Ex Parte Yun

Patent Trial and Appeal Board

Mar 23, 2017

13946759 (P.T.A.B. Mar. 23, 2017)

United States Patent and Trademark Office
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O.Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
13/946,759 07/19/2013 Anthony Joonkyoo Yun PALO-026 3695
93726 7590 03/27/2017
EPA - Bozicevic Field & Francis LLP
Bozicevic, Field & Francis
201 REDWOOD SHORES PARKWAY
SUITE 200
REDWOOD CITY, CA 94065
EXAMINER
CARLSON, KAREN C
ART UNIT PAPER NUMBER
1656
NOTIFICATION DATE DELIVERY MODE
03/27/2017 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
docket@bozpat.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte ANTHONY JOONKYOO YUN
Appeal 2016-002460
Application 13/946,759
Technology Center 1600
Before MELANIE L. McCOLLUM, JEFFREY N. FREDMAN, and
TIMOTHY G. MAJORS, Administrative Patent Judges.
FREDMAN, Administrative Patent Judge.
DECISION ON APPEAL
This is an appeal1 under 35 U.S.C. § 134 involving claims to a method
for producing a biological product from a cell. The Examiner rejected the
claims as indefinite and as obvious. We have jurisdiction under 35 U.S.C.
§ 6(b). We reverse and enter a New Ground of Rejection.
Statement of the Case
Background
“A variety of useful biological materials may be produced by
organisms, including therapeutics (e.g. peptides and antibodies), research
tools, and nutritional products” (Spec. 12). “Aspects of the methods include
1 Appellant identifies the Real Party in Interest as Palo Alto Investors (see
App. Br. 3).
Appeal 2016-002460
Application 13/946,759
modulating the stress conditions of the cells and/or organism to produce
biological materials having one or more desired properties” (Spec. 13).
The Claims
Claims 1—8, 10-12, 14—17, 20, 25, 27, and 53—55 are on appeal.2
Claim 1, the only independent claim, is representative and reads as follows:
1. A method for producing a biological product from a cell, the
method comprising:
culturing the cell containing a nucleic acid encoding at least a
portion of the biological product, under conditions that modulate a
stressed phenotype of the cell and allow for expression of the
biological product, wherein the produced biological product is
different from the biological product produced in a control cell having
a stressed phenotype as compared to the cell; and
recovering the biological product.
The issues
A. The Examiner rejected claims 14—17 and 20 under 35 U.S.C. § 112, second
paragraph as indefinite (Ans. 2).
B. The Examiner rejected claims 1—8, 10-12, 14—17, 20, 25, 27, and 53—55 under
35 U.S.C. § 102(b) as anticipated by Galbraith3 as evidenced by Hamsters4 (Ans. 3—
5).
2 Claims 9, 13, 18, 19, 21—24, 26, and 29-42, and 44—52 were identified as
cancelled and claims 28 and 43 as withdrawn from examination (see Resp.
After Final, dated April 30, 2015 and Advisory Action, dated May 11,
2015).
3 Galbraith et al., Control of Culture Environment for Improved
Polyethylenimine-Mediated Transient Production of Recombinant
Monoclonal Antibodies by CHO Cells, 22 Biotechnology Progress 753—62
(2006) (“Galbraith”).
2
Appeal 2016-002460
Application 13/946,759
A. 35 U.S.C. § 112, second paragraph
The Examiner finds “Claims 14—17 and 20 do not further limit Claim
1 because these claims refer to parent cells/organisms which is not defining
to the cell of Claim 1” (Ans. 2).
Appellant contends “the Examiner has provided no evidence to
support the allegation that the stress state of the organism from which the
cell is derived does not impact the cell producing the biological product”
(Reply Br. 3). Appellant contends “the specification and the method are not
limited to the use of commercially available or even immortalized cell lines.
For example, the specification describes that primary cells may be used . . .
[that] would be expected to maintain characteristics of the host, such as,
e.g., stress characteristics” (Id. ). Appellant contends “that Claim 14
requires that the source organism is unstressed does, in fact, further limit
Claim 1 because it requires that the organism from which the cell is derived
is unstressed” (Id. at 4).
We find that Appellant has the better position. As Appellant points
out, claims 14—17 and 20 further limit claim 1 by placing a specific
limitation on the source of cells used in the method of claim 1. The
Specification teaches stresses that impact organisms (see Spec. 26—33), and
there are stresses that may impact the genetic material within cells, for
example, by modifying methylation of DNA that would result in cells
derived from organisms that differ based on stress of the organism source.
4 Hamsters, http://www.smallanimalchannel.com/hamsters/, 13 (accessed
Feb. 26, 2015).
3
Appeal 2016-002460
Application 13/946,759
We reverse the rejection under 35U.S.C. § 112, second paragraph.
B. 35 U.S.C. § 102(b) over Galbraith evidenced by Hamsters
The Examiner finds Galbraith teaches “culturing mammalian . . .
CHO [Chinese Hamster Ovary] cells transformed with nucleic acid encoding
IgG4 monoclonal antibody . . . optimized conditions for transfection
efficiency comprised maintaining the cells at 37 or 32°C” (Ans. 3). The
Examiner finds “Claim 1 does not state what that stressed phenotype of the
control cell is, or what the difference is between the biological product
produced in the non-stressed cell and the stressed phenotype cell may be”
(Ans. 4).
The issue with respect to these rejections is: Does the evidence of
record support the Examiner’s conclusion that Galbraith evidenced by
Hamsters anticipates the claims?
Findings of Fact
1. Galbraith teaches “we observed no significant difference in the
glycosylation of Mabs produced at any stage post-transfection, and the
glycosylation of Mabs derived from the transient process was also not
significantly different from that produced by stably transfected GS-CHO
cells” (Galbraith 758, col. 1).
Principles of Law
The Examiner bears the initial burden of establishing a prima facie
case of anticipation. In re King, 801 F.2d 1324, 1326—27 (Fed. Cir. 1986).
Anticipation under 35 U.S.C. § 102 requires that “‘each and every element
as set forth in the claim is found, either expressly or inherently described, in
4
Appeal 2016-002460
Application 13/946,759
a single prior art reference.”’ In re Robertson, 169 F.3d 743, 745 (Fed. Cir.
1999) (quoting VerdegaalBros., Inc. v. Union Oil Co., 814 F.2d 628, 631
(Fed. Cir. 1987).
Analysis
Appellant contends “regardless of whether any of the cells described
may be considered stressed or unstressed Galbraith concludes, in the only
comparison made, that there was no difference between the produced
products” (App. Br. 13).
We agree with Appellant. Claim 1 requires “the produced biological
product is different from the biological product produced in a control cell
having a stressed phenotype as compared to the cell.” Galbraith specifically
states that they “observed no significant difference” in glycosylation of the
products (FF 1). Therefore, the Examiner has failed to establish that
Galbraith anticipates this limitation either directly or inherently because the
Examiner fails to demonstrate any difference in biological products
produced under different conditions.
