition comprises a pharmaceutically acceptable surfactant and
IL-18BP having a glycoform profile comprising about 0% to about 15%
of basic glycoforms, about 15% to about 30% of less acidic
glycoforms, about 45% to about 65% of acidic glycoforms, and about
10% to about 25% of highly acidic glycoforms.
3. The IL-18BP composition according to claim 1, wherein said
composition comprises a pharmaceutically acceptable excipient and
IL-18BP having a glycoform profile comprising about 0% to about 15%
of basic glycoforms, about 15% to about 30% of less acidic
glycoforms, about 45% to about 65% of acidic glycoforms, and about
10% to about 25% of highly acidic glycoforms.
4. The IL-18BP composition according to claim 1, wherein said
composition comprises a pharmaceutically acceptable carrier and
IL-18BP having a glycoform profile comprising about 0% to about 15%
of basic glycoforms, about 15% to about 30% of less acidic
glycoforms, about 45% to about 65% of acidic glycoforms, and about
10% to about 25% of highly acidic glycoforms.
5. The IL-18BP composition according to claim 1, wherein said
composition comprises a pharmaceutically acceptable diluent and
IL-18BP having a glycoform profile comprising about 0% to about 15%
of basic glycoforms, about 15% to about 30% of less acidic
glycoforms, about 45% to about 65% of acidic glycoforms, and about
10% to about 25% of highly acidic glycoforms.
6. The IL-18BP composition according to claim 1, wherein said
composition comprises a pharmaceutically acceptable vehicle and
IL-18BP having a glycoform profile comprising about 0% to about 15%
of basic glycoforms, about 15% to about 30% of less acidic
glycoforms, about 45% to about 65% of acidic glycoforms, and about
10% to about 25% of highly acidic glycoforms.
7. An IL-18BP composition comprising a pharmaceutically acceptable
surfactant, excipient, carrier, diluent or vehicle and IL-18BP
having a sialylation profile comprising about 15% to about 25% of
unsialylated N-glycans, about 15% to about 30% of mono-sialylated
glycans, about 35% to about 55% of di-sialylated N-glycans, about
5% to about 15% of tri-sialylated N-glycans and about 1% to about
5% of tetra-sialylated N-glycans.
8. The IL-18BP composition according to claim 7, wherein said
composition comprises a pharmaceutically acceptable surfactant and
IL-18BP having a sialylation profile comprising about 15% to about
25% of unsialylated N-glycans, about 15% to about 30% of
mono-sialylated glycans, about 35% to about 55% of di-sialylated
N-glycans, about 5% to about 15% of tri-sialylated N-glycans and
about 1% to about 5% of tetra-sialylated N-glycans.
9. The IL-18BP composition according to claim 7, wherein said
composition comprises a pharmaceutically acceptable excipient and
IL-18BP having a sialylation profile comprising about 15% to about
25% of unsialylated N-glycans, about 15% to about 30% of
mono-sialylated glycans, about 35% to about 55% of di-sialylated
N-glycans, about 5% to about 15% of tri-sialylated N-glycans and
about 1% to about 5% of tetra-sialylated N-glycans.
10. The IL-18BP composition according to claim 7, wherein said
composition comprises a pharmaceutically acceptable carrier and
IL-18BP having a sialylation profile comprising about 15% to about
25% of unsialylated N-glycans, about 15% to about 30% of
mono-sialylated glycans, about 35% to about 55% of di-sialylated
N-glycans, about 5% to about 15% of tri-sialylated N-glycans and
about 1% to about 5% of tetra-sialylated N-glycans.
11. The IL-18BP composition according to claim 7, wherein said
composition comprises a pharmaceutically acceptable diluent and
IL-18BP having a sialylation profile comprising about 15% to about
25% of unsialylated N-glycans, about 15% to about 30% of
mono-sialylated glycans, about 35% to about 55% of di-sialylated
N-glycans, about 5% to about 15% of tri-sialylated N-glycans and
about 1% to about 5% of tetra-sialylated N-glycans.
12. The IL-18BP composition according to claim 7, wherein said
composition comprises a pharmaceutically acceptable vehicle and
IL-18BP having a sialylation profile comprising about 15% to about
25% of unsialylated N-glycans, about 15% to about 30% of
mono-sialylated glycans, about 35% to about 55% of di-sialylated
N-glycans, about 5% to about 15% of tri-sialylated N-glycans and
about 1% to about 5% of tetra-sialylated N-glycans.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 11/915,453, filed Nov. 26, 2007, which is the U.S. national
stage application of International Patent Application No.
PCT/EP2006/062851, filed Jun. 1, 2006, which claims the benefit of
U.S. Provisional Patent Application No. 60/687,631, filed Jun. 3,
2005, the disclosures of which are hereby incorporated by reference
in their entireties, including all figures, tables and amino acid
or nucleic acid sequences.
FIELD OF THE INVENTION
[0002] The present invention is in the field of protein production.
More specifically, it relates to a process for the production of
recombinant IL-18 binding protein (IL-18BP). The invention further
relates to an IL-18BP composition characterized by a specific
glycosylation profile.
BACKGROUND OF THE INVENTION
[0003] Proteins have become commercially important as drugs that
are also generally called "biologicals". One of the greatest
challenges is the development of cost effective and efficient
processes for the production of recombinant proteins on a
commercial scale.
[0004] The biotech industry makes an extensive use of mammalian
cells for the manufacturing of recombinant glycoproteins for human
therapy.
[0005] Today, fed-batch and perfusion cultures are the two dominant
modes of industrial operation for the mammalian cell culture
processes that require large amount of proteins (Hu and Aunins
1997). Whatever the production technology of choice is, development
efforts aim at obtaining production processes that warrant high
volumetric productivity, batch-to-batch consistency, homogenous
product quality at low costs.
[0006] The decision between fed-batch or perfusion production mode
is mainly dictated by the biology of the clone and the property of
the product, and is done on a case-by-case basis during the course
of the development of a new drug product (Kadouri and Spier
1997).
[0007] When the selection is a perfusion process, one of the
culture systems of choice is stationary packed-bed bioreactor in
which cells are immobilized onto solid carriers. This system is
easy to operate and with appropriate carriers and culture
conditions very high cell density (of .about.10.sup.7-10.sup.8
cellml.sup.-1) can be achieved.
[0008] A consequence of this high cell density is the need for an
intensive medium perfusion rate (feed and harvest) that should be
used in order to keep the cells viable and productive. It appears
that the perfusion rate is one of the central parameters of such a
process: it drives the volumetric protein productivity, the protein
product quality and has a very strong impact on the overall
economics of the process.
[0009] Therefore at industrial scale the optimal stationary
packed-bed bioreactor process should operate with a perfusion rate
as low as possible without compromising on quantity and quality of
the product.
[0010] In the course of reducing the perfusion rate, several
studies were conducted where the concentration of glucose (Wang et
al. 2002) (Dowd et al. 2001) is used as an indicator of other
nutrients level in the feed medium in order to operate the
bioreactor at a low perfusion rate without accumulating in the
culture high levels of toxic by-products such as lactate and
ammonia (Sugiura and Kakuzaki 1998) (Racher et al. 1993).
Modification of culture parameters such as pH or temperature
(Chuppa et al. 1997) is also a common strategy to optimize culture
conditions and reduce medium perfusion needs.
[0011] In order to achieve optimal medium perfusion rate, three
approaches can be considered:
[0012] a) To fix perfusion rate at a constant value during the
entire production run.
[0013] This approach is usually preferred in industrial production
processes, since it is simpler to operate in a robust and
consistent way. It also it has the advantage to define medium costs
of the process as there is no variation in perfusion rate from run
to run.
[0014] b) To adjust perfusion rate in response to cell number
and/or nutrient consumption such as glucose (Oh et al. 1994) (Dowd
et al. 2001) (Gorenflo et al. 2003), glutamine (Gorenflo et al.
2002), or oxygen (Kyung et al. 1994). Although this approach
provides a more scientific rationale for adjusting the perfusion
rate, it may lead to an overgrown culture and an "out-of-control"
increase of the perfusion rate. When cells are cultured in
suspension mode, a "culture bleed" is done to avoid overgrowing the
culture, but this is not possible when cells are immobilized on a
carrier. So in general, this approach is not preferred for
manufacturing operations as it is difficult to operate in a robust
and consistent manner and the medium perfusion rate needs to be
readjusted on a daily basis.
[0015] c) To combine both strategies a) and b) with an initial cell
propagation phase (or "growth phase") where the perfusion rate is
progressively increased according to cell growth requirements
during the growth phase followed by a shift of culture conditions
such as temperature and/or pH in order to stabilize and keep cell
metabolism at a relatively low and constant level. At this stage,
the perfusion rate can be reduced to a fixed value, matching the
reduced need of the cells throughout the production phase.
[0016] It is known that modification of the perfusion rate during a
perfusion process, as well as modification of other bioprocess
factors, can influence the recombinant protein quality and in
particular its glycosylation pattern (Jenkins et al. 1996)
(Andersen et al. 2000). Glycosylation is usually recognized as an
important function in the solubility, immunogenicity, and
pharmacokinetic properties of human glycoproteins and those are key
parameters in the safety and clinical efficacy of a product
(Goochee et al. 1991). In particular, glycosylation affects folding
and secretion of many glycoproteins, as well as their plasma
half-life, thus having an important impact on in vivo biology and
activity of glycosylated proteins.
[0017] In general, the term "glycosylation" of a protein refers to
the formation of the sugar-amino acid linkage. Glycosylation is a
crucial event in the biosynthesis of the carbohydrate units of
(secreted) glycoproteins. It sets into motion a complex series of
posttranslational enzymatic steps that lead to the formation of a
host of protein-bound oligosaccharides with diverse biological
functions.
