Ex Parte YunDownload PDFPatent Trial and Appeal BoardMar 23, 201713946759 (P.T.A.B. Mar. 23, 2017) Copy Citation 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. 10 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 * Document Number Country Code-Number-Kind Code Date MM-YYYY Name CPC Classification US Classification A us- B us- C US- D US- E US- F US- G US- H US- 1 US- J US- K US- L US- M US- FOREIGN PATENT DOCUMENTS * Document Number Country Code-Number-Kind Code Date MM-YYYY Country Name CPC Classification N O P Q R S T 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 teins. Curr. Opin. Biotechnol. 1994, 5, 546—549. Barnabe, N.; Butler, M. The relationship between intracellular UDP-lV-acetyl hexosamine pool and monoclonal antibody production in a mouse hybridoma. J. Biotechnol. 1998, 60, 67-80. Bond, A.; Jones, M. G.; Hay, F. C. Human IgG preparations isolated by ion-exchange or protein G affinity chromatography differ in their glycosylation profiles. J. Immunol. Methods 1993, 166, 27-33. Borys, M. C.; Linzer, D. I. H.; Papoutsakis, E. T. Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-I by Chinese hamster ovary cells in a pH- dependent manner. Biotechnol. Bioeng. 1994, 43, 505—514. Boyd, P. N.; Lines, A. C.; Patel, A. K. The effect of the removal of sialic acid, galactose and total carbohydrate on the func tional activity of Campath-IH. Mol. Immunol. 1995, 32, 1311-1318. Burton, D. R. Structure and function of antibodies. In Molecular Genetics of Immunglobulin; Calabi, F., Neuberger, M. S., Eds.; Elsevier: New York, 1987; pp 1—50. Chotigeat, W.; Watanapokasin, Y.; Mahler, S.; Gray, P. P. Role of environmental conditions on the expression levels, glyco- form pattern and levels of sialyltransferase for hFSH pro duced by recombinant CHO cells. Cytotechnology 1994, 15, 217-221. deZengotita, V. M.; Kimura, R.; Miller, W. M. Effects of CO2 and osmolality on hybridoma cells: growth, metabolism, and monoclonal antibody production. Cytotechnology 1998, 28, 213-227. Endo, T.; Wright, A.; Morrison, S. L.; Kobata, A. Glycosylation of the variable region of immunoglobulin G—site specific maturation of the sugar chains. Mol. Immunol. 1995, 32, 931-940. Gawlitzek, M.; Valley, U.; Nimtz, M.; Wagner, R.; Conradt, H. S. Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions. J. Biotechnol. 1995, 42, 117—131. Gawlitzek, M.; Valley, U.; Wagner, R. Ammonium ion and glucosamine dependent increases of oligosaccharide complex ity in recombinant glycoproteins secreted from cultivated BHK-21 cells. Biotechnol. Bioeng. 1998, 57, 518—528. Goochee, C. F.; Monica, T. Environmental effects on protein glycosylation. Bio / Technology 1990, 8, 421—427. Goochee, C. F.; Gramer, M. J.; Andersen, D. C.; Bahr, J. B.; Rasmussen, J. R. The oligosaccharides of glycoproteins: bio process factors affecting oligosaccharide structure and their effect on glycoprotein properties. Bio/Technology 1991, 9, 1347-1355. Gray, D. R.; Chen, S.; Howarth, W.; Inlow, D.; Maiorella, B. L. CO2 in large-scale and high-density CHO cell perfusion culture. Cytotechnology 1996, 22, 65—78. Jan, D. C. H.; Petch, D. A.; Huzel, N.; Butler, M. The effect of dissolved oxygen on the metabolic profile of a murine hybri doma grown in serum-free medium in continuous culture. Biotechnol. Bioeng. 