Conclusion of Law
The evidence of record does not support the Examiner’s conclusion
that Galbraith evidenced by Hamsters anticipates the claims.
5
Appeal 2016-002460
Application 13/946,759
New Ground of Rejection
Under the provisions of 37 C.F.R. § 41.50(b), we enter the following
new grounds of rejection.
Claims 1, 4, 6, 11, 12, 25, and 27 are rejected under 35 U.S.C.
§ 102(b) as anticipated by Kunkel.5 As the Board’s function is primarily one
of review and not search, we leave to the Examiner the determination of
whether there is additional prior art to address the remaining dependent
claims, either in view of Kunkel as applied to independent claim 1 or other
prior art (see, e.g., Galbraith 757, col. 2, citing Kunkel and other references).
Findings of Fact
2. Kunkel teaches the “murine-murine B-lymphocyte hybridoma
cell line, CC9C10 . . . were adapted for growth in serum-free medium . . .
Chemostat cultures were established with DO [dissolved oxygen]
concentrations of 10%, 50%, and 100% of air saturation” (Kunkel 464, col.
1).
3. Kunkel teaches: “Cell culture parameters in the two bioreactors
at each of the three DO setpoints were therefore nominally identical”
(Kunkel 464, col. 1).
4. Kunkel teaches: “Monoclonal antibody was prepared once from
each of the three DO concentration cultures (10%, 50%, and 100%) from
each of the two bioreactors” (Kunkel 464, col. 2).
5 Kunkel et al., Comparisons of the Glycosylation of a Monoclonal Antibody
Produced under Nominally Identical Cell Culture Conditions in Two
Different Bioreactors, 16 Biotechnol. Prog. 462-470 (2000).
6
Appeal 2016-002460
Application 13/946,759
5. Kunkel teaches “[approximately 1.0 L of eluted cell culture
medium, containing about 40-50 mg mL'1 of secreted mAb, was collected
and centrifuged at 10 000 x g for 15 min to remove cells. The mAb was
then purified from the supernatant” (Kunkel 464, col. 2).
6. Kunkel teaches:
In 10% DO, the chains were mainly agalactosyl or monogalactosyl,
with a small contribution of digalactosyl chains. In 50% and 100%
DO, there was a significant reduction in the amount of agalactosyl
chains and a corresponding increase in the amount of monogalactosyl
and digalactosyl chains, principally the latter. The effect of DO on
galactosylation is comparatively less pronounced in the NBS
bioreactor, particularly the shift between the relative amounts of
agalactosyl and digalactosyl chains in 10% and 50% DO.
(Kunkel 464, col. 1).
7. Kunkel teaches “an obvious shift in galactosylation of the core-
fucosylated asialo biantennary chains from agalactosyl to digalactosyl as the
DO concentration was increased from 10% to 50% to 100%” (Kunkel 468,
col. 1).
Principles of Law
Whether the rejection is based on “inherency” under 35 U.S.C. § 102,
on “prima facie obviousness” under 35 U.S.C. § 103, jointly or
alternatively, the burden of proof is the same, and its fairness is
evidenced by the PTO’s inability to manufacture products or to obtain
and compare prior art products.
In re Best, 562 F.2d 1252, 1255 (CCPA 1977).
Analysis
We begin with claim interpretation because before a claim is properly
interpreted, its scope cannot be compared to the prior art. We address two
7
Appeal 2016-002460
Application 13/946,759
specific limitations in claim 1, “culturing a cell containing a nucleic acid
encoding at least a portion of the biological product” and “conditions that
modulate a stressed phenotype.” We first look to the Specification to
interpret these phrases, but recognize that “during patent prosecution when
claims can be amended, ambiguities should be recognized, scope and
breadth of language explored, and clarification imposed.” In re Zletz, 893
F.2d 319, 322 (Fed. Cir. 1989).
“culturing a cell ”
Regarding the “culturing a cell” limitation, the Specification teaches
the “biological material may or may not be a natural product of the cell or
organism, so long as the product or material is produced by the cell or
organism” (Spec. 119). The Specification also teaches “a biological product
that is produced by a cell or organism and subsequently modified or altered
in one or more ways (e.g. glycosylation, radiolabeling, and the like) is still
considered a biological product” (Id.).
Therefore, the broadest reasonable interpretation, consistent with the
Specification, of the “culturing a cell containing a nucleic acid encoding at
least a portion of the biological product” limitation encompasses the
situation of an antibody produced by a cell using the natural nucleic acid
already present in the cell where the antibody product is modified by
glycosylation.
“conditions that modulate a stressed phenotype ”
Regarding the “stressed phenotype” limitation, the Specification
teaches the “‘environment’ refers broadly to the overall set of conditions in
which the cell or organism is present, including temperature, light, energy
8
Appeal 2016-002460
Application 13/946,759
source availability, etc. As such, environmental modulation can be
accomplished in a variety of different ways” {Id. 133).
Therefore, the broadest reasonable interpretation, consistent with the
Specification, of the “conditions that modulate a stressed phenotype”
limitation encompasses environmental changes to culture conditions for
cultured cells, including changes in the amount of dissolved oxygen
available to the cells.
Prima Facie Anticipation
Kunkel teaches a method for producing a biological product from a
cell, specifically an antibody from a mouse cell (FF 2, 5). Kunkel teaches
culturing the mouse cells that contain a nucleic acid encoding antibodies (FF
2, 4) under three different conditions, 10%, 50%, and 100% dissolved
oxygen (FF 4). The different dissolved oxygen concentrations represent
conditions that differentially modulate stress on the cells consistent with our
claim interpretation above, and Kunkel teaches that the recovered biological
product (FF 5) differs in glycosylation patterns between the three different
conditions (FF 6—7).
Therefore, Kunkel anticipates claim 1.
With regard to claims 4 and 6, Kunkel teaches altering the
environmental condition of dissolved oxygen concentration to produce
unstressed phenotypes in the cell (FF 4).
With regard to claim 11, Kunkel performs a proteomic assessment by
analyzing the antibody glycosylation of the cell (FF 6—7).
With regard to claim 12, Kunkel teaches the use of mouse cells, which
are mammalian (FF 2).
9
Appeal 2016-002460
Application 13/946,759
With regard to claims 25 and 27, Kunkel teaches an antibody
biological product that comprises a peptide chain (FF 4—5).
SUMMARY
In summary, we reverse the rejection of claims 14—17 and 20 under 35
U.S.C. § 112, second paragraph as indefinite.
We reverse the rejection of claims 1—8, 10-12, 14—17, 20, 25, 27, and
53—55 under 35 U.S.C. § 102(b) as anticipated by Galbraith as evidenced by
Hamsters.
We reject claims 1, 4, 6, 11, 12, 25, and 27 under 35 U.S.C. § 102(b)
as anticipated by Kunkel.
This decision contains a new ground of rejection pursuant to 37
C.F.R. § 41.50(b). Section 41.50(b) provides “[a] new ground of rejection
pursuant to this paragraph shall not be considered final for judicial review.”