[0018] Mammalian glycoproteins commonly contain three types of
constituent glycans; the N-linked glycans which are attached to
asparagine via an N-acetylglucosamine (GlcNAc) residue in an
Asn-Xxx-(Ser, Thr) motif, where Xxx can be any amino acid except
praline, those attached to serine or threonine, referred to as
O-linked glycans and the carbohydrate components of
glycosylphosphatidylinositol. Although many variations are
possible, the antennae of mature glycans usually consist of one or
more N-acetyllactosamine units with the chains terminating in
either sialic acid or .alpha.-linked galactose. Fucose is
frequently found attached to the asparagine-linked GlcNAc residue
and often, additionally on the antennae. Other common modifications
to the basic structure include a GlcNAc residue attached to the
4-position of the core branching mannose residue, referred to as a
"bisecting" GlcNAc residue and sulphate groups which can be found
in a variety of locations, both on the core and the antennae.
[0019] The biosynthesis of these compounds involves attachment to
the asparagine of a glycan containing the trimannosyl-chitobiose
core together with an additional six mannose and three glucose
residues followed by removal of the glucose and four mannose
residues. Various other glycosyl transferases and glycosidases then
process the (GlcNAc)2(Man)5 structure to the mature glycan. This
process results in three general types of N-linked glycan depending
on the extent of processing; "high-mannose" glycans in which only
mannose resides on the two antennae, "hybrid glycans" in which one
antenna is processed and "complex" glycans where both antennae are
modified. O-linked glycans, on the other hand, are much more
diverse, ranging from monosaccharides to large sulphated
polysaccharides with no common core structure or consensus sequence
of amino acids at the attachment site (Harvey, 2001).
[0020] One such glycosylated protein of therapeutic interest is
interleukin-18 binding protein.
[0021] Interleukin-18 binding protein (IL-18BP) is a naturally
occurring soluble protein that was initially affinity purified, on
an IL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes
IL-18 induction of IFN-.gamma. and IL-18 activation of NF-.kappa.B
in vitro. In addition, IL-18BP inhibits induction of IFN-.gamma. in
mice injected with LPS.
[0022] The IL-18BP gene was localized to the human chromosome 11,
and no exon coding for a transmembrane domain could be found in the
8.3 kb genomic sequence comprising the IL-18BP gene. Four isoforms
of IL-18BP generated by alternative mRNA splicing have been
identified in humans so far. They were designated IL-18BP a, b, c,
and d, all sharing the same N-terminus and differing in the
C-terminus (Novick et al 1999). These isoforms vary in their
ability to bind IL-18 (Kim et al. 2000). Of the four human IL-18BP
(hIL-18BP) isoforms, isoforms a and c are known to have a
neutralizing capacity for IL-18. The most abundant IL-18BP isoform,
isoform a, exhibits a high affinity for IL-18 with a rapid on-rate
and a slow off-rate, and a dissociation constant (Kd) of
approximately 0.4 nM (Kim et al. 2000).
[0023] IL-18BP belongs to the immunoglobulin superfamily.
[0024] The residues involved in the interaction of IL-18 with
IL-18BP have been described through the use of computer modelling
(Kim et al. 2000) and based on the interaction between the similar
protein IL-1.beta.with the IL-1R type I (Vigers et al. 1997).
[0025] IL-18BP is constitutively present in many cells (Puren et
al. 1999) and circulates in healthy humans, representing a unique
phenomenon in cytokine biology. Due to the high affinity of IL-18BP
to IL-18 (Kd=0.4 nM) as well as the high concentration of IL-18BP
found in the circulation (20 fold molar excess over IL-18), it has
been speculated that most, if not all of the IL-18 molecules in the
circulation are bound to IL-18BP. Thus, the circulating IL-18BP
that competes with cell surface receptors for IL-18 may act as a
natural anti-inflammatory and an immunosuppressive molecule.
[0026] IL-18BP has been suggested as a therapeutic protein in a
number of diseases and disorders, such as psoriasis, Crohn's
Disease, rheumatoid arthritis, psoriatic arthritis, liver injury,
sepsis, atherosclerosis, ischemic heart diseases, allergies, etc.,
see e.g. WO9909063, WO0107480, WO0162285, WO0185201, WO02060479,
WO02096456, WO03080104, WO02092008, WO02101049, WO03013577.
[0027] The prior art does not describe a process for the production
of recombinant IL-18BP in CHO cells, nor IL-18BP compositions
characterized by a specific glycosylation profile.
SUMMARY OF THE INVENTION
[0028] The present invention relates to the development of a
process for producing recombinant Interleukin-18 binding protein
(IL-18BP) in mammalian cells in a bioreactor under serum-free
culture conditions comprising a cell propagation phase at about
37.degree. C. and a production phase at a temperature ranging from
about 29.degree. C. to about 34.degree. C.
[0029] In particular, an efficient perfusion production process has
been developed for IL-18 binding protein (IL-18BP) that involves
reduction of the perfusion rate during the production phase of a
recombinant protein from mammalian cells in a bioreactor without
significantly reducing the productivity of the recombinant protein
product by the cells.
[0030] The perfusion process for producing recombinant
Interleukin-18 binding protein (IL-18BP) in mammalian cells in a
bioreactor under serum-free culture conditions comprising: [0031]
a) A cell propagation phase at 37.degree. C. with a given perfusion
rate (100%); [0032] b) A production phase I at 33.5.degree. C. at a
perfusion rate that ranges from about 85 to about 65% or about 80
to about 70% or about 75% of the perfusion rate of the perfusion
rate of step (a); [0033] c) A production phase II at 32.5.degree.
C. at a perfusion rate that that ranges from about 85 to about 65%
or about 80 to about 70% or about 75% of the perfusion rate of the
perfusion rate of step (a).
[0034] Additionally, an efficient fed-batch process has been
developed for the production of IL-18BP, under serum-free culture
conditions, comprising: [0035] a. A cell propagation phase at
37.degree. C.; [0036] b. Optionally, an intermediate phase at
33.degree. C.; [0037] c. A production phase at 29.degree. C.
[0038] The invention also relates to a composition comprising
IL-18BP that is characterized by a specific glycosylation profile
comprising about 15 to about 25% of unsialylated N-glycans, about
15 to about 25% of unsialylated N-glycans, about 15 to about 30% of
mono-sialylated glycans, about 35 to about 55% of di-sialylated
N-glycans, about 5 to about 15% of tri-sialylated N-glcyans and
about 1 to about 5% of tetra-sialylated N-glycans.
[0039] The invention further relates to a composition comprising an
IL-18BP that is characterized by a glycoform profile comprising
about 0 to about 15% of basic glycoforms, about 15 to about 30% of
less acidic glycoforms, about 40 to about 65% of acidic glycoforms,
and about 10 to about 30% of highly acidic glycoforms.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1: Medium perfusion rate during continuous cultures of
CHO cells in a packed-bed bioreactor according to example 1, with
perfusion levels of 2.6 vvd ( ), 2.0 vvd (.smallcircle.) and 1.3
vvd (.tangle-solidup.). The bioreactor runs have been labelled as
run-100 for 100% perfusion ( ), run-75 for 75% perfusion
(.smallcircle.) and run-50 for 50% perfusion (.tangle-solidup.).
The maximum perfusion rate of 100% corresponds to 2.6 vvd that were
used as the reference conditions.
[0041] FIG. 2: Glucose (A) and lactate (B) concentration profiles
during continuous cultures of CHO cells in a packed-bed bioreactor
according to example 1, for medium perfusion rates of 100% ( ) 75%
(.smallcircle.) and 50% (.tangle-solidup.).
[0042] FIG. 3: Glucose Consumption Rate (GCR) profile during
continuous cultures of CHO cells in a packed-bed bioreactor
according to example 1, for medium perfusion rates of 100% ( ) 75%
(.smallcircle.) and 50% (.tangle-solidup.). GCR is expressed in
grams of glucose consumed per day and per kg of Fibra-Cel
carrier.
[0043] FIG. 4: Apparent lactate from glucose molar conversion ratio
profile during continuous cultures of CHO cells in a packed-bed
bioreactor according to example 1, for medium perfusion rates of
100% ( ) 75% (.smallcircle.) and 50% (.tangle-solidup.).
[0044] FIG. 5: Normalized productivity data (A) volumetric
productivity in units of product per total culture volume per day,
(B) cumulated product in units of product, (C) titre in units of
product per total culture volume, for r-IL-18BP which is produced
during continuous cultures of CHO cells in a packed-bed bioreactor
according to example 1, for medium perfusion rates of 100% ( ) 75%
(.smallcircle.) and 50% (.tangle-solidup.). To normalize the data,
the average value obtained at 100% perfusion rate over the 60-day
production phase was taken as 100% productivity (dotted line).
[0045] FIG. 6A-6C: Sialylation by N-Glycan mapping (RP-HPLC
profiles) in intermediate bulk samples at production day 47-48 of
run-50 (FIG. 6A), run-75 (FIG. 6B) and run-100 (FIG. 6C).
[0046] FIG. 7: Process performance of the fed-batch process
according to example 2 in terms of (A) total cell-density, (B)
viability, (C) residual glucose concentration, and (D) r-hIL-18BP
titer.
[0047] FIG. 8: Sialylation by N-Glycan mapping (RP-HPLC profiles)
in purified IL-18BP prepared in a perfusion process (upper panel)
or fed-batch (lower panel) process.
[0048] FIG. 9: Comparison of Capillary Zone Electrophoresis (CZE)
profiles obtained from two separate analytical sequences, for: a
(pre-treated) crude harvest sample from the fed-batch process
(top), and a (pre-treated) harvest sample from the perfusion
process (bottom).
[0049] FIG. 10: Distribution of isoforms (given as % of all forms
of the target molecule) by Capillary Zone Electrophoresis (CZE) in
pre-treated crude harvest samples (dotted and grey) and purified
(hatched and black) samples of IL-18BP for perfusion runs (dotted
and hatched) and fed-batch runs (grey and black). The error bars
correspond to the .+-.10% variability of the CZE method.
DETAILED DESCRIPTION OF THE INVENTION
[0050] The present invention is based on the development of
efficient processes for the production of recombinant IL-18BP in a
bioreactor under serum-free cell culture conditions. Therefore, the
present invention relates to a process for producing recombinant
Interleukin-18 binding protein (IL-18BP) in mammalian cells in a
bioreactor under serum-free culture conditions comprising a cell
propagation phase at about 37.degree. C. and a production phase at
a temperature ranging from about 29.degree. C. to about 34.degree.