1997, 54, 153—164. Jefferis, R.; Lund, J.; Goodall, M. Recognition sites on human IgG for Fey receptors: the role of glycosylation. Immunol. Lett. 1995, 44, 111-117. Jenkins, N.; Parekh, R. B.; James, D. C. Getting the glycosy lation right: implications for the biotechnology industry. Nature Biotechnol. 1996, 14, 975—981. Kimura, R.; Miller, W. M. Effects of elevated PCO2 and/or osmolality on the growth and recombinant tPA production of CHO cells. Biotechnol. Bioeng. 1996, 52, 152—160. Kimura, R.; Miller, W. M. Glycosylation of CHO-derived recom binant tPA produced under elevated PCO2. Biotechnol. Prog. 1997, 13, 311-317. Kumpel, B. M.; Rademacher, T. W.; Rook, G. A. W.; Williams, P. J.; Wilson, I. B. H. Galactosylation of human IgG mono clonal anti-D produced by EBV-transformed B-lymphoblastoid cell lines is dependent on culture method and affects Fc receptor-mediated functional activity. Hum. Antibod. Hybri- domas 1994, 5, 143—151. Kunkel, J. P.; Jan, D. C. H.; Jamieson, J. C.; Butler, M. Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal anti body. J. Biotechnol. 1998, 62, 55—71. Leatherbarrow, R. J.; Dwek, R. A. The effect of aglycosylation on the binding of mouse IgG to staphylococcal protein A. FEBS Lett. 1983, 164, 227-230. Lee, S.-O.; Connolly, J. M.; Ramirez-Soto, D.; Poretz, R. D. The polypeptide of immunoglobulin G influences its galactosyla tion in vivo. J. Biol. Chem. 1990, 265, 5833—5839. Lee, Y. C. High-performance anion-exchange chromatography for carbohydrate analysis. Anal. Biochem. 1990, 189, 151— 162. Leibiger, H.; Wiinstner, D.; Stigler, R.-D.; Marx, U. Variable domain-linked oligosaccharides of a human monoclonal IgG: structure and influence on antigen binding. Biochem. J. 1999, 338, 529-538. Lifely, M. R.; Hale, C.; Boyce, S.; Keen, M. J.; Phillips, J. Glycosylation and biological activity of CAMPATH-IH ex pressed in different cell lines and grown under different culture conditions. Glycobiology 1995, 5, 813—822. Lin, A. A.; Kimura, R.; Miller, W. M. Production of tPA in recombinant CHO cells under oxygen-limited conditions. Biotechnol. Bioeng. 1993, 42, 339—350. Liideman, I.; Portner, R.; Markl, H. Effect of NH3 on the cell growth of a hybridoma cell line. Cytotechnology 1994,14,11— 20. Lund, J.; Takahashi, N.; Nakagawa, H.; Goodall, M.; Bentley, T. ; Hindley, S. A.; Tyler, R.; Jefferis, R. Control of IgG/Fc glycosylation: a comparison of oligosaccharides from chimeric human/mouse and mouse subclass immunoglobulin Gs. Mol. Immunol. 1993, 30, 741—748. 470 Biotechnol. Prog., 2000, Vol. 16, No. 3 Lund, J.; Takahashi, N.; Pound, J. D.; Goodall, M.; Jefferis, R. Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc gamma receptor I and influence the synthesis of its oligosaccharide chains. J. Immunol. 1996, 157, 4963—4969. Maiorella, B. L.; Winkelhake, J.; Young, J.; Moyer, B.; Bauer, R.; Hora, M.; Andya, J.; Thompson, J.; Patel, T.; Parekh, R. Effect of culture conditions on IgM antibody structure, pharmacokinetics and activity. Bio / Technology 1993, 11, 387-392. Malhotra, R.; Wormald, M. R.; Rudd, P. M.; Fischer, P. B.; Dwek, R. A.; Sim, R. B. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the man- nose-binding protein. Nature Med. 1995, 1, 237—243. Matsuda, H.; Nakamura, S.; Ichikawa, Y.; Kozai, K.; Takano, R.; Nose, M.; Endo, S.; Nishimura, Y.; Arata, Y. Proton nuclear magnetic resonance studies of the structure of the Fc fragment of human immunoglobulin Gl: comparisons of native and recombinant proteins. Mol. Immunol. 1990, 27, 571-579. McQueen, A.; Bailey, J. E. Effect of ammonium ion and extracellular pH on hybridoma cell metabolism and antibody production. Biotechnol. Bioeng. 1990, 35, 1067—1077. Miller, W. M.; Wilke, C. R.; Blanch, H. W. Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture. J. Cell Physiol. 1987, 132, 524—530. Mizuochi, T.; Hamako, J.; Titani, K. Structures of the sugar chains of mouse immunoglobulin G. Arch. Biochem. Biophys. 1987, 257, 387-394. Monica, T. J.; Goochee, C. F.; Maiorella, B. L. Comparative biochemical characterization of a human IgM produced in both ascites and in vitro cell culture. Bio / Technology 1993, 11, 512-515. Ogawa, T.; Kamihira, M.; Yoshida, H.; Iijima, S.; Kobayashi, T. Effect of dissolved oxygen concentration on monoclonal antibody production in hybridoma cell cultures. J. Ferment. Bioeng. 1992, 74, 372 -378. Oiler, A. R.; Buser, C. W.; Tyo, M. A.; Thilly, W. G. Growth of mammalian cells at high oxygen concentration. J. Cell. Sci. 1989, 14, 43-49. Parekh, R. B.; Dwek, R. A.; Sutton, B. J.; Fernandes, D. L.; Leung, A.; Stanworth, D.; Rademacher, T. W.; Mizuochi, T.; Taniguchi, T.; Matsuta, K.; Takeuchi, F.; Nagano, Y.; Miya moto, T.; Kobata, A. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 1985, 316, 452—457. Parekh, R.; Roitt, I.; Isenberg, D.; Dwek, R.; Rademacher, T. Age-related galactosylation of the N-linked oligosaccharides of human serum IgG. J. Exp. Med. 1988, 167, 1731—1736. Parekh, R.; Isenberg, D.; Rook, G.; Roitt, I.; Dwek, R.; Radema cher, T. A comparative analysis of disease-associated changes in galactosylation of serum IgG. J. Autoimmun. 1989,2, 101— 114. Patel, T. P.; Parekh, R. B.; Moellering, B. J.; Prior, C. P. Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody. Biochem. J. 1992, 285, 839-845. Putnam, F. W. Immunoglobulins: structure, function, and genes. In The Plasma Proteins, 3rd ed.; Putnam, F. W., Ed.; Academic Press: New York, 1987; Vol. V, pp 49—140. Rademacher, T. W.; Jones, R. H. V.; Williams, P. J. Significance and molecular basis for IgG glycosylation changes in rheu matoid arthritis. Adv. Exp. Med. Biol. 1995, 376, 193—204. Rademacher, T. W.; Jaques, A.; Williams, P. J. The defining characteristics of immunoglobulin glycosylation. In Abnor malities of IgG Glycosylation and Immunological Disorders', Isenberg, D. A.; Rademacher, T. W., Eds.; John Wiley & Sons: New York, 1996; pp 1—44. Robinson, D. K.; Chan, C. P.; Yu Ip, C.; Tsai, P. K.; Tung, J.; Seamans, T. C.; Lenny, A. B.; Lee, D. K.; Irwin, J.; Silberk- lang, M. Characterization of a recombinant antibody produced in the course of a high yield fed-batch process. Biotechnol. Bioeng. 1994, 44, 727-735. Rook, G. A. W.; Steele, J.; Brealey, R.; Whyte, A.; Isenberg, D.; Sumar, N.; Nelson, J. L.; Bodman, K. B.; Young, A.; Roitt, I. M. ; Williams, P.; Scragg, I.; Edge, C. J.; Arkwright, P. D.; Ashford, D.; Wormald, M.; Rudd, P.; Redman, C. W. G.; Dwek, R. A.; Rademacher, T. W. Changes in IgG glycoform levels are associated with remission of arthritis during pregnancy. J. Autoimmun. 