Section 41.50(b) also provides:
When the Board enters such a non-final decision, the appellant,
within two months from the date of the decision, must exercise one of
the following two options with respect to the new ground of rejection
to avoid termination of the appeal as to the rejected claims:
(1) Reopen prosecution. Submit an appropriate amendment of
the claims so rejected or new Evidence relating to the claims so
rejected, or both, and have the matter reconsidered by the examiner, in
which event the prosecution will be remanded to the examiner. The
new ground of rejection is binding upon the examiner unless an
amendment or new Evidence not previously of Record is made which,
in the opinion of the examiner, overcomes the new ground of rejection
designated in the decision. Should the examiner reject the claims,
appellant may again appeal to the Board pursuant to this subpart.
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Appeal 2016-002460
Application 13/946,759
(2) Request rehearing. Request that the proceeding be reheard
under § 41.52 by the Board upon the same Record. The request for
rehearing must address any new ground of rejection and state with
particularity the points believed to have been misapprehended or
overlooked in entering the new ground of rejection and also state all
other grounds upon which rehearing is sought.
Further guidance on responding to a new ground of rejection can be
found in the Manual of Patent Examining Procedure § 1214.01.
REVERSED; 37 C.F.R, $ 41.50(b)
11
Notice of References Cited
Application/Control No.
13/946,759
Applicant(s)/Patent Under
Reexamination
Examiner Art Unit
1656
Page 1 of 1
U.S. PATENT DOCUMENTS
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NON-PATENT DOCUMENTS
* Include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages)
U Kunkel et al., Comparisons of the Glycosylation of a Monoclonal Antibody Produced under Nominally Identical Cell Culture
Conditions in Two Different Bioreactors, 16 Biotechnol. Prog. 462-470 (2000).
V
w
X
*A copy of this reference is not being furnished with this Office action. (See MPEP § 707.05(a).)
Dates in MM-YYYY format are publication dates. Classifications may be US or foreign.
U.S. Patent and Trademark Office
PTO-892 (Rev. 01-2001) Notice of References Cited Part of Paper No.
462 Biotechnol. Prog. 2000, 16, 462-470
Comparisons of the Glycosylation of a Monoclonal Antibody
Produced under Nominally Identical Cell Culture Conditions in Two
Different Bioreactors
Jeremy P. Kunkel,' David C. H. Jan,1,5 Michael Butler,1 and James C. Jamieson* ̂
Departments of Chemistry and Microbiology, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada
The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free
continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%,
and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific
(NBS) CelliGen bioreactors. All culture parameters were monitored and controlled
and were nominally identical at steady state in the two bioreactors. The secreted
monoclonal antibody (mAb), an immunoglobulin Gi, was purified and subjected to
enzymatic deglycosylation using peptide Al-glycosidase F (PNGase F). Asparagine-
linked (N-linked) oligosaccharide pools released from mAb samples cultured in each
bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange
chromatography with pulsed amperometric detection (HPAEC—PAD). The predomi­
nant N-linked structures were core-fucosylated asialo biantennary chains with varying
galactosylation. There were also minor amounts of monosialyl oligosaccharides and
trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation
of this mAb. A definite reduction in the level of galactosylation of N-glycan chains
was observed at lower DO in both bioreactors, as evidenced by prominent increases
in the relative amounts of agalactosyl chains and decreases in the relative amounts
of digalactosyl chains—with the relative amounts of monogalactosyl chains being
comparatively constant. However, the quantitative results are not precise matches
between the two bioreactors. The effect of DO on galactosylation is less pronounced
in the NBS bioreactor than in the LH bioreactor, particularly the shift between the
relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. There
are also perceptibly higher levels of sialylation of the mAb glycans in the NBS
bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate
that the DO effect is not bioreactor specific and that nominally identical steady-state
conditions in different chemostat bioreactors may still lead to some incongruities in
glycosylation, possibly due to the particular architectures of the bioreactors and the
design of their respective monitoring and control systems. The observed differences
in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and
NBS bioreactors may be explained by the differences in oxygen supply and control
strategies between the two bioreactors.
Introduction
Immunoglobulin G (IgG) contains 2.3 asparagine-
linked (N-linked) biantennary oligosaccharide chains per
molecule in mice (Mizuochi et al., 1987) and 2.8 in
humans (Parekh et al., 1985). Two of these represent the
conserved glycosylation sites at Asn-297 in each of the
two heavy chain Ch2 domains of the Fc portion, and the
remainder are found in the hypervariable regions of the
Fab section with a frequency and position dependent on
the chance occurrence of the N-glycosylation consensus
sequence (Asn-Xaa-Thr/Ser). A general schematic of the
IgG molecule is illustrated in Figure la. About 30
variants of biantennary chains occur, resulting in many
* Tel: (204) 474-9253. Fax: (204) 474-7608. E-mail: jamies®
ms.umanitoba.ca.
1 Department of Chemistry.
* Department of Microbiology.
§ Present address: Covance Biotechnology Services Inc., Re­
search Triangle Park, Cary, NC 27513.
different glycoforms of IgG (Parekh et al., 1985). Figure
lb represents a composite of the most common IgG
N-linked structures. The majority of these oligosaccha­
rides are core-fucosylated in both humans and mice
(Parekh et al., 1985; Mizuochi et al., 1987). Normally,
Fc glycans are core-fucosylated and varyingly galactosy-
lated with none, one, or two terminal galactose residues.
Those glycans containing at least one galactose may also
be sialylated, but sialylation is restricted in Fc glycans
as a result of their internal sequestering between the two
Ch2 domains. Fc oligosaccharides therefore have a low
incidence of monosialylation and no disialylation. Fab
glycans are similar to Fc glycans but are more completely
galactosylated and characterized by a higher incidence
of monosialylated and disialylated structures. Bisecting
N-acetylglucosamine in murine IgG is uncommon (Mi­
zuochi et al., 1987). In humans, Fc glycans have a low
incidence of bisecting N-acetylglucosamine, while those
of the Fab have a high incidence (Wormald et al., 1997).