C.
[0051] Any temperature between approximately 29 to approximately
34.degree. C. will be suitable in the frame of the present
invention such as e.g. 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32,
32.5, 33, 33.5, 34, 34.5, 35.degree. C.
[0052] Preferably, the invention relates to a perfusion process for
producing recombinant Interleukin-18 binding protein (IL-18BP) in
mammalian cells in a bioreactor under serum-free culture conditions
comprising: [0053] a) A cell propagation phase at 37.degree. C.
with a given perfusion rate (100%); [0054] b) A production phase I
at 33.5.degree. C. at a perfusion rate that ranges from about 85 to
about 65% or about 80 to about 70% or about 75% of the perfusion
rate of the perfusion rate of step (a); [0055] c) A production
phase II at 32.5.degree. C. at a perfusion rate that that ranges
from about 85 to about 65% or about 80 to about 70% or about 75% of
the perfusion rate of the perfusion rate of step (a).
[0056] It will be appreciated by the person skilled in the art that
the perfusion rate of production phase (either I or II or both) may
be e.g. at about 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75,
74, 73, 72, 71, 70, 69, 68, 67, 66 or 65, or 64% of the initial
perfusion rate in step (a).
[0057] As shown in the examples below, it has been surprisingly
found that in spite of the reduced perfusion rate, the cellular
productivity of IL-18BP was substantially preserved.
[0058] It will also be appreciated by the person skilled in the art
that the temperature may vary within certain limits, so as to be
e.g. about 37.5.degree. C. in step (a) or 34-33.degree. C. in step
(b) or 32-33 in step (c).
[0059] Typically, the Dissolved Oxygen concentration (DO) of a cell
culture is maintained at about 50 to 70% of air saturation, and pH
values vary between 6.5 and 7.5, preferably around 7.0.
[0060] The term "bioreactor", as used herein, refers to an
apparatus or closed container that is used for generating
biomolecules such as secreted proteins using the synthetic or
chemical conversion capacity of a cell. Bioreactors include
classical fermenters and cell culture perfusion systems.
Bioreactors allow controlling various parameters during the cell
culture process such as, e.g., the circulation loop flow, the
temperature, the overpressure and/or the medium perfusion rate.
[0061] The term "serum-free medium", as used herein, refers to any
medium that is free from components derived from animal serum such
as e.g. fetal calf serum. Examples for commercially available
serum-free media that can be used in accordance with the present
invention include, e.g., SFM 90 (JRH, 67350), SFM 90.1 (JRH,
67350), Supmed300 or Supmed300 modified (JRH, 67350), DMEM (Gibco,
7490571), DMEM/F12 (Gibco, 99.5043), SFM CHO 3a (BioWhittaker), CHO
PFM (Sigma, C6970), ProCHO 5, EX-CELL media such as EX-CELL 302
(JRH, Catalogue No. 14312-1000M) or EX-CELL 325 (JRH, Catalogue No.
14335-1000M), CHO--CD3 (Sigma, Catalogue No. C-1490), CHO III PFM
(Gibco, Catalogue No. 96-0334SA), CHO--S-SFM II (Gibco, Catalogue
No. 12052-098), CHO-DHFR (Sigma, Catalogue No. C-8862), ProCHO 5
(Cambrex, Catalogue No. BE12-766Q), SFM4CHO (HyClone, Catalogue No.
SH30549.01), Ultra CHO (Cambrex, Catalogue No. 12-724Q), HyQ PF CHO
(HyClone, Catalogue No. SH30220.01), HyQ SFX CHO (HyClone,
Catalogue No. SH30187.01), HyQ CDM4CHO (HyClone, Catalogue No.
SH30558.01), IS CHO-CD (Irvine Scientific, Catalogue No. #91119),
IS CHO-V (Irvine Scientific, Catalogue No. #9197) and derivatives
thereof. The composition of SFM 90, SFM 90.1, SupMed300, DMEM,
DMEM/F12, SFM CHO 3.sup.a and CHP PFM is shown in Table I
below.
[0062] The serum-free medium may preferably be a chemically defined
medium, i.e., a medium prepared from purified ingredients and
therefore whose exact composition is known. Specifically,
chemically defined media do neither contain animal derived
components nor undefined hydrolysates.
[0063] In accordance with the perfusion process of the invention,
in a preferred embodiment the perfusion rate of step (a) has a
dilution rate in the range of about 2 to 3 vvd, preferably about
2.5 vvd.
[0064] The term "dilution rate", as defined herein, refers to the
dilution rate D expressed in vvd, calculated as liter of medium per
liter of total system working volume per day (total
volume=packed-bed+conditioning tank volume).
[0065] It will be appreciated by the person skilled in the art that
the perfusion rate of step (a) may be e.g. about 1.9, 2.0, 2.1,
2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 or 3.1 vvd.
[0066] Initial perfusion rates of about 2.6 or 2.5 or 2.75 vvd have
shown to be particularly advantageous.
[0067] The length of the cell propagation phase of step (a), the
production phase I of step (b) and the production phase II of step
(c) will be readily determined by the person skilled in the art on
the basis of parameters such as the initial cell seed, the type of
cells used, the daily measured glucose consumption rate, the
dilution rate, and process time. In accordance with the present
invention, it is preferred that the cells are associated to
carriers in the bioreactor, and that production phase I of step (b)
is started at a glucose consumption rate of about 250 to 350 g of
Glucose per kilogram of carrier.
[0068] The carrier that may be used in accordance with the
processes of present invention may e.g. be a microcarrier.
Microcarriers are small solid particles on which cells may be grown
in suspension culture. Cells are capable of adhering and
propagating on the surface of microcarriers. Typically,
microcarriers consist of beads, the diameter of which is comprised
between 90 .mu.m and 300 .mu.m. Microcarriers can be made of
various materials that have proven successful for cell attachment
and propagation such as, e.g., glass, polystyrene, polyethylene,
dextran, gelatin and cellulose. In addition, the surface of
microcarriers may be coated with a material promoting cell
attachment and growth such as, e.g., e.g., N,N-diethylaminoethyl,
glass, collagen or recombinant proteins. Both macroporous and
non-porous microcarriers do exist. Macroporous surfaces give the
cells easy access to the interior of the microcarrier after
inoculation, and once inside of the microcarrier, the cells are
protected from the shear forces generated by mechanical agitation
and aeration in the bioreactor.
[0069] A further solid carrier that may be used in accordance with
the present invention may e.g. be a Fibra-Cel.RTM. disk.
Fibra-Cel.RTM. disks are disks of 6 mm in diameter that are
composed of polyester non-woven fiber bonded to a sheet of
polypropylene mesh (see, e.g., U.S. Pat. No. 5,266,476 and world
wide web pages nbsc.com/products/miscellaneous/fibracel/ and
nbsc.com/support/faqs/#fibra). Fibra-Cel.RTM. disks are usually
treated electrostatically to facilitate suspension cells adhering
to the disks and becoming trapped in the fiber system, where they
remain throughout the cultivation process. Cell density and
productivity achieved with cells grown on Fibra-Cel.RTM. disks can
be up to ten times higher than with cells growing on
microcarriers.
[0070] The cells expressing IL-18BP, that may be cultured in the
bioreactor in accordance with the processes of the present
invention may be any mammalian cell, including animal or human
cells, such as e.g. 3T3 cells, COS cells, human osteosarcoma cells,
MRC-5 cells, BHK cells, VERO cells, CHO cells, rCHO-tPA cells,
rCHO--Hep B Surface Antigen cells, CHO-S cells, HEK 293 cells, rHEK
293 cells, rC127--Hep B Surface Antigen cells, Normal Human
fibroblast cells, Stroma cells, Hepatocytes cells and PER.C6
cells.
[0071] It is preferred to use Chinese Hamster Ovary (CHO) cells for
expression of IL-18BP in a process according to the invention.
[0072] The processes for production of IL-18BP of the invention
preferably further comprises a step of collecting the cell culture
supernatant (harvest).
[0073] In a further preferred embodiment, the processes further
comprise one or more steps of purifying IL-18BP. Any suitable
method may be used for the purification of IL-18BP, such as e.g.
the purification processes described for IL-18BP in WO 2006/003134
or WO 2005/049649.
[0074] The purified IL-18PB product may then preferably be
formulated into a pharmaceutical composition.
[0075] The invention also relates to an IL-18BP composition
characterized by a sialylation profile comprising about 15 to about
25%, preferably about 19 to about 21% of unsialylated N-glycans,
about 15 to about 30%, preferably about 20 to about 25% of
mono-sialylated glycans, about 35 to about 55%, preferably about
39% to about 44% of di-sialylated N-glycans, about 5 to about 15%,
preferably about 7 to about 10% of tri-sialylated N-glcyans and
about 1 to about 5%, preferably about 2% to about 3% of
tetra-sialylated N-glycans.
[0076] Such an IL-18BP composition is preferably obtained by the
production processes of the invention.
[0077] The present invention further relates to a fed-batch process
for producing recombinant Interleukin-18 binding protein (IL-18BP)
in mammalian cells in a bioreactor under serum-free culture
conditions comprising the steps of: [0078] a. A cell propagation
phase at 37.degree. C.; [0079] b. Optionally, an intermediate phase
at 33.degree. C.; [0080] c. A production phase at 29.degree. C.
[0081] The length of phases (a), optionally (b), and (c) can be
readily determined by the person skilled in the art. Phase (a) will
continue until an adequate number of cells has been generated, such
as e.g. in the range of 10.sup.5 to 10.sup.6. Phase (b) will
preferably be short, as it is a transient phase serving at
adaptation of the cells to lower temperatures.
[0082] In a preferred embodiment, the total cell density in the
production phase ranges between 4 to 8.times.10.sup.6 cells per ml
per day, preferably over at least 10 days of cell culture.
[0083] In a further preferred embodiment, the viability ranges
between 100 and 80%, preferably over at least 10 days of cell
culture.
[0084] It is further preferred that the protein productivity is
higher than about 150 mg or about 250 mg or about 350 mg per 1 per
day.