1991, 4, 779—794. Rothman, R. J.; Warren, L.; Vliegenthart, J. F. G.; Hard, K. J. Clonal analysis of the glycosylation of immunoglobulin G secreted by murine hybridomas. Biochemistry 1989, 28, 1377-1384. Routier, F. H.; Davies, M. J.; Bergemann, K.; Hounsell, E. F. The glycosylation pattern of a humanized IgGi antibody (D1.3) expressed in CHO cells. Glycoconjugate J. 1997, 14, 201-207. Sawidou, G.; Klein, M.; Grey, A. A.; Dorrington, K. J.; Carver, J. P. Possible role for peptide-oligosaccharide interactions in differential oligosaccharide processing at asparagine-107 of the light chain and asparagine-297 of the heavy chain in a monoclonal IgGi kappa. Biochemistry 1984, 23, 3736—3740. Schweikart, F.; Jones, R.; Jaton, J.-C.; Hughes, G. J. Rapid structural characterisation of a murine monoclonal IgA a chain: heterogeneity in the oligosaccharide structures at a specific site in samples produced in different bioreactor systems. J. Biotechnol. 1999, 69, 191—201. Tarentino, A. L.; Gomez, C. M.; Plummer, T. H., Jr. Deglycos- ylation of asparagine-linked glycans by peptide: N-glycosi- dase F. Biochemistry 1985, 24, 4665—4671. Townsend, R. R.; Hardy, M. R.; Hindsgaul, O.; Lee, Y. C. High- performance anion-exchange chromatography of oligosaccha rides using pellicular resins and pulsed amperometric detec tion. Anal. Biochem. 1988, 174, 459—470. van der Valk, P.; Gille, J. J. P.; Oostra, A. B.; Roubos, E. W.; Sminia, T.; Joenje, H. Characterization of an oxygen-tolerant cell line derived from Chinese hamster ovary. Cell Tissue Res. 1985, 239, 61-68. Weitzhandler, M.; Hardy, M.; Co, M. S.; Avdalovic, N. Analysis of carbohydrates on IgG preparations. J. Pharm. Sci. 1994, 83, 1670-1675. Wormald, M. R.; Rudd, P. M.; Harvey, D. J.; Chang, S.-C.; Scragg, I. G.; Dwek, R. A. Variations in oligosaccharide- protein interactions in immunoglobulin G determine the site- specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides. Biochemistry 1997, 36, 1370-1380. Wright, A.; Morrison, S. L. Antibody variable region glycosyla tion: biochemical and clinical effects. Springer Semin. Im- munopathol. 1993, 15, 259—273. Wright, A.; Morrsion, S. L. Effect of glycosylation on antibody function: implications for genetic engineering. Trends Bio technol. 1997, 15, 26—32. Yamada, E.; Tsukamoto, Y.; Sasaki, R.; Yagyu, R.; Takahashi, N. Structural changes of immunoglobulin G oligosaccharides with age in healthy human serum. Glycoconjugate J. 1997, 14, 401-405. Yet, M.-G.; Shao, M.-C.; Wold, F. Effects of the protein matrix on glycan processing in glycoproteins. FASEB J. 1988,2, 22— 31. Zanghi, J. A.; Mendoza, T. P.; Knop, R. H.; Miller, W. M. Ammonia inhibits neural cell adhesion molecule polysialyla- tion in Chinese hamster ovary and small cell lung cancer cells. J. Cell. Physiol. 1998, 177, 248-263. Zanghi, J. A.; Schmelzer, A. E.; Mendoza, T. P.; Knop, R. H.; Miller, W. M. Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCOs or pH. Biotechnol. Bioeng. 1999, 65, 182-191. Accepted for publication March 20, 2000. BP000026U Copy with citationCopy as parenthetical citation