10.1021/bp000026u CCC: $19.00 © 2000 American Chemical Society and American Institute of Chemical Engineers
Published on Web 05/10/2000
Biotechnol. Prog., 2000, Vol. 16, No. 3 463
a b
±SA* ±SA*
| (a2-3/6) | (a2-3/<
±Gal ±Gal
[(Pl-4) |(PI-4)
GlcNAc GlcNAc
|(PI-2) | (Pl-2)
Man ±GlcNAc
|(Pl-4)
Man
(al-3)^^ Man ^
|(Pl-4)
GlcNAc
|(Pl-4)
(al-6)
GlcNAc — ±Fuc
Asn
*SA = Neu5Ac, Neu5Gc
Figure 1. (a) Schematic representation of IgG showing the domain structure, intra- and interchain disulfide bonds, and glycosylation
sites. The conserved N-linked oligosaccharides are attached to Asn-297 in the Ch2 domains of the Fc and are generally asymmetric
to establish the required bridge between the two opposing Ch2 domains (Rademacher et al., 1996). Additional N-linked sites may
occur in the Fab hypervariable regions (shaded). While the diagram shows murine IgGi, human IgGi is very similar but possesses
only two inter-heavy-chain disulfide bonds in the hinge region (Burton, 1987; Putnam, 1987). (b) Composite structure of the N-linked
oligosaccharides most typical of human and murine IgG (Parekh et al., 1985; Mizuochi et al., 1987). The terminal sialic acids, while
infrequent in Fc glycans, are common in Fab glycans. GlcNAc, Af-acetylglucosamine; Fuc, fucose; Man, mannose; Gal, galactose; SA,
sialic acid; Neu5Ac, 5(/V)-acetylneuraminic acid; Neu5Gc, 5(/V)-glycolylneuraminic acid. (Reprinted with permission from Elsevier
Science.)
The Fc glycans of IgG are essential to the structural
integrity of the antibody (Matsuda et al., 1990; Malhotra
et al., 1995; Wormald et al., 1997). Alterations of these
oligosaccharides have been reported to affect susceptibil­
ity to proteolytic degradation, clearance rate, Fc receptor
binding, antibody-dependent cellular cytotoxicity (ADCC),
monocyte binding, protein G binding, and Clq component
binding/C 1 whole complement activation (Bond et al.,
1993; Boyd et al., 1995; Jefferis et al., 1995; Lund et al.,
1996; Wright and Morrison, 1997). Interestingly, protein
A binding is not affected by Fc glycosylation (Leather-
barrow and Dwek, 1983). Fab N-linked glycosylation in
the hypervariable regions, while occurring much less
frequently, has been reported to influence the binding
affinity of antigens (Wright and Morrison, 1993; Endo
et al., 1995; Leibiger et al., 1999). The consequences of
these factors are critical for the development and produc­
tion of IgG for diagnostic and therapeutic use. The
oligosaccharide structures of glycoprotein biopharmaceu­
ticals and the position of their relative glycosylation sites
need to be determined not only to understand funda­
mental structure—function relationships but also to
ensure consistent production quality and possibly for
regulatory and patent protection purposes (Jenkins et al.,
1996).
Factors that are known to affect N-linked glycosylation
are the availability of the various processing enzymes and
their kinetic characteristics and compartmentalization
within the endoplasmic reticulum and Golgi complex. In
turn, these factors are all dependent on the age, health,
species, and type of cell. In addition, because of clonal
variation in glycosylation capabilities, each cell within a
given population of like cells may express a subset of
potential structures (Rothman et al., 1989; Lifely et al.,
1995; Routier et al., 1997). The protein milieu near a
potential glycosylation site can affect the extent of
oligosaccharide processing and lead to site-specific gly­
cosylation (Yet et al., 1988). This has been shown to be
the case for IgG (Savvidou et al., 1984; Lee et al., 1990;
Lund et al., 1996). The shielding of Fc glycans between
the two Ch2 domains may limit their accessibility to
processing enzymes, resulting in the distinctions between
Fc and Fab glycosylation. Peptide, disulfide, and glycosyl
bond formation involved in the biosynthesis of IgG are
not discrete events but are temporally intertwined and
influence the level of IgG Fc-oligosaccharide galactosy-
lation (Rademacher et al., 1995, 1996). The physicochem­
ical environment of a cell also affects glycosylation
(Goochee and Monica, 1990; Goochee et al., 1991; Ander­
sen and Goochee, 1994; Gawlitzek et al., 1995). It is the
dynamic interplay of various genetic, biological, and
environmental factors that accounts for cell-dependent,
site-dependent, and physiologically influenced glycosy­
lation.
Our earlier work described the effect of dissolved
oxygen (DO) concentration on the growth and monoclonal
antibody (mAh) production of a murine hybridoma cell
line at steady state in serum-free continuous culture in
DO concentrations between 10% and 150% of air satura­
tion (Jan et al., 1997). It was found that increases in DO
concentration caused changes in energy metabolism, with
a higher proportion of glucose utilized at higher oxygen
concentrations. This increase in specific glucose utiliza­
tion at higher DO could be explained entirely by an
increase in anaerobic metabolism. The flux of glucose
through the glycolysis and pentose phosphate pathways
increased considerably, whereas flux through the tricar­
boxylic acid cycle decreased substantially at high DO. The
degree of aerobic metabolism in these cultures was highly
sensitive to the glucose concentration. The cultures did
464 Biotechnol. Prog., 2000, Vol. 16, No. 3
not appear to be limited by glucose, although it was not
possible to establish conclusively a single limiting sub­
strate. There was also a small but significant increase
in glutamine utilization at higher DO. The specific mAb
production rate was relatively constant from 10% to 100%
DO but rose sharply in 125%. This was in contrast to
the cell growth rate, which was relatively constant from
10% to 100% DO but dropped in 125%. As in another
report (Miller et al., 1987), this suggested a differential
effect of DO on the growth and specific mAb production
rates. Increases in DO were associated with the induction
of antioxidant enzymes, which serve to reduce the cyto­
toxic effect of reactive oxygen species. Other prior work
established a link between steady-state DO concentration
and the structure of the N-linked oligosaccharide chains
of the mAb produced by the hybridoma cells in an LH
Series 210 (LH) bioreactor (Kunkel et al., 1998). The level
of galactosylation of the N-glycans decreased as the DO
setpoint was lowered. There was no evidence for threo­
nine- or serine-linked (O-linked) glycosylation of the mAb,
and all N-linked glycans were associated with the heavy
chains, presumably in the Fc at Asn-297 (Barnabe and
Butler, 1998; Kunkel et al., 1998).
We have now examined the effect of varying DO
concentration in an New Brunswick Scientific (NBS)
CelliGen bioreactor under nominally identical cell culture
conditions. As in the LH bioreactor, the main effect of
DO concentration on the mAb produced by the CC9C10
hybridoma cell line in the NBS bioreactor was a shift in
the level of galactosylation of the chains. In 10% DO, the
chains were mainly agalactosyl or monogalactosyl, with
a small contribution of digalactosyl chains. In 50% and
100% DO, there was a significant reduction in the
amount of agalactosyl chains and a corresponding in­
crease in the amount of monogalactosyl and digalactosyl
chains, principally the latter. The effect of DO on galac­
tosylation is comparatively less pronounced in the NBS
bioreactor, particularly the shift between the relative
amounts of agalactosyl and digalactosyl chains in 10%
and 50% DO.
Materials and Methods
Cell Line and Cell Culture. The murine-murine
B-lymphocyte hybridoma cell line, CC9C10, was obtained
from the American Type Culture Collection (ATCC HB-
123). The cell line is derived from the Sp2/0 myeloma and
secretes a mAb of class IgGi,. The cells were shown to
be mycoplasma-free by routine testing in an independent
laboratory (Rh Pharmaceuticals, Winnipeg, MB, Canada).