[0085] Preferred mammalian cells to be used in the frame of the
process of the present invention are Chinese Hamster Ovary (CHO)
cells.
[0086] The process preferably further comprises the steps of
collecting the cell culture supernatant and/or purifying IL-18BP
and/or formulating the IL-18BP into a pharmaceutical
composition.
[0087] The invention also relates to an IL-18BP composition having
a glycoform profile comprising about 0 to about 15% of basic
glycoforms, about 15 to about 30% of less acidic glycoforms, about
45 to about 65% of acidic glycoforms, and about 10 to about 25% of
highly acidic glycoforms.
[0088] The glyocoform profile is characterized by way of capillary
zone electrophoresis (CZE), indicating the extent of basic, less
acidic, acidic and highly acidic glycoforms of IL-18BP. The method,
and a definition of basic, less acidic, acidic and highly acidic
glycoforms, can be taken from the examples below.
[0089] The hypothetical charge number Z is a parameter calculated
according to a known formula (Hermentin et al., 1996, Gervais et
al., 2003, see also the examples below), and characterizes the
extent of sialylation of glycoproteins.
[0090] The IL-18BP composition of the present invention is
characterized by a Z-number ranging between about 130 to about 160,
preferably between about 140 and 155, more preferably between about
145 and about 150. Preferred Z-numbers for IL-18BP are e.g. 139,
140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,
153, 154, 155, or 156.
[0091] Although the IL-18BP compositions of the present invention
may be produced by any process, such as e.g. perfusion processes
operated with different parameter settings than described herein,
e.g. at higher perfusion rates, the IL-18BP compositions of the
present invention are preferably produced in the perfusion process
or the fed-batch process of the present invention.
[0092] In accordance with the present invention, IL-18BP to be
produced may be any IL-18BP from any species. Preferably, it is
human IL-18BP.
[0093] Since IL-18BP is a soluble, secreted protein, it is released
into the cell culture supernatant, either by means of its natural
signal peptide, or by means of a heterologous signal peptide, i.e.
a signal peptide derived from another secreted protein which may be
more efficient in the particular expression system used, such as
e.g. the GH signal peptide.
[0094] The term "IL-18 binding protein" is used herein synonymously
with "IL-18BP". This term relates IL-18 binding proteins such as
the ones defined in WO 99/09063 or in Novick et al., 1999. The term
IL-18BP includes splice variants and/or isoforms of IL-18 binding
proteins, as the ones defined in Kim et al., 2000, in particular
human isoforms a and c of IL-18BP.
[0095] The term "IL-18PB", as used herein, further includes
muteins, functional derivatives, active fractions, fused proteins,
circularly permutated proteins and salts of IL-18BP as defined in
WO 99/09063.
[0096] As used herein the term "muteins" refers to analogs of an
IL-18BP, or analogs of a viral IL-18BP, in which one or more of the
amino acid residues of a natural IL-18BP or viral IL-18BP are
replaced by different amino acid residues, or are deleted, or one
or more amino acid residues are added to the natural sequence of an
IL-18BP, or a viral IL-18BP, without changing considerably the
activity of the resulting products as compared with the wild type
IL-18BP or viral IL-18BP. These muteins are prepared by known
synthesis and/or by site-directed mutagenesis techniques, or any
other known technique suitable therefor.
[0097] Muteins in accordance with the present invention include
proteins encoded by a nucleic acid, such as DNA or RNA, which
hybridizes to DNA or RNA, which encodes an IL-18BP or encodes a
viral IL-18BP (WO9909063) under stringent conditions. The term
"stringent conditions" refers to hybridization and subsequent
washing conditions, which those of ordinary skill in the art
conventionally refer to as "stringent". See Ausubel et al., Current
Protocols in Molecular Biology, supra, Interscience, N.Y.,
.sctn..sctn.6.3 and 6.4 (1987, 1992). Without limitation, examples
of stringent conditions include washing conditions 12-20.degree. C.
below the calculated Tm of the hybrid under study in, e.g.,
2.times.SSC and 0.5% SDS for 5 minutes, 2.times.SSC and 0.1% SDS
for 15 minutes; 0.1.times.SSC and 0.5% SDS at 37.degree. C. for
30-60 minutes and then, a 0.1.times.SSC and 0.5% SDS at 68.degree.
C. for 30-60 minutes. Those of ordinary skill in this art
understand that stringency conditions also depend on the length of
the DNA sequences, oligonucleotide probes (such as 10-40 bases) or
mixed oligonucleotide probes. If mixed probes are used, it is
preferable to use tetramethyl ammonium chloride (TMAC) instead of
SSC. See Ausubel, supra.
[0098] Identity reflects a relationship between two or more
polypeptide sequences or two or more polynucleotide sequences,
determined by comparing the sequences. In general, identity refers
to an exact nucleotide to nucleotide or amino acid to amino acid
correspondence of the two polynucleotides or two polypeptide
sequences, respectively, over the length of the sequences being
compared.
[0099] For sequences where there is not an exact correspondence, a
"% identity" may be determined. In general, the two sequences to be
compared are aligned to give a maximum correlation between the
sequences. This may include inserting "gaps" in either one or both
sequences, to enhance the degree of alignment. A % identity may be
determined over the whole length of each of the sequences being
compared (so-called global alignment), that is particularly
suitable for sequences of the same or very similar length, or over
shorter, defined lengths (so-called local alignment), that is more
suitable for sequences of unequal length.
[0100] Methods for comparing the identity and homology of two or
more sequences are well known in the art. Thus for instance,
programs available in the Wisconsin Sequence Analysis Package,
version 9.1 (Devereux J et al., 1984), for example the programs
BESTFIT and GAP, may be used to determine the % identity between
two polynucleotides and the % identity and the % homology between
two polypeptide sequences. BESTFIT uses the "local homology"
algorithm of Smith and Waterman (1981) and finds the best single
region of similarity between two sequences. Other programs for
determining identity and/or similarity between sequences are also
known in the art, for instance the BLAST family of programs
(Altschul S F et al, 1990, Altschul S F et al, 1997, accessible
through the home page of the NCBI at www.ncbi.nlm.nih.gov) and
FASTA (Pearson W R, 1990).
[0101] Any such mutein preferably has a sequence of amino acids
sufficiently duplicative of that of an IL-18BP, or sufficiently
duplicative of a viral IL-18BP, such as to have substantially
similar activity to IL-18BP. One activity of IL-18BP is its
capability of binding 1L-18. As long as the mutein has substantial
binding activity to IL-18, it can be used in the purification of
IL-18, such as by means of affinity chromatography, and thus can be
considered to have substantially similar activity to IL-18BP. Thus,
it can be determined whether any given mutein has substantially the
same activity as IL-18BP by means of routine experimentation
comprising subjecting such a mutein, e.g., to a simple sandwich
competition assay to determine whether or not it binds to an
appropriately labeled IL-18, such as radioimmunoassay or ELISA
assay.
[0102] In a preferred embodiment, any such mutein has at least 40%
identity or homology with the sequence of either an IL-18BP or a
virally encoded IL-18BP homologue, as defined in WO 99/09063. More
preferably, it has at least 50%, at least 60%, at least 70%, at
least 80% or, most preferably, at least 90% identity or homology
thereto.
[0103] Muteins of IL-18BP polypeptides or muteins of viral
IL-18BPs, which can be used in accordance with the present
invention, or nucleic acid coding therefor, include a finite set of
substantially corresponding sequences as substitution peptides or
polynucleotides which can be routinely obtained by one of ordinary
skill in the art, without undue experimentation, based on the
teachings and guidance presented herein.
[0104] Preferred changes for muteins in accordance with the present
invention are what are known as "conservative" substitutions.
Conservative amino acid substitutions of IL-18BP polypeptides or
proteins or viral IL-18BPs, may include synonymous amino acids
within a group which have sufficiently similar physicochemical
properties that substitution between members of the group will
preserve the biological function of the molecule (Grantham, 1974).
It is clear that insertions and deletions of amino acids may also
be made in the above-defined sequences without altering their
function, particularly if the insertions or deletions only involve
a few amino acids, e.g., under thirty, and preferably under ten,
and do not remove or displace amino acids which are critical to a
functional conformation, e.g., cysteine residues. Proteins and
muteins produced by such deletions and/or insertions come within
the purview of the present invention.
[0105] Preferably, the synonymous amino acid groups are those
defined in Table 4. More preferably, the synonymous amino acid
groups are those defined in Table 5; and most preferably the
synonymous amino acid groups are those defined in Table 6.
TABLE-US-00001 TABLE I Preferred Groups of Synonymous Amino Acids
Amino Acid Synonymous Group Ser Ser, Thr, Gly, Asn Arg Arg, Gln,
Lys, Glu, His Leu Ile, Phe, Tyr, Met, Val, Leu Pro Gly, Ala, Thr,
Pro Thr Pro, Ser, Ala, Gly, His, Gln, Thr Ala Gly, Thr, Pro, Ala
Val Met, Tyr, Phe, Ile, Leu, Val Gly Ala, Thr, Pro, Ser, Gly Ile
Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met, Tyr, Ile, Val, Leu, Phe
Tyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser, Thr, Cys His Glu,
Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, Thr, Arg, Gln Asn
Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu, Asn, Asp
Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu, Met
Trp Trp
TABLE-US-00002 TABLE II More Preferred Groups of Synonymous Amino
Acids Amino Acid Synonymous Group Ser Ser Arg His, Lys, Arg Leu
Leu, Ile, Phe, Met Pro Ala, Pro Thr Thr Ala Pro, Ala Val Val, Met,
Ile Gly Gly Ile Ile, Met, Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe
Tyr Phe, Tyr Cys Cys, Ser His His, Gln, Arg Gln Glu, Gln, His Asn
Asp, Asn Lys Lys, Arg Asp Asp, Asn Glu Glu, Gln Met Met, Phe, Ile,
Val, Leu Trp Trp
TABLE-US-00003 TABLE III Most Preferred Groups of Synonymous Amino
Acids Amino Acid Synonymous Group Ser Ser Arg Arg Leu Leu, Ile, Met
Pro Pro Thr Thr Ala Ala Val Val Gly Gly Ile Ile, Met, Leu Phe Phe
Tyr Tyr Cys Cys, Ser His His Gln Gln Asn Asn Lys Lys Asp Asp Glu
Glu Met Met, Ile, Leu Trp Met
[0106] Examples of production of amino acid substitutions in
proteins which can be used for obtaining muteins of IL-18BP
polypeptides or proteins, or muteins of viral IL-18BPs, for use in
the present invention include any known method steps, such as
presented in U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, to
Mark et al; U.S. Pat. No. 5,116,943 to Koths et al., U.S. Pat. No.
