Impact of hydrolysates on monoclonal antibody productivity, purification and quality in Chinese hamster ovary cells

Ho, Soo Yei; Nian, Rui; Woen, Susanto; Chng, Jake; Zhang, Peiqing

doi:10.1016/j.jbiosc.2016.03.003

Cited by 15 publications

(9 citation statements)

References 26 publications

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“…**p \ 0.01; ***p \ 0.001 commonly observed modifications that form basic variants. Moreover, the addition of hydrolysate to culture medium or feed has been reported to significantly enhance mAb production and glycosylation (Ho et al 2016;Kim and Lee 2009), and in the current study, the production of anti-IgE mAb was also enhanced in the presence of hydrolysate. However, the effect of the addition of hydrolysate on charge heterogeneity during anti-IgE mAb production has been relatively unknown, so further investigation of how charge heterogeneity can be regulated based on temperature shift was needed.…”

Section: Discussionsupporting

confidence: 60%

Combination of temperature shift and hydrolysate addition regulates anti-IgE monoclonal antibody charge heterogeneity in Chinese hamster ovary cell fed-batch culture

et al. 2018

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Charge heterogeneity has been broadly studied as a critical quality attribute during monoclonal antibody (mAb) production that may subsequently affect product stability and biopotency. However, the charge variation distribution is poorly controlled, so methods of more effective control need to be explored. In this study, the combined effects of temperature shift (37-34, 37-32, or 37-30 °C) and hydrolysate addition (0.100 g/L) to culture feed on the charge heterogeneity of anti-IgE mAb were investigated. The results showed that the distribution of charge variation was significantly regulated by the combination of hydrolysate addition with a highly sub-physiological temperature (34 °C). In addition, under this condition, the main peak content significantly increased, and the acidic peak content significantly decreased. Furthermore, we explored Lys variant content, which is the major basic variant content, as well as its relationship with temperature shift and hydrolysate addition. Lys variant levels were positively related to the Lys and Arg concentrations in the medium and negatively related to carboxypeptidase B and carboxypeptidase H transcript levels. The combination of temperature shift and hydrolysate addition can thus effectively improve anti-IgE mAb charge heterogeneity and significantly increase main variant levels and decrease acidic variant levels.

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Section: Discussionsupporting

confidence: 60%

Combination of temperature shift and hydrolysate addition regulates anti-IgE monoclonal antibody charge heterogeneity in Chinese hamster ovary cell fed-batch culture

et al. 2018

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“…Cell growth media developed for mammalian cells has been shown to include hydroxyproline in the formulation [ 41 , 42 ]. Additionally, bioreactor feed formulations can include hydrolysates derived from plant sources such as soybean which is a rich source of hydroxyproline [ 43 – 45 ].…”

Section: Discussionmentioning

confidence: 99%

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines

et al. 2020

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With the advent of highly sensitive technologies such as tandem mass spectrometry and next-generation sequencing, recombinant antibodies are now routinely analyzed for the presence of low-level sequence variants including amino acid misincorporations. During mAb cell culture process development, we found that proline was replaced with the non-canonical amino acid, hydroxyproline, in the protein sequence. We investigated the relationship between proline content in the cell culture media and proline sequence variants and found that the proline concentration was inversely correlated with the amount of sequence variants detected in the protein sequence. Hydroxyproline incorporation has been previously reported in recombinant proteins produced in mammalian expression systems as a post-translational modification. Given the dependency on proline levels, the mechanism was then investigated. To address the possibility of co-translational misincorporation of hydroxyproline, we used tandem mass spectrometry to measure incorporation of stable-isotope labelled hydroxyproline added to the feed of a production bioreactor. We discovered co-translational misincorporation of labelled hydroxyproline in the recombinant antibody. These findings are significant, since they underscore the need to track non-canonical amino acid incorporation as a co-translational event in CHO cells. Understanding the mechanism of hydroxyproline incorporation is crucial in developing an appropriate control strategy during biologics production.

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“…Application of tyrosine‐, histidine‐, or glutamine‐containing dipeptides, substitution of glutamine with alpha‐ketoglutarate (alpha‐KG), or altering the glutamine levels in media have been reported to lower lactate and ammonia production, thus providing better pH maintenance, improved q p , and overall process yield . Yeast extracts produced a beneficial effect on q p by maintaining the mammalian target of rapamycin pathway activity and enhancing protein translation .…”

Section: Introductionmentioning

confidence: 99%

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines

Tian

Oliveira

et al. 2020

Engineering in Life Sciences

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Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS−/− CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10–19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500‐L and 1000‐L bioreactors replicated laboratory results using 5‐L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

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Impact of hydrolysates on monoclonal antibody productivity, purification and quality in Chinese hamster ovary cells

Cited by 15 publications

References 26 publications

Combination of temperature shift and hydrolysate addition regulates anti-IgE monoclonal antibody charge heterogeneity in Chinese hamster ovary cell fed-batch culture

Combination of temperature shift and hydrolysate addition regulates anti-IgE monoclonal antibody charge heterogeneity in Chinese hamster ovary cell fed-batch culture

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines

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