Influence of cell‐ and media‐derived factors on the integrity of a human monoclonal antibody after secretion into serum‐free cell culture supernatants

Ackermann, M.; Jäger, Volker; Marx, Uwe

doi:10.1002/bit.260450202

Cited by 12 publications

(4 citation statements)

References 38 publications

(5 reference statements)

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“…Also cell attachment to the surface was lower (the proportion of suspended cells increased with decreasing serum content) and in most cases led to cell aggregation, similar to that reported for BHK21 pSVII cells producing interleukin-2 (Jager et al, 1988). This is also in agreement with the reported fact that both BHK21 and CHO cells formed spheroids of up to 500 m in diameter after transfer to serum-free culture conditions (Ackermann et al, 1995).…”

Section: Multi-step Adaptation To Serum-free Mediumsupporting

confidence: 89%

Untitled

et al. 1998

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In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.

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Section: Multi-step Adaptation To Serum-free Mediumsupporting

confidence: 89%

Untitled

et al. 1998

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“…In contrast to chemical synthesis processes that can be maintained consistently with precision between batches for the manufacturing of small molecule therapeutics, it is practically impossible to maintain identical conditions for the animal cell-based production systems, even when batches were manufactured at the same site with the same procedure. Variations can occur during the upstream bioreactor culturing process, in terms of pH, DO, and temperature, which will cause variations in cell growth, viability and cell density, which will then cause variations in the downstream purification process [ 6 , 7 , 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Lee

Choi²,

Hwang³

et al. 2022

IJMS

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The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately.

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“…When hybridoma cells were cultured in medium containing either fructose, mannose, or galactose instead of glucose, galactose elevated the antigen binding activity of the antibody more than the other sugars (Tachibana et al, 1994). Shifts of the structural or functional antibody heterogeneity due to variations in production parameters are not caused by extracellular cell-and media-derived factors in mammalian cultures once the product has been secreted into the supernatant (Ackermann et al, 1995). It is concluded that the glycosylation pattern of a secreted monoclonal antibody is dependent on the culture method employed to obtain it (Patel et al, 1992).…”

Section: Discussionmentioning

confidence: 98%

Production and Molecular Characterization of Clinical Phase I Anti-Melanoma Mouse IgG3 Monoclonal Antibody R24

Kemminer

Conradt²,

Nimtz³

et al. 2001

Biotechnol. Prog.

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R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 microg sialic acid per mg protein, which splits into 0.243 microg Neu5Gc and 0.217 microg Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy.

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Influence of cell‐ and media‐derived factors on the integrity of a human monoclonal antibody after secretion into serum‐free cell culture supernatants

Cited by 12 publications

References 38 publications

Untitled

Untitled

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Production and Molecular Characterization of Clinical Phase I Anti-Melanoma Mouse IgG3 Monoclonal Antibody R24

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