4,965,195 to Namen et al; U.S. Pat. No. 4,879,111 to Chong et al;
and U.S. Pat. No. 5,017,691 to Lee et al; and lysine substituted
proteins presented in U.S. Pat. No. 4,904,584 (Shaw et al).
[0107] The term "fused protein" refers to a polypeptide comprising
an IL-18BP, or a viral IL-18BP, or a mutein or fragment thereof,
fused with another protein, which, e.g., has an extended residence
time in body fluids. An IL-18BP or a viral IL-18BP, may thus be
fused to another protein, polypeptide or the like, e.g., an
immunoglobulin or a fragment thereof.
[0108] "Functional derivatives" as used herein cover derivatives of
IL-18BPs or a viral IL-18BP, and their muteins and fused proteins,
which may be prepared from the functional groups which occur as
side chains on the residues or the N- or C-terminal groups, by
means known in the art, and are included in the invention as long
as they remain pharmaceutically acceptable, i.e. they do not
destroy the activity of the protein which is substantially similar
to the activity of IL-18BP, or viral IL-18BPs, and do not confer
toxic properties on compositions containing it.
[0109] These derivatives may, for example, include polyethylene
glycol side-chains, which may mask antigenic sites and extend the
residence of an IL-18BP or a viral IL-18BP in body fluids. Other
derivatives include aliphatic esters of the carboxyl groups, amides
of the carboxyl groups by reaction with ammonia or with primary or
secondary amines, N-acyl derivatives of free amino groups of the
amino acid residues formed with acyl moieties (e.g. alkanoyl or
carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl
groups (for example that of seryl or threonyl residues) formed with
acyl moieties.
[0110] As "active fractions" of an IL-18BP, or a viral IL-18BP,
muteins and fused proteins, the present invention covers any
fragment or precursors of the polypeptide chain of the protein
molecule alone or together with associated molecules or residues
linked thereto, e.g., sugar or phosphate residues, or aggregates of
the protein molecule or the sugar residues by themselves, provided
said fraction has substantially similar activity to IL-18BP.
[0111] The term "salts" herein refers to both salts of carboxyl
groups and to acid addition salts of amino groups of IL-18
inhibitor molecule, or analogs thereof. Salts of a carboxyl group
may be formed by means known in the art and include inorganic
salts, for example, sodium, calcium, ammonium, ferric or zinc
salts, and the like, and salts with organic bases as those formed,
for example, with amines, such as triethanolamine, arginine or
lysine, piperidine, procaine and the like. Acid addition salts
include, for example, salts with mineral acids, such as, for
example, hydrochloric acid or sulfuric acid, and salts with organic
acids, such as, for example, acetic acid or oxalic acid. Of course,
any such salts must retain the biological activity of the IL-18
inhibitor, such as induction of IFN-gamma in blood cells.
[0112] The sequences of IL-18BP and its splice variants/isoforms
can be taken from WO99/09063 or from Novick et al., 1999, as well
as from Kim et al., 2000.
[0113] Functional derivatives of IL-18BP may be conjugated to
polymers in order to improve the properties of the protein, such as
the stability, half-life, bioavailability, tolerance by the human
body, or immunogenicity. To achieve this goal, IL18-BP may be
linked e.g. to Polyethlyenglycol (PEG). PEGylation may be carried
out by known methods, described in WO 92/13095, for example.
[0114] A fusion protein of IL-18BP may e.g. comprise an
immunoglobulin fusion, i.e. the inhibitor of IL-18 is a fused
protein comprising all or part of an IL-18 binding protein, which
is fused to all or a portion of an immunoglobulin. Methods for
making immunoglobulin fusion proteins are well known in the art,
such as the ones described in WO 01/03737, for example. The person
skilled in the art will appreciate that the resulting fusion
protein of the invention substantially retains the biological
activity of IL-18BP, such as e.g. the binding to IL-18 which can be
measured in in vitro assays described in the prior art such as e.g.
WO 99/09063. The fusion may be direct, or via a short linker
peptide which can be as short as 1 to 3 amino acid residues in
length or longer, for example, 13 amino acid residues in length.
Said linker may be a tripeptide of the sequence E-F-M
(Glu-Phe-Met), for example, or a 13-amino acid linker sequence
comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met
introduced between the IL-18BP sequence and the immunoglobulin
sequence. The resulting fusion protein has improved properties,
such as an extended residence time in body fluids (half-life),
increased specific activity, increased expression level, or the
purification of the fusion protein is facilitated.
[0115] In a preferred embodiment, IL-18BP is fused to the constant
region of an Ig molecule, e.g. an Fc portion of an Immunoglobulin.
Preferably, it is fused to heavy chain regions, like the CH2 and
CH3 domains, optionally with the hinge region of human IgG1, for
example. The Fc part may e.g. be mutated in order to prevent
unwanted activities, such as complement binding, binding to Fc
receptors, or the like.
[0116] The generation of specific fusion proteins comprising
IL-18BP and a portion of an immunoglobulin are described in example
11 of WO 99/09063, for example. Other isoforms of Ig molecules are
also suitable for the generation of fusion proteins according to
the present invention, such as isoforms IgG.sub.2 or IgG.sub.4, or
other Ig classes, like IgM or IgA, for example. Fusion proteins may
be monomeric or multimeric, hetero- or homomultimeric.
[0117] Further fusion proteins of IL-18BP may be prepared by fusing
domains isolated from other proteins allowing the formation or
dimers, trimers, etc. Examples for protein sequences allowing the
multimerization of the polypeptides of the Invention are domains
isolated from proteins such as hCG (WO 97/30161), collagen X (WO
04/33486), C4BP (WO 04/20639), Erb proteins (WO 98/02540), or
coiled coil peptides (WO 01/00814).
[0118] The IL-18BP produced according to the process of the
invention, or the composition according to the invention, may be
intended for therapeutic use, i.e. for administration to patients.
If IL-18BP is administered to patients, it is preferably
administered systemically, and preferably subcutaneously or
intramuscularly, or topically, i.e. locally. Rectal or intrathecal
administration may also be suitable, depending on the specific use
of IL-18BP.
[0119] For this purpose, produced IL-18BP may be formulated as a
pharmaceutical composition, i.e. together with a pharmaceutically
acceptable carrier, excipients or the like.
[0120] The definition of "pharmaceutically acceptable" is meant to
encompass any carrier, which does not interfere with effectiveness
of the biological activity of the active ingredient and that is not
toxic to the host to which it is administered. For example, for
parenteral administration, the active protein(s) may be formulated
in a unit dosage form for injection in vehicles such as saline,
dextrose solution, serum albumin and Ringer's solution.
[0121] The active ingredients of the pharmaceutical composition
according to the invention can be administered to an individual in
a variety of ways. The routes of administration include
intradermal, transdermal (e.g. in slow release formulations),
intramuscular, intraperitoneal, intravenous, subcutaneous, oral,
intracranial, epidural, topical, rectal, and intranasal routes. Any
other therapeutically efficacious route of administration can be
used, for example absorption through epithelial or endothelial
tissues or by gene therapy wherein a DNA molecule encoding the
active agent is administered to the patient (e.g. via a vector),
which causes the active agent to be expressed and secreted in vivo.
In addition, the protein(s) according to the invention can be
administered together with other components of biologically active
agents such as pharmaceutically acceptable surfactants, excipients,
carriers, diluents and vehicles.
[0122] For parenteral (e.g. intravenous, subcutaneous,
intramuscular) administration, the active protein(s) can be
formulated as a solution, suspension, emulsion or lyophilized
powder in association with a pharmaceutically acceptable parenteral
vehicle (e.g. water, saline, dextrose solution) and additives that
maintain isotonicity (e.g. mannitol) or chemical stability (e.g.
preservatives and buffers). The formulation is sterilized by
commonly used techniques.
[0123] The therapeutically effective amounts of the active
protein(s) will be a function of many variables, including the type
of antagonist, the affinity of the antagonist for IL-18, any
residual cytotoxic activity exhibited by the antagonists, the route
of administration, the clinical condition of the patient (including
the desirability of maintaining a non-toxic level of endogenous
IL-18 activity).
[0124] A "therapeutically effective amount" is such that when
administered, the IL-18 inhibitor results in inhibition of the
biological activity of IL-18. The dosage administered, as single or
multiple doses, to an individual will vary depending upon a variety
of factors, including IL-18 inhibitor pharmacokinetic properties,
the route of administration, patient conditions and characteristics
(sex, age, body weight, health, size), extent of symptoms,
concurrent treatments, frequency of treatment and the effect
desired. Adjustment and manipulation of established dosage ranges
are well within the ability of those skilled in the art, as well as
in vitro and in vivo methods of determining the inhibition of IL-18
in an individual.
[0125] IL-18BP may be used in amounts in the ranges of about 0.001
to 100 mg/kg or about 0.01 to 10 mg/kg or body weight, or about 0.1
to 5 mg/kg of body weight or about 1 to 3 mg/kg of body weight or
about 2 mg/kg of body weight.
[0126] IL-18BP may be administered daily or every other day or
three times per week or once per week, at similar doses, or at
doses increasing or decreasing with the time.
[0127] The daily doses are usually given in divided doses or in
sustained release form effective to obtain the desired results.
Second or subsequent administrations can be performed at a dosage
which is the same, less than or greater than the initial or
previous dose administered to the individual. A second or
subsequent administration can be administered during or prior to
onset of the disease.
[0128] IL-18BP may be administered prophylactically or
therapeutically to an individual prior to, simultaneously or
sequentially with other therapeutic regimens or agents (e.g.
multiple drug regimens), in therapeutically effective amounts.