The cells were adapted for growth in serum-free
medium based on a 1:1 volume ratio of Dulbecco’s
Modified Eagle’s Medium (D-MEM) and Ham’s F-12
media (Gibco, Grand Island, NY) containing 17.5 mM
glucose, 6 mM glutamine, and supplements (Sigma, St.
Louis, MO). Continuous cultures at each of three DO
setpoints were carried out at a working volume of 1.0—
1.5 L, a dilution rate of 1 vol day-1, a temperature of 37
°C, a pH of 7.1, and an agitation rate of 100 rpm in both
an LH Series 210 bioreactor (Inceltech, Toulouse, France)
and in an NBS CelliGen bioreactor (New Brunswick
Scientific, Edison, NJ). Chemostat cultures were estab­
lished with DO concentrations of 10%, 50%, and 100% of
air saturation. Cell culture parameters in the two biore­
actors at each of the three DO setpoints were therefore
nominally identical. DO concentration was monitored
using polarographic DO sensors (Ingold Electrodes, Wilm­
ington, MA) and controlled by the respective bioreactor
controller. Eluted cell culture mixture was collected
following the establishment of steady-state conditions,
which was assumed after at least five volume changes
with constant viable cell concentration and nutrient
levels over 3 days.
Monoclonal Antibody Purification. Monoclonal
antibody was prepared once from each of the three DO
concentration cultures (10%, 50%, and 100%) from each
of the two bioreactors. Approximately 1.0 L of eluted cell
culture medium, containing about 40—50 mg mL-1 of
secreted mAb, was collected and centrifuged at 10 000
x g for 15 min to remove cells. The mAb was then
purified from the supernatant by protein A-affinity
chromatography with an alternate buffer system as
described elsewhere (Kunkel et al., 1998). Protein A was
chosen over protein G because protein G may select for
certain oligosaccharide profiles (Bond et al., 1993), which
does not seem to be the case for protein A (Leatherbarrow
and Dwek, 1983; Boyd et al., 1995).
Monoclonal antibody preparations, approximately 40—
50 mg, from each of the two bioreactors at each of the
three DO setpoints, were dialyzed extensively against
distilled—deionized water in 12 000 D MWCO cellulose
dialysis tubing (Sigma). Solubility problems were not
encountered. The dialyzates were then filtered through
0.2-«m nylon 25-mm syringe filters (Nalge Nunc, Roch­
ester, NY), divided into 5-mg aliquots for glycosylation
analysis, and lyophilized.
Deglycosylation/PNGase F Digestion. Samples of
each mAb preparation were enzymatically deglycosylated
using the recombinant amidohydrolase peptide N-gly-
cosidase F (PNGase F) (Tarentino et al., 1985) in the
supplied buffer (Oxford GlycoSciences, Abingdon, U.K.).
Briefly, 5-mg samples of each mAb were reconstituted
in 250 fiL of 20 mM sodium phosphate buffer pH 7.5
containing 50 mM EDTA and 0.02% sodium azide (w/v)
in 2.0-mL polypropylene microcentrifuge tubes (VWR,
West Chester, PA) to which was added 5 U (5 IUBMB
mU) of PNGase F. These mixtures, with an enzyme
concentration of 20 U mL-1 and an enzyme to mAb ratio
of 1 U mg-1, were incubated at 37 °C for 24 h. No attempt
was made to first denature the antibody before the
addition of enzyme. After incubation, the enzyme digests
were cooled on ice, and three volumes (750 fiL) of ice-
cold absolute ethanol were added. After 10 min on ice,
the tubes were centrifuged at 12 000 x g for 10 min. The
supernatants were set aside, and the remaining pro­
teinaceous pellets were washed with 250 fiL of distilled—
deionized water. A second precipitation with three vol­
umes of ethanol, cooling, and centrifugation followed.
Finally, the pooled N-linked oligosaccharide-containing
supernatants (approximately 2.0 mL total) for each mAb
sample were dialyzed extensively against distilled—
deionized water in Spectra/Por 500 MWCO cellulose ester
dialysis tubing (Spectrum, Laguna Hills, CA), filtered
through 0.2-Min nylon 25-mm syringe filters, and lyo-
philized. Deglycosylation of this mAb by this PNGase F
protocol and by hydrazinolysis (a nonspecific chemical
method) yield essentially identical oligosaccharide pro­
files in fluorophore-assisted carbohydrate electrophoresis
(FACE) and high-pH anion-exchange chromatography
with pulsed amperometric detection (HPAEC—PAD)
(Kunkel et al., 1998).
Glycan Analysis/HPAEC—PAD. Oligosaccharides
released by PNGase F digestion from multiple samples
of each of the mAb preparations were analyzed by
HPAEC—PAD (Townsend et al., 1988). Glycans were
separated using the DX-300 system (Dionex, Sunnyvale,
CA) equipped with 4 mm x 50 mm guard and 4 mm x
250 mm analytical CarboPac PA100 columns (Dionex) at
a flow rate of 1 mL min-1 at ambient temperature.
Biotechnol. Prog., 2000, Vol. 16, No. 3 465
a
70
nA
201
10% DO
10 15 20 25 30 35 40 45 50 55 60 65 70
Minutes
10% DO
10 15 20 25 30 35 40 45 50 55 60 65 70
Minutes
70T
riA_
20L
50% DO
4a 4b
10 15 20 25 30 35 40 45 50 55 60 65 70
Minutes
70
nA
20L
100% DO
l \ 7 U
7a
nA
20L
Minutes
100% DO
10 15 20 25 30 35 40 45 50 55 60 65 70
Minutes
10 15 20 25 30 35 40 45 50 55 60 65 70
Minutes
Figure 2. HPAEC—PAD chromatograms of the PNGase F-released N-linked oligosaccharides from 150 fig of the mAh produced in
10%, 50%, and 100% DO serum-free continuous culture in two different bioreactors, (a) LH Series 210 bioreactor, (b) New Brunswick
Scientific CelliGen bioreactor. See Figure 3 for structures of identified peaks. (Figure 2a reprinted with permission from Elsevier
Science.)
Electrochemical detection was by the PAD-2 (Dionex)
using the basic PAD cell (Dionex) with a 1.4-mm gold
working electrode and a 0.005-in. cell gasket at 300 mA
full-scale. The triple-pulse PAD sequence was set at 0.05
V for 480 ms, 0.65 V for 180 ms, and —0.30 V for 120 ms
with a response time of 1 s and a 0.20-s sampling rate.
The elution scheme employed was as follows: isocratic
at 0.10 M NaOH for 0.5 min, a linear gradient to 0.04 M
sodium acetate in 0.10 M NaOH in 49.5 min, a linear
gradient to 0.20 M sodium acetate in 0.10 M NaOH in
30 min, a 15-min hold at these conditions to wash the
columns, a return to initial conditions by a linear
gradient to 0.10 M NaOH in 5 min, and finally, reequili­
bration at the initial conditions for 15 min. Postcolumn
predetector addition of NaOH was not exercised. Eluants
were prepared with NANOpure II distilled—deionized
water (Barnstead Thermolyne, Chicago, IL) with a
background resistivity of not less than 17.0 MQ, 50%
NaOH solution (Mallinckrodt, Paris, KY), and sodium
acetate trihydrate (Mallinckrodt). Eluants were filtered
through 0.2-«m nylon 47-mm filters (Nalge Nunc) prior
to use.
