Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning

He, Qiaojun; Rehmann, Matthew S.; Tian, Jun; Xu, Jinying; Sabino, Luzmary; Vandermark, Erik; Basson, Ziev; Po, Iris; Bierilo, Kathleen; Tremml, Gabi; Rizzi, Giovanni; Langsdorf, Erik; Qian, Nan‐Xin; Borys, Michael; Khetan, Anurag; Li, Zheng Jian

doi:10.3390/bioengineering9040173

Cited by 4 publications

(6 citation statements)

References 37 publications

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“…We presumed that the exceptional stability of our MCB for product substrate is due to the acquired fitness of the clone associated with the sequence of multiple stringent screening methods during clonal isolation. This notion is supported by a recent study, which has reported that a secondary screening of an established MCB produced high-titer stable clones without affecting the quality attributes of the desired product [50].…”

Section: Discussionmentioning

confidence: 78%

Development and qualification of a high-yield recombinant human Erythropoietin biosimilar

Nag¹,

Islam²,

Khan³

et al. 2023

Preprint

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Recombinant human erythropoietin (rhEPO) has been saving millions of lives worldwide as a potent and safe treatment for the lack of erythrocyte, which is caused by chronic kidney disease (CKD) and other issues. Several biosimilars of rhEPO have been approved since the expiry of the relevant patents to provide cost-effective options but the price of rhEPO is still high for the affordability of global community. Therefore, development of biosimilar of rhEPO at a lower price is highly necessary. Here we report the development and characterization of a biosimilar of rhEPO with high-yield satisfying regulatory requirements. The hEPO-expressing cDNA was stably expressed in CHO cells with successive transfection. The master cell bank (MCB) and working cell bank (WCB) were established from the best selected clone and characterized for 50 passages. The rhEPO was expressed from the WCB in single-use suspension culture system with a high-titer (1.24 +/- 0.16 g/L). To the best of our knowledge this is the highest reported rhEPO titer to date. The rhEPO was purified using a series of validated chromatography unit processes including virus inactivation and filtration. The purified EPO was formulated in serum-free buffer, sterile filtered, and analyzed as the biosimilar of reference product Eprex(R). Physicochemical analysis strongly suggested similarities between the developed rhEPO (GBPD002) and the reference. The in vitro and in vivo functional assays confirmed the similar biofunctionality of the GBPD002 and Eprex(R). GBPD002 could provide a less-expensive solution to the needful communities as an effective and safe biosimilar where rhEPO treatment is necessary.

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Section: Discussionmentioning

confidence: 78%

Development and qualification of a high-