[0129] IL-18BP produced in accordance with the present invention
may be used for preparation of a medicament for treatment and/or
prevention of a number of diseases or disorders. Such diseases or
disorders may e.g. be IL-18 mediated disorders. For instance,
IL-18BP may be used for treatment and/or prevention of psoriasis,
psoriatic arthritis, Crohn's Disease, inflammatory bowel disease,
rheumatoid arthritis, liver injury such as alcoholic liver
cirrhosis, sepsis, atherosclerosis, ischemic heart diseases,
allergies, in particular delayed-type hypersensitivity, and closed
head injury.
[0130] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations and conditions without departing from the spirit and
scope of the invention and without undue experimentation.
[0131] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended
claims.
[0132] All references cited herein, including journal articles or
abstracts, published or unpublished U.S. or foreign patent
application, issued U.S. or foreign patents or any other
references, are entirely incorporated by reference herein,
including all data, tables, figures and text presented in the cited
references. Additionally, the entire contents of the references
cited within the references cited herein are also entirely
incorporated by reference.
[0133] Reference to known method steps, conventional methods steps,
known methods or conventional methods is not any way an admission
that any aspect, description or embodiment of the present invention
is disclosed, taught or suggested in the relevant art.
[0134] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art (including
the contents of the references cited herein), readily modify and/or
adapt for various application such specific embodiments, without
undue experimentation, without departing from the general concept
of the present invention. Therefore, such adaptations and
modifications are intended to be within the meaning range of
equivalents of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance presented herein, in
combination with the knowledge of one of ordinary skill in the
art.
EXAMPLES
Example 1
Perfusion Process for the Production of Recombinant, HumanIL-18BP
from Serum-Free CHO Cell Harvest in Packed-Bed Bioreactor
[0135] This example describes a process based on the high cell
density culture of recombinant CHO cells in a packed-bed
bioreactor, in which the perfusion rate was adjusted according to
cell growth requirements during the growth phase and then
pronouncedly reduced during the production phase without
compromising process productivity or protein quality.
[0136] The protein product produced by this process, IL-18BP, was
characterized and turned out to have an advantageous
N-glycan-profile.
[0137] A first-generation process had originally been designed with
the aim to rapidly produce material for pre-clinical and early
clinical trials.
[0138] This process was designed with a high perfusion rate of 2.6
vvd in order to supply the high cell density (.about.2.510.sup.7
cellml.sup.-1 of packed-bed) with fresh medium during production
phase. In this first-generation process, product degradation was
not a concern since the high dilution rate imposed to the culture
maintained a low residence time of the product, IL-18BP, in the
bioreactor environment.
[0139] At a later stage of the development, a reduction of the
medium perfusion rate by -25% and -50% was tested in order to
improve the process. A small-scale system was used to run the
tests, and the selected conditions were then implemented at pilot
scale in order to further produce material for clinical trials with
an improved second-generation process.
[0140] Materials and Methods
[0141] Cell Culture--Experimental System
[0142] A packed-bed bioreactor (Ducommun et al. 2002a; Ducommun et
al. 2002b) with Fibra-Cel.RTM. carrier (Bibby Sterilin, U.K.) was
used to cultivate CHO cells (Laboratoires Serono S.A.,
Corsier-sur-Vevey, Switzerland) that express and secrete IL-18BP in
a serum free medium (Sigma C-9486). In the small-scale system that
was used to investigate a reduction of the perfusion rate, the
bioreactor and packed-bed had a working volume of 15 and 5 litres
respectively.
[0143] The bioreactor was perfused with 2.6 vvd (as defined in
Table 1) during growth and production phase. This basic perfusion
rate is chosen as the reference 100%. stated as run-100
TABLE-US-00004 TABLE 1 Perfusion rates tested during the production
phase for run-100, run-75 and run-50. Average of n replicates
(.+-.2 standard deviations for run-100). Perfusion rate Dilution
rate Replicates (l kg.sub.Fibra-Cel.sup.-1 day.sup.-1) (vvd*) (n)
run-100 100 .+-. 3.5 2.6 .+-. 0.1 8 run-75 75 2.0 3 run-50 50 1.3 2
*The dilution rate D expressed in vvd is calculated as litre of
medium per litre of total system working volume per day (total
volume = packed-bed + conditioning tank volume).
[0144] These conditions were applied for all sets of bioreactor
experiments: medium was perfused at 100% during growth phase at
37.degree. C. The temperature was regulated at 37.0.degree. C.
during growth phase, and then reduced in two steps down to
32.5.degree. C. The pH was regulated at 7.00 and Dissolved Oxygen
concentration (DO) was maintained at 70% of air saturation
throughout the culture.
[0145] Due to the fact that counting of cells and cell number
determination in a packed-bed bioreactor is a complex and
inaccurate analysis, we used Glucose Consumption Rate (GCR) as an
indirect method to estimate cell growth and density in the
packed-bed bioreactor. In our system, we have determined by direct
cell counts on a number of packed-beds cultures that a GCR of 300
grams of glucose per kilogram of Fibra-Cel.RTM. disks per day
corresponds to approximately 2.510.sup.7 cells per ml of packed-bed
bioreactor volume (data not shown).
[0146] This stage has been defined as the end of the cell
propagation phase at 37.degree. C., and when GCR reached a level of
300 grams of glucose per kilogram of Fibra-Cel.RTM. disks per day,
the cultures were switched from 37.0.degree. C. to the production
mode by lowering the temperature to 33.5.degree. C. At this stage,
in one set of bioreactors the perfusion was kept at 100% (run-100)
and the two other sets were performed with a medium perfusion rate
of 75% (run-75) and 50% (run-50) of the maximal level, as
summarized in Table 1. The temperature was further decreased to
32.5.degree. C. at a later stage of the production phase to prevent
further cell growth and to promote production.
[0147] In the results section, the bars displayed on the figures
represent an interval of 4 standard deviations (.+-.2 standard
deviation) measured for the 8 replicates ran under the conditions
of run-100.
[0148] Assays
[0149] Samples were removed daily during the culture. Glucose and
lactate concentrations were quantified with an EMI, 105 analyzer
(Radiometer Medical A/S, Bronshoj, Denmark).
[0150] The produced recombinant IL18BP was quantified with an ELISA
test.
[0151] The quality of the recombinant protein was assessed with an
RP-HPLC method, in combination with SDS-PAGE methods (ExcelGel SDS
Homogeneous 12.5% (Cat #80-1261-01, Pharmacia). The gels were
stained specifically by Western Blot in order to detect high
molecular weight (i.e. dimers, aggregates) or low molecular weight
variants of the IL-18BP. Non-specific staining by Silver Stain was
also used to assess the relative intensity of the IL-18BP compared
to impurities after scanning (Scanner ARCUS 2, Afga) of individual
bands in order to determine their relative intensity.
[0152] Protein sialylation, and in particular the abundance of
neutral, mono-, di-, tri- and tetra-sialylated N-glycans was
analysed by separation of the N-glycans according to their charge,
as described in Gervais et al. 2003. The N-glycans were
specifically cleaved from the IL-18BP by hydrazinolysis
(N-glycanase E-5006B or E-5006C, Glyko Inc.), labelled with the
2-Aminobenzamide fluorescent dye (Signal 2-AB labelling kit K404,
Glyko Inc.), and separated by a chromatography column before
passing through a fluorescent detector. The proportions of
N-glycans species could then be determined after integration of the
HPLC peaks corresponding to neutral, mono-, di-, tri-, and
tetra-sialylated forms.
[0153] Results
[0154] Reduction of Perfusion Rate
[0155] The first attempt was to reduce the perfusion rate from 2.6
vvd to 2.0 vvd and 1.3 vvd during the production phase (FIG. 1) and
to follow its effect on cell metabolism, volumetric productivity
and product quality.
[0156] The concentration of glucose and lactate were measured for
all three perfusion rates (FIGS. 2A and B), and the residual
glucose level remained above 0.5 g/L.
[0157] The results in FIG. 3 show the glucose consumption rate
(GCR) levels for the three sets of medium perfusion rate tested.
These results indicate that a reduced perfusion rate induces a
lower GCR of the culture. However, this effect was barely
significant and when the medium perfusion rate was reduced by -25%
and -50%, the average GCR measured over the 60-day production phase
were reduced by -8% and -15% respectively (FIG. 3).
[0158] In parallel, the apparent molar ratio (FIG. 4) of glucose
conversion to lactate Y.sub.lac/glc slightly decreased in response
to lower perfusion rate but remained in a range of 1.55 to 1.65
mole of lactate produced per mole of glucose consumed. The
differences observed for the Y.sub.lac/glc ratio were not
statistically significant since all data points measured for the
test runs at reduced perfusion rate are comprised within .+-.2
standard deviations of values obtained with the reference runs.
[0159] Process Productivity
[0160] The results presented in FIGS. 5A, B and C show a comparison
of the recombinant protein produced (volumetric productivity, total
production, and titre) in the reference run-100 and in the runs
with reduced medium perfusion.
[0161] When the medium perfusion rate was reduced by -25% and -50%,
the average volumetric productivity was reduced by -3% and -30%
respectively (FIG. 5A). The lower productivity result obtained for
run-50 are statistically different from the reference
conditions.
[0162] Another difference observed in FIG. 5A is the decline of the
volumetric productivity over time along the production phase. A
stable volumetric productivity was observed for run-100 and run-75,
but in run-50 productivity declined over the duration of the 60-day
production phase. More specifically, the productivity level of
run-50 was 60% of the reference value at the end of the 60-day
production phase.
[0163] The lower productivity of run-50 is also shown on FIG. 5B,
which represents the cumulated amount of product made over the
60-days production phase.
[0164] The corresponding titers measured for the different
perfusion rates are presented in FIG. 5C, which shows that
recombinant protein titers were increased by +25% and +50%
respectively as opposed to the control when the perfusion was
decreased by -25% and -50%.
[0165] Product Quality
[0166] With the reduction of the perfusion rate from 2.6 vvd, to
2.0 vvd and 1.3 vvd that was tested for run-100 run-75 and run-50,
the residence time (t) of the recombinant protein was increased
from 0.4 to 0.5 and 0.8 day respectively (t=1/D).