N-Linked glycans (approximately 2 nmol) from 150 fig
of mAh were injected automatically from a 50-fiL sample
loop. Retention times of eluting oligosaccharide peaks
were compared to the following five N-linked oligosac­
charide standards: core-fucosylated disialo digalacto
biantennary, core-fucosylated asialo digalacto bianten-
nary, asialo digalacto biantennary, core-fucosylated asialo
agalacto biantennary, and asialo agalacto biantennary
(Oxford GlycoSciences). Chromatographic data analysis
and integration were accomplished using the AI-450
chromatography automation software interface (Dionex).
Results
The N-linked oligosaccharides released enzymatically
from the mAh using PNGase F were separated and
detected by HPAEC—PAD. The HPAEC—PAD chromato­
grams indicate that all mAh N-linked oligosaccharide
preparations gave three main peaks of varying relative
areas eluting at approximately 18.5, 21, and 23.5 min
(Figure 2a and b). These peaks were identified by the
use of the five N-glycan standards to correspond to
structures 1—3 in Figure 3. The trace peaks eluting after
peak 3 in the range of 25—35 min may be structures
resulting from the presence, however unlikely in murine-
derived cells, of a bisecting N-acetylglucosamine in one
or more of structures 1—3. Because of the comparative
paucity of these peaks, their ultimate identification was
not pursued.
There were also three groups of minor peaks eluting
at about 47—51, 53—57, and 62—66 min. These peaks owe
their delayed elution to their relative acidity due to
monosialylation of the core-fucosylated biantennary chains.
Two of these three groups of peaks were a consequence
of their different sialic acid content: either N-acetyl-
neuraminic acid (Neu5Ac, 47—51 min) or N-glycolyl-
neuraminic acid (Neu5Gc, 62—66 min) in a2,6-linkages
(and perhaps a2,3-linkages) to either one or the other,
but not both, of the antennae of the core-fucosylated
biantennary structures (structures 4a and 4b, Figure 3).
These variations in monosialylation could yield up to 4
different monosialylated structures for each of the two
sialic acids, with minor differences in retention times in
this HPAEC gradient. The third group of peaks at 53—
57 min could result from variants of structure 4a
containing a bisecting N-acetylglucosamine. It is also
possible that a minor proportion of the peaks eluting at
53—57 min was due to coelution of core-fucosylated
disialo digalacto biantennary chains (structure 4a, but
disialylated with Neu5Ac). Again, because these peaks
represented only a small measure of the total oligosac­
charide content, their definite assignment was not en­
deavored.
466 Biotechnol. Prog., 2000, Vol. 16, No. 3
GlcNAc(pi-2)Man(al-6)\ ̂ Fuc(al-6)^
Man(pi-4)GlcNAc(pi-4)GlcNAc-
GlcNAc(pl-2)Man(al-3)///{
Gal(pl-4)GlcNAc(pl-2)Man(al-6)s^ Fuclal^)^
Man(pi-4)GlcNAc(pi-4)GlcNAc-
Gal(pi-4)GlcNAc(pi-2)Man(al-3)//^
4a Gal(pi-4)GlcNAc(pl-2)Man(al-6)\
Neu5Ac(a2-3/6) {
Gal(pi-4)GlcNAc(pi-2)Man(al-3)/
Fuc(al-6)\^
Man(pi-4)GlcNAc(pl-4)GlcNAc —
Gal(pi-4)GlcNAc(pi-2)Man(al-6)v Fuc(al-6)v
/ \ \
Neu5Gc(a2-3/6) < Man(pi-4)GlcNAc(pi-4)GlcNAc —
»■ /
Gal(pi-4)GlcNAc(pl-2)Man(al-3)'
Figure 3. Structures for N-linked oligosaccharides removed from the IgG preparations. The structure numbering corresponds to
the peak numbering in HPAEC—PAD chromatograms (Figure 2) and the corresponding table (Table 1) and bar graph (Figure 4).
Structures 1 and 3 were determined by the use of standards; structure 2 by inference; structures 4a and 4b by comparison with
Weitzhandler et al. (1994). Stucture 1, core-fucosylated asialo agalacto biantennary; structure 2, core-fucosylated asialo monogalacto
biantennary; structure 3, core-fucosylated asialo digalacto biantennary; structure 4a, core-fucosylated monosialo (Neu5Ac) digalacto
biantennary; structure 4b, core-fucosylated monosialo (Neu5Gc) digalacto biantennary. (Reprinted with permission from Elsevier
Science.)
The HPAEC—PAD analyses of N-linked oligosaccha­
rides enzymatically removed from multiple samples of
monoclonal IgGi preparations from 10%, 50%, and 100%
DO cultures in both LH and NBS bioreactors presented
a conspicuous trend. In both the LH (Figure 2a) and NBS
(Figure 2b) bioreactors, only two prominent peaks were
obtained in 10% DO, corresponding to the core-fucosy­
lated asialo agalacto biantennary chain (peak 1, Figure
2a and 2b; structure 1, Figure 3) and the core-fucosylated
asialo monogalacto biantennary chain (peak 2, Figure 2 a
and b; structure 2, Figure 3). Peak 3, the core-fucosylated
asialo digalacto biantennary chain, was less pronounced.
The oligosaccharides from mAh produced at 50% and
100% DO in both bioreactors showed a shift toward
increased amounts of the core-fucosylated asialo digalacto
biantennary chain (peak 3, Figure 2a and 2b; structure
3, Figure 3) and a reduction in the amount of the core-
fucosylated asialo agalacto biantennary chain (peak 1).
Most of the increase in peak 3 appears to be at the
expense of peak 1, as peak 2 appears largely unaffected.
The results also show that the small amount of sialyla-
tion increased with the increase in galactosylation. This
is reasonable since sialic acid attaches to galactose in
these N-glycans. There were marginally higher levels of
sialylation of the mAh glycans from the NBS bioreactor
than from the LH bioreactor at all three DO setpoints,
but particularly in 10% DO.
The total detector response (sum of all peak areas) from
each chromatogram was approximately equal. Thus, the
level of glycosylation of the mAh (glycosylation site
occupancy rate) in either bioreactor at each DO setpoint
was relatively consistent. Note that peak areas from PAD
integration are not directly proportional to the relative
amounts of each oligosaccharide, since it is well estab­
lished that the PAD response varies from glycan to glycan
(Lee, 1990). However, if glycans of similar size and
composition are considered, as they are here, it is
reasonable to postulate that the peak areas approximate
their relative quantity (Townsend et al., 1988).
The relative peak areas of these three neutral oligosac­
charides, presented in Table 1, were calculated by
comparing peaks 1—3 shown in Figure 2a and b. As is
indicated by the standard deviation in relative peak
areas, repeated PNGase F digestions of different samples
of the same mAh preparations on different dates and
their analyses by HPAEC—PAD were remarkably con­
sistent (n = 6 for the LH preparation and n = 5 for the
NBS preparation). There is extremely close agreement
in the quantitation of each of the three main peaks of
each mAh preparation produced in 10%, 50%, and 100%
DO culture in each bioreactor. The general trend ob­
served in the LH and NBS bioreactors of decreased
galactosylation of mAh N-linked oligosaccharides with
reduced DO is most easily appreciated by inspection of a
Biotechnol. Prog., 2000, Vol. 16, No. 3 467
60
Peak 1 Peak 1 Peak 2 Peak 2 Peak 3 Peak 3
LH NBS LH NBS LH NBS
Figure 4. Bar graph comparison of the relative peak areas from HPAEC—PAD of the three major neutral N-linked oligosaccharides
from the mAh produced in 10%, 50%, and 100% DO serum-free continuous culture in two different bioreactors. See Figure 3 for
structures of identified peaks. LH, LH Series 210 bioreactor; NBS, New Brunswick Scientific CelliGen bioreactor. See Table 1 for
further discussion of data and errors.
bar graph of the relative peak area data (Figure 4).
However, while the tendency for decreased galactosyla-
tion with lower DO is common to both LH and NBS
bioreactors, there are quantitative differences between
the two bioreactors. The effect of DO on galactosylation
is less pronounced in the NBS bioreactor than in the LH
bioreactor, particularly the shift between the amounts
of peaks 1 and 3 in 10% and 50% DO.
Discussion
Serum IgG from patients with rheumatoid arthritis
and a small number of other rheumatological disorders
contains the same set of N-linked biantennary oligosac­
charides found in normal individuals, although in very
different and characteristic proportions (Parekh et al.,
1985, 1989). The incidence of structures lacking galactose
is dramatically increased. Interestingly, in women with
rheumatoid arthritis and elevated levels of agalactosyl
glycoforms, decreases in agalactosyl glycoforms are cor­
related with remission of the disease during gestation
followed by postpartum recurrence (Rook et al., 1991).
The glycoform distribution of serum IgG has also been
shown to change with age (Parekh et al., 1988; Yamada
et al., 1997).
Since IgG glycosylation is responsive to normal physi­
ological changes and certain disease states, it is not
surprising that different cell culture systems and condi­
tions may affect the glycosylation of mAb’s and other
recombinant proteins (Goochee and Monica, 1990; Goochee
et al., 1991; Andersen and Goochee, 1994). For example,
a monoclonal IgGi produced from murine cells in fed-
batch culture contained more high-mannose and trun­
cated complex glycans with increasing age of the culture
(Robinson et al., 1994). Monoclonal antibodies are gly­
cosylated differently when produced in ascites or by a
variety of cell culture techniques with serum-free and
serum-supplemented media (Patel et al., 1992; Maiorella
et al., 1993; Monica et al., 1993). The Fc N-glycans of
Table 1. Relative Peak Areas of Major Neutral
Oligosaccharides: Multiple Analyses of mAb
Glycosylation from Different CC9C10 Cultures'1
%DO
peak 10 50 100
LH Series 210 BioreactoP
1 45.1 ± 0.1 21.1 ± 0.2 17.6 ± 0.4
2 46.1 ± 0.1 54.0 ± 0.1 52.6 ± 0.2
3 8.8 ± 0.2 24.9 ± 0.2 29.8 ± 0.5
NBS CelliGen Bioreactor
1 32.4 ± 0.2 25.6 ± 0.1 20.6 ± 0.1
2 52.9 ± 0.1 54.9 ± 0.3 56.2 ± 0.2
3 14.7 ± 0.2 19.5 ± 0.2 23.2 ± 0.1
° Data are presented as the average relative peak areas (%) and
standard deviation with re — 1 degrees of freedom. There were re
= 6 replicate analyses for each mAb preparation from the LH
Series 210 bioreactor and re = 5 replicate analyses for each mAb
preparation from the NBS CelliGen bioreactor. Replicate analyses
were performed over a 6-month period on multiple samples from
each mAb preparation (which were prepared once from each of
the three DO concentration cultures from each bioreactor). b Data
for the LH Series 210 bioreactor are as reported in Kunkel et al.
(1998) (reprinted with permission from Elsevier Science).
murine monoclonal IgG2b and various chimeric murine-
human antibodies expressed in a murine cell line were
more highly galactosylated in static batch cultures than
in hollow fiber bioreactors or ascites (Lund et al., 1993).
Various human monoclonal IgG antibodies, produced by
virally transformed B-cell lines in serum-free media, were
more highly galactosylated in low-density batch culture
than in high-density hollow fiber bioreactors (Kumpel et
al., 1994). Similarly, the heavy chain glycosylation of a
murine mAb exhibited marked decreases in complexity
as culture intensity was increased from continuous
stirred tank bioreactor to fluidized bed bioreactor to
hollow fiber bioreactor (Schweikart et al., 1999). Changes
in the biological activities of mAb’s as a consequence of
altered glycosylation have been observed when they are
468 Biotechnol. Prog., 2000, Vol. 16, No. 3
expressed in different cell lines (Lifely et al., 1995) and
under different culture conditions (Maiorella et al., 1993)
Mammalian cell cultures in controlled bioreactors are
typically supplied with 20% to 50% DO to maintain
optimal growth, although some cell lines may be adapted
to grow at much lower (Miller et al., 1987) and much
higher (van der Valk et al., 1985; Oiler et al., 1989) DO
concentrations. The effects of DO on the glycosylation of
some recombinant proteins have been previously studied.
For instance, in the production of human follicle stimu­
lating hormone from CHO cells in perfusion culture, the
DO concentration was varied from 10% to 90% (Chotigeat
et al., 1994). Sialyltransferase activity, sialic acid content,
and specific productivity all increased with greater DO.
By contrast, there was little change in the N-glycosylation
of tissue plasminogen activator from CHO cells in per­
fusion culture under normal, mildly hypoxic, severely
hypoxic, and anoxic conditions (Lin et al., 1993).
The murine hybridoma, CC9C10, chosen for this study
has been grown routinely with a relatively high mAb
productivity in a simple serum-free formulation. Serum-
free continuous cultures at steady state are a useful
means of studying the metabolism of cells because they
are held in an equilibrium under constant and defined
physiological conditions, where no variations in culture
parameters or cell metabolism occur. The effect of a single
parameter, such as the DO concentration, can be dis­
tinctly studied by the perturbation and reestablishment
of steady state. Our previous work with continuous
cultures of this hybridoma showed that steady-state
viable cell concentration, cell viability, and the specific
rates of mAb production and oxygen utilization did not
significantly change in DO concentrations of 10%, 50%,
and 100% in either the LH or NBS bioreactor (Jan et al.,
1997). These results are concordant with another report,
which also calculated that the specific ATP production
rate is essentially constant in this DO range (Miller et
al., 1987).