[0167] As a longer exposure to the environment in the bioreactor
could potentially lead to a degradation of the recombinant protein,
a stability study was done before initiating the tests in
bioreactors. A sample of IL-18BP was spiked into cell culture
medium, and the quality attributes of the IL-18BP were monitored
after 1, 2 and 5 days of incubation at 37.degree. C. Since no sign
of product degradation was detected by the stability indicating
method (data not shown), it was decided to proceed with the
bioreactor experiments.
[0168] During the tests in bioreactors, the recombinant protein was
purified to homogeneity at three points of the bioreactor runs (day
20, 40 and 60) in order to verify that the product quality was
maintained for each perfusion rate investigated. The conditions of
run-50 were considered as a worst case for protein degradation
since this run had the lowest perfusion rate and the longest
product residence time in the bioreactor.
[0169] To evaluate if product quality was affected by the reduced
perfusion rate, final bulks of run-50 were analyzed by SDS-PAGE
Silver Stain and SDS-PAGE Western Blot, and compared to the profile
obtained in the reference conditions of run-100. The corresponding
data obtained for one bulk produced with IL-18BP harvested at days
47-48 of the production phase of run-50 resulted in the expected
single bands at a molecular weight of about 40 kDa (not shown). No
modification of the IL-18BP quality attributes was detected for
run-50 compared to run-100. The electrophoretic purity measured by
SDS-PAGE Silver Stain was higher than 99% purity for all lanes, and
no presence of aggregates or truncated forms could be detected as
shown on the SDS-PAGE Western Blot (not shown).
[0170] The high level of purity (.about.100%) of the IL-18BP
produced was confirmed by RP-HPLC for run-100, run-75 and
run-50.
[0171] Finally, the amount of impurities derived from the host CHO
cell (Host Cell Proteins,
[0172] HCP) in the produced protein was quantified by HCP-ELISA
(data not shown) and was found consistent between run-50, run-75
and run-100.
[0173] N-Glycan Profile
[0174] Samples of the produced recombinant IL-18BP were submitted
to N-glycan mapping according to the method reported above, in
order to quantify the proportion of the different sialylated forms
of the protein of interest. The N-Glycan mapping results summarized
in Table 2 show that comparable proportions of N-glycans are
obtained for all perfusion rates tested. This is also illustrated
by the corresponding HPLC profiles reported in FIG. 6.
TABLE-US-00005 TABLE 2 Fraction of differently sialylated N-Glycan
molecules (given as % of the N-glycan groups) in semi-produced
samples of drug substance for run-100, run-75 and run-50. Fraction
of differently sialylated N-Glycan molecules Mono- Tetra- Neutrals
sialylated Di-sialylated Tri-sialylated sialylated (%) (%) (%) (%)
(%) run-100 19-21 21-29 39-44 7-11 2-4 run-75 18-22 20-23 44-50 8-9
3-4 run-50 19-21 20-25 45-48 8-10 2-4
[0175] A comparison of these data demonstrates that the product
sialylation is not altered by the reduced perfusion rate, and the
data obtained for run-75 and run-50 are clearly within the range
obtained under the standard conditions of run-100.
[0176] Summary and Conclusions
[0177] In the present study, the optimal medium perfusion rate to
be used for the continuous culture of a recombinant CHO cell line
in a packed-bed bioreactor made of FibraCel.RTM. disk carriers was
determined.
[0178] A first-generation process had originally been designed with
a high perfusion rate, originally operated with a perfusion of 2.6
vvd during production phase in order to supply the high cell
density (.about.2.510.sup.7 cellml.sup.-1 of packed-bed) with
sufficient fresh medium.
[0179] In order to improve the economics of this process, a
reduction of the medium perfusion rate by -25% and -50% was
investigated at small-scale. The best option was then implemented
at pilot scale in order to further produce material for clinical
trials with an improved second-generation process.
[0180] With a -25% reduction of the perfusion rate, the volumetric
productivity was maintained compared to the first-generation
process, but a -30% loss of productivity was obtained when further
reducing the medium perfusion rate to -50% of its original
level.
[0181] The protein quality under reduced perfusion rate conditions
was analysed for purity, N-glycan sialylation level, abundance of
dimers or aggregates, and showed that the quality of the final drug
substance was comparable to that obtained in high perfusion
conditions.
[0182] In this study it was established that the product quality
was maintained upon reduction of the medium perfusion rate. The run
with the lowest perfusion, run-50, has the longest residence time
(t=0.8 day) and this is the worst case for protein stability since
it leaves the product exposed to all potentially degrading
activities present in the bioreactor environment for the longest
time. Under these conditions we can consider that 99% of the
protein will have a residence time in the bioreactor system shorter
than 4 days (5t=4 day for run-50).
[0183] Since a stability study demonstrated that the IL-18BP could
be stored for up to 4 days at 37.degree. C. in crude harvest
without significant alterations, it was anticipated that under the
range of perfusion rates tested the product would not be degraded.
This was confirmed by the results obtained in the bioreactor runs,
as all lots of produced protein generated at production day-20,
day-40 and day-60 of each perfusion conditions tested met the
specifications established with reference material from run-100.
Thus, in the study reported here, no sign of product degradation
could be detected.
[0184] Glucose starvation is a typical cause of incorrect product
sialylation (Goochee and Monica 1990). This effect has been studied
for IFN-g produced from CHO cells, and the product glycosylation
was found affected under low glucose residual levels below 0.1 g/L
(Hayter et al. 1993), (Hooker et al. 1995).
[0185] In the conditions reported here, the IL-18BP sialylation was
not affected due to the lower glucose concentrations reached during
the production phase of run-75 and run-50. Without wishing to be
limited to a specific explanation, this could be explained by the
fact that the range of residual glucose concentrations (2.6 g/L to
0.5 g/L) reported here is still much higher than the value of less
than 0.1 g/L glucose level (probably inducing some glucose
starvation effect) for which incomplete sialylation of IFN-g was
observed.
[0186] Based on the results obtained at small-scale, a reduction of
-25% medium perfusion was implemented at pilot scale in the
second-generation process, which enabled to maintain the same
productivity and the same quality of the molecule, while reducing
costs of media, material and manpower of the production
process.
[0187] The -25% reduction on medium translated directly into a -25%
saving on: the powder medium and side ingredients, the pre-filters
and sterilizing filters, the sterile bags used for media storage
after filtration, and the labour costs associated with medium
preparation as fewer medium batches were needed.
[0188] As the IL-18BP titer in the crude harvest was increased by
+25% in the second-generation process, the downstream processing
benefited from similar savings: reduced manpower needs due to
smaller volumes to handle, reduced production cycle time,
optimisation of the equipment, etc (data not shown).
[0189] Conclusion
[0190] From the results obtained at small-scale it is clear that
reduction of -25% in perfusion rate combined both benefits of
maximizing productivity with a saving of -25% on medium
consumption.
[0191] Further reduction of the perfusion rate to -50% led to
reduced process productivity, which declined by -30% under such
conditions. With the -50% perfusion rate conditions, volumetric
productivity declined over the duration of the 60-day production
phase whereas a stable productivity was maintained with the higher
perfusion rates tested. Product stability remained comparable,
irrespective of the perfusion rate used in the process.
Example 2
Fed-Batch Process for the Production of Recombinnt, Human IL-18BP
from Serum-Free CHO Cell Culture
[0192] A fed-batch process with recombinant human IL-18BP
expressing cells in suspension culture was developed as well. Three
runs were performed in total, using bioreactors of 5 L (n=2) or 300
L (n=1) nominal volume.
[0193] In summary, the culture set points were: oxygen
concentration of 50% air saturation, pH 7.0 and 6.90, temperature
of 37.0.degree. C. during growth phase and then reduced in two
steps down to 29.0.degree. C. During the course of the fed-batch
culture, the serum-free basal medium (Sigma, S-9942) was gradually
supplemented with a concentrated feed solution.
[0194] The parameters of the fed-batch process are summarized in
Table 3.
TABLE-US-00006 TABLE 3 Fed-batch Manufacturing process scheme
Time/WD* T pH Feed [day] [.degree. C.] [pHU] Feed-1 Comments -3
Transfer the wave-bag inoculum pool to VCs** = 0.20 .+-. 0.05
mioCs/mL the seed bioreactor.sup.1) with a target VCs of: 0
Transfer the seed bioreactor inoculum VCs = 0.60 .+-. 0.10 mioCs/mL
pool to the production bioreactor.sup.2) with a target VCs of: 0 37
6.90 .dwnarw. N/A N/A +80 g/kg.sub.Supernatant c(glucose) < 1.0
g/L.sup.3) 4 .fwdarw. 33 N/A .dwnarw. N/A N/A +30
g/kg.sub.Supernatant c(glucose) < 5.5 g/L.sup.4) 6 .fwdarw. 29
N/A .dwnarw. N/A N/A 20 Crude harvest clarification .sup.1)5 L and
75 L bioreactor for small-scale (5 L) and pilot-scale (300 L)
operation, respectively .sup.2)5 L and 300 L bioreactor for
small-scale (5 L) and pilot-scale (300 L) operation, respectively
.sup.3)The first addition of Feed-1 occurs when c(glucose) < 1.0
g/L (+80 g/kg) .sup.4)The subsequent additions of Feed-1 occur each
time when c(glucose) < 5.5 g/L (+30 g/kg) *WD = working days
**VCs = viable cells
[0195] Important performance indicators, namely total cell-density,
viability, residual glucose concentration, and protein titer were
analysed. As shown in FIG. 7(A) to (D), the final fed-batch process
allowed growing up the clone to a maximum total cell-density of
approximately 7.0 mioCs/mL and maintaining cell viability above 80%
until working day 19, where the 300 L culture was harvested,
clarified and captured. FIG. 7(C) corresponds to the residual
glucose concentration profiles, based on daily off-line
measurements. These profiles illustrate the applied feeding
strategy. For example, the first bolus addition of 80 g/kg of
supernatant occurred in all three batches on working day 4 in order
to raise residual glucose concentration up to more than 5.5 g/L. In
a similar way, each subsequent addition of 30 g/kg of supernatant
can easily be identified. Further, process productivity is shown in
FIG. 7(D), with a consistent final r-hIL-18BP titre of 400 mg/L on
working day 19. Protein quality was assessed on all three batches
on working day 19.
Example 3
Sialylation Profile of RHIL-18BP Produced in Perfusion and the
Fed-Batch Process for CHO Cell Culture
[0196] A further perfusion process for production of IL-18BP was
set up. Perfusion runs were performed with a perfusion rate 2.75
vvd in a bioreactor containing a total volume of 160 L (including
external column of 40 L) packed with 4.4 kg of Fibracel.RTM.-disks,
at production temperatures of 33.5 or 32.5.degree. C.
[0197] The IL-18BP produced in this perfusion process was compared
to the material derived from the fed-batch process described in
example 2.
[0198] Either perfusion or fed-batch supernatant were subjected to
a capture step using affinity chromatography.
[0199] The post-capture IL-18BP material was analyzed for
N-glycanation as described in Example 1 above.
[0200] The hypothetical charge number, the so-called Z-number, was
calculated as described in Gervais et al. (2003). Briefly, the Z
number is defined as the sum of the products of the respective
areas (A) in the neutral, mono-, di-, tri-, tetra-, and
pentasialylated region of the N-glycan species, each multiplied by
the corresponding charge:
Z=A.sub.(neutral).times.0+A.sub.(mono).times.1+A.sub.(di).times.2+A.sub.-
(tri).times.3+A.sub.(tetra).times.4+A(penta).times.5
[0201] The results are depicted in FIG. 8, showing that despite
some qualitative differences, mainly in the di-sialylated groups,
the N-glycan mapping data indicate that the product sialylation was
comparable for the perfusion (Z=149) and the fed-batch (Z=145)
process conditions in terms of quantity.
[0202] The IL-18BP material ("crude harvest material"), either
derived from the fed-batch or the perfusion process, was also
analyzed by Capillary Zone Electrophoresis (CZE) for differentially
glycosylated isoforms ("glycoforms"), according to the protocol
described in detail below.
[0203] The isoform distribution was further compared to purified
IL-18BP, either derived from fed-batch or the perfusion
process.
[0204] Crude harvest material underwent a microcapture step on a
Sep-Pak.RTM. tC2 cartridge (Waters) followed by a desalting step by
Centricon.RTM. 10 (Millipore) ultrafiltration before being injected
into the capillary. Purified IL-18BP was obtained essentially as
described in patent application WO 2005/049649, albeit with a
different capture step based on affinity chromatography. Briefly,
the purification steps included metal ion affinity chromatography
(on Chelating Sepharose FF), hydrophobic charge-induction
chromatography (on Mep Hypercel), ion exchange chromatography (on
CM-Sepharose FF, using the flow-through), hydrophobic interaction
chromatography (on Phenyl Sepharose Fast Flow HS), and reverse
phase chromatography (on Reverse Phase-Source 30 RPC). The finally
purified material was directly applied to capillary without any
further desalting step.
[0205] Method:
[0206] Solutions for CZE [0207] 5 mM phosphate CZE wash/run buffer:
[0208] Prepare by 1:10 dilution of 50 mM phosphate stock solution
pH 7.0. Filter through 0.22 .mu.m filter. Prepare fresh. [0209] 0.5
M NaOH (CZE washing solution): [0210] Add 26.2 .mu.L 50% NaOH to
water, 1 mL total volume. Prepare fresh. [0211] 1M NaOH (CZE
regeneration solution): [0212] Add 52.4 .mu.L 50% NaOH to water, 1
mL total volume. Prepare fresh. [0213] Neutral Marker (dilution
1:10000) [0214] Add 10 .mu.L neutral marker stock solution to
water, 1 mL total volume. [0215] Add 10 .mu.L of this neutral
marker 1:100 dilution to water, 1 mL total volume. [0216] Store for
three months at 4.degree. C.
[0217] CZE Analysis
[0218] .gtoreq.20 .mu.L sample/reference are transferred into PCR
vials containing 1/10 volume of neutral marker, and mixed by
reverse pipetting, avoiding generating bubbles.
[0219] Electrophoretic Parameters:
[0220] The following reagents are added into separate vial holders,
avoiding generating bubbles.
TABLE-US-00007 TABLE 4 Electrophoretic parameters for CZE Reagent
Reagent Inlet reagents volume Outlet reagents volume Wash buffer
1.2 mL Run Buffer 1.2 mL Run Buffer (3.3.1) 1.2 mL Purified water
1.2 mL Purified water 1.2 mL CZE washing solution 1.0 mL CZE
regenerating solution* 1.0 mL Sample >22 .mu.L/PCR Vial Waste
(purified 0.2 mL water) 1 Waste (purified 0.2 mL water) 2 Air*
Empty Vial *use only if required
TABLE-US-00008 TABLE 5 CZE Analysis time table Event Value Duration
Inlet Vial Volume reagent Outlet Vial Rinse pressure 20.0 psi 2.00
min Wash buffer 1.2 mL Waste 1 (0.2 mL) Inject-pressure 0.5 psi 5.0
sec Sample .gtoreq.22 .mu.L Run Buffer (3.3.1) Separate- 25 KV
30.00 min Run Buffer 1.2 mL Run Buffer Voltage (3.3.1) Rinse
pressure 20.0 psi 1.00 min Wash buffer 1.2 mL Waste 1 (0.2 mL)
Rinse pressure 20.0 psi 1.00 min Purified water 1.2 mL Waste 1 (0.2
mL) Rinse pressure 20.0 psi 1.00 min CZE washing 1.0 mL Waste 2
(0.2 mL) solution Rinse pressure 40.0 psi 2.00 min Purified water
1.2 mL Waste 2 (0.2 mL) Rinse pressure 40.0 psi 2.00 min Wash
buffer 1.2 mL Waste 2 (0.2 mL) Wait Wash buffer 1.2 mL Run Buffer
(3.3.1)
TABLE-US-00009 TABLE 6 CZE capillary regeneration timetable Volume
Event Value Duration Inlet Vial reagent Outlet Vial Rinse pressure
40.0 psi 1.00 min Purified water 1.2 mL Waste 2 (0.2 mL) Rinse
pressure 40.0 psi 10.00 min CZE regenerating 1.0 mL Waste 2 (0.2
mL) solution Rinse pressure 40.0 psi 4.00 min Purified water 1.2 mL
Waste 2 (0.2 mL) Rinse pressure 40.0 psi 1.00 min Wash buffer 1.2
mL Waste 2 (0.2 mL) Rinse pressure 0.5 psi 30.00 min Wash buffer
1.2 mL Waste 2 (0.2 mL) Wait Run Buffer 1.2 mL Run Buffer (3.3.1)
Capillary length to detector/total length 50/60 cm Polarity
positive to negative (forward) Temperature capillary = 25 .+-. 2
C..degree. Sample tray = 10 .+-. 2 C..degree. Detection 214 nm
[0221] Injection Protocols
[0222] There should be at least three injections of the Standard
Reference material for the purpose of capillary conditioning.
[0223] Standard Reference 1 (start)
[0224] Single injection of sample 1
[0225] Single injection of sample 2
[0226] Single injection of sample 3
[0227] Single injection of sample 4
[0228] Standard Reference 2 (end)
[0229] NOTE: To increase reproducibility, a maximum of 4 samples
can be analyzed in one sequence between reference 1 and reference 2
by using the same CZE running buffer.
[0230] Alternatively two bracketing references can be used for each
sample as described below.
[0231] At least three injections of the Standard Reference material
are done for the purpose of capillary conditioning.
[0232] Standard Reference (start 1)
[0233] Single injection of sample 1
[0234] Standard Reference (end1/start2)
[0235] Single injection of sample 2
[0236] Standard Reference replicate (end2/start3)
[0237] Single injection of sample 3
[0238] Standard Reference replicate (end3)
[0239] Data Analysis
[0240] The Standard Reference material is used for comparison of
sample data.
[0241] The overlaid and stacked electroferograms of sample/s and
both bracketing Reference standard (start/end) are printed out and
archived.
[0242] Determination of Migration Times MT2 and MT3
[0243] The migration times MT2 and MT3 at the left and right
valleys of -3 and +3 peaks of the Reference Standard (Start) are
determined. The 0 peak is the principal peak of the reference.
[0244] Isoform Classification
[0245] Due the high acidity of IL-18BP glycoprotein profile, the
isoforms between MT2 and MT3 are named "acidic isoforms". Isoforms
with migration times higher than MT3 are named "highly acidic
isoforms". Isoforms with migration times lower than MT2 are named
"less acidic isoforms".
[0246] In some circumstances, it might be necessary to add the
class of "basic isoforms" defined as isoforms with migration times
lower then MT1.
[0247] Isoforms Abundance Estimation
[0248] The reference and each sample are analyzed by using the
functions: manual peak between MT1-M2, MT2-MT3 and MT3-MT4; the
manual baseline between 5 and 28 minutes and integration OFF
between 0 and MT1 and between MT4 and 30 minutes. Manually modify
the functions Width and Threshold to obtain an integration of three
groups of peaks between MT1-M2 (less acidic isoforms), MT2-MT3
(acidic isoforms) and MT3-MT4 (highly acidic isoforms) similar to
that showed above for the Reference Standard.
% isoform abundance = area ( MT 1 - MT 2 or MT 2 - MT 3 or MT 3 -
MT 4 ) Total area ( MT 1 - MT 4 ) ##EQU00001##
[0249] When necessary, the group of peaks corresponding to "basic
isoforms" defined as isoforms with migration times lower than MT1,
is added, and the above formula corrected accordingly.
[0250] Results:
[0251] This CZE method was applied to crude harvest samples (FIG.
9) and to purified samples for both perfusion and fed-batch
process. Despite some differences observed for the pre-treated
harvest samples (e.g. higher proportion of basic isoforms with the
perfusion process), these basic isoforms were successfully removed
by ion exchange chromatography during the purification process
(data not shown). Hence, for the purified product, a comparable
isoforms profile was obtained for both processes (FIG. 10).
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