In this report, glycosylation analysis of the monoclonal
IgGi samples by HPAEC—PAD indicate the predominant
N-linked structures were core-fucosylated asialo bian-
tennary chains with varying galactosylation. There were
also minor amounts of monosialyl oligosaccharides and
trace amounts of afucosyl oligosaccharides. Integration
and statistical analysis of the data indicate an obvious
shift in galactosylation of the core-fucosylated asialo
biantennary chains from agalactosyl to digalactosyl as
the DO concentration was increased from 10% to 50% to
100% (Table 1). The level of monogalactosyl chains
remained relatively constant. The effect of DO on galac­
tosylation of the mAb N-glycans appears to be a general
result but is evidently less pronounced in the NBS
bioreactor than in the LH bioreactor (Figure 4). In the
LH bioreactor, the effect of DO on peaks 4a and 4b, which
are sialylated structures, was observed to correspond to
that of peak 3. As the level of peak 3 (and therefore
structure 3) increased, the levels of peaks 4a and 4b
increased (Figure 2a). This is to be expected, since
galactose is required for the attachment of terminal sialic
acid in these oligosaccharides. However, the NBS biore­
actor showed a more constant and slightly greater level
of sialylation of mAb glycans than the LH bioreactor at
all three DO setpoints, especially in 10% DO (Figure 2b).
This indicates that a greater proportion of monogalactosyl
and digalactosyl structures were sialylated in the NBS
bioreactor.
Several possible mechanistic explanations for the effect
of DO on galactosylation of the secreted mAb have been
considered elsewhere (Kunkel et al., 1998) and will not
be discussed further here. However, the observed differ­
ences in glycosylation of the mAb between the LH and
NBS bioreactors at the three DO setpoints may be
explained by differences in the gassing regimes between
the two bioreactors. Temperature, pH, agitation rate,
dilution rate, and DO were monitored and controlled at
specific setpoints and were nominally equivalent at
steady state in the two bioreactors. Identical DO sensors
were used in both the LH and NBS bioreactors, capable
of DO control within ±1% of each DO setpoint (10 ± 1%,
50 ± 1%, and 100 ± 1% DO). Although steady-state
cultures were established at identical DO setpoints in the
two bioreactors, it is possible that distinctions in oxygen
monitoring and control contributed to the observed
differences in mAb glycosylation.
The LH bioreactor provides a base level of culture
aeration by constant headspace gassing. Additionally, the
culture is intermittently sparged with oxygen as required
via a ring sparger. The sparging is directed by an
independent DO module with proportional-integral-dif­
ferential (PID) control activated by the DO sensor. The
NBS bioreactor uses an alternative system for oxygen­
ation based on a combined four-gas interactive pH and
DO controller with proportional-integral (PI) control
activated by pH and DO sensors. Proportionally mea­
sured gases are sparged continuously and sequentially
(air, oxygen, nitrogen, and carbon dioxide) into a stainless
steel mesh aeration cage separated from the bulk culture.
The aeration cage prevents bubble formation in the
culture in an attempt to reduce cell damage. The absence
of serum and low protein content in these cultures
reduced their attributed protective effects and rendered
the cells more susceptible to any potential toxic or stress
effects from the culture media. Hybridomas grown in
serum-free media have shown enhanced sensitivity to DO
levels, resulting in altered metabolic patterns compared
to those grown in serum-based media (Ogawa et al.,
1992).
Carbon dioxide can accumulate in poorly ventilated
cultures, particularly batch and fed-batch cultures, caus­
ing an increase in the partial pressure of C02 (pC02) and
a concomitant increase in osmolality due to an increase
in dissolved HC03-. The increase in osmolality is exac­
erbated by pH control with NaHC03 or NaOH. It has
been shown that an increase in osmolality can affect
glycoprotein glycosylation to varying degrees in a pH-
dependent manner (Kimura and Miller, 1997; Zanghi et
al., 1999). The /jC02 and osmolality were not monitored
in our experiments and, as a result of the differences in
gassing regimes, may have been slightly different at
steady state in the two bioreactors. However, since the
changes in glycosylation that are reported in the litera­
ture are from batch or perfusion cultures over relatively
large variations in /jC02 or pH, the high influx of fresh
media (1 vol day-1) in our chemostat cultures would
largely mitigate their effects. As mentioned earlier, we
observed that viable cell concentration, cell viability, and
the specific rate of mAb production did not significantly
change in DO concentrations of 10%, 50%, and 100% in
either the LH or NBS bioreactor (Jan et al., 1997). These
data support the assertion that /jC02 and osmolality were
not factors in the observed DO and bioreactor effects on
glycosylation, since elevated /jC02 and osmolality are also
known to affect specific cell growth rate and viable cell
density (Kimura and Miller, 1996; deZengotita et al.,
1998) and specific glycoprotein production (Gray et al.,
1996; Kimura and Miller, 1996). Interestingly, specific
antibody production of a hybridoma was not appreciably
Biotechnol. Prog., 2000, Vol. 16, No. 3 469
affected by pC02 and osmolality in the range tested
(deZengotita et al., 1998).
Ammonia has also been reported to affect hybridoma
cell growth (Liideman et al., 1994), antibody production
(McQueen and Bailey, 1990), and glycoprotein glycosy-
lation (Borys et al., 1994; Gawlitzek et al., 1998; Zanghi
et al., 1998). We observed no significant variations in
steady-state ammonia concentration or specific ammonia
production in these cultures (Jan et al., 1997).
Conclusions
The fact that culture conditions can affect the glycos-
ylation of glycoprotein biopharmaceuticals and, conse­
quentially, their immunological functions and other
aspects of their efficacy in vivo has obvious implications
for the development and production of mAb’s for diag­
nostic and therapeutic use. Here we reported that mAb
produced in 10%, 50%, and 100% DO in both the LH and
NBS bioreactors all possessed the same types of bian-
tennary N-glycans. However, the proportions of these
oligosaccharide structures were strongly dependent on
the DO concentration and, to a lesser extent, the produc­
tion bioreactor. There are likely other significant cell
culture parameters in the production of mAb’s and other
clinically useful glycoproteins that require consideration
and investigation to ensure that structural features of
the oligosaccharide chains either correspond to those of
the native molecule or are consistently reproducible.
Acknowledgment
This work was partially supported by grants to M.B.
and J.C.J. from the Natural Sciences and Engineering
Research Council of Canada (NSERC). Figures 1, 2a, and
3 and the data for the LH Series 210 bioreactor in Table
1 are reprinted from Kunkel et al. (1998) with kind
permission from Elsevier Science.
References and Notes
Andersen, D. C.; Goochee, C. F. The effect of cell-culture condi­
tions on the oligosaccharide structures of secreted glycopro­
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Accepted for publication March 20, 2000.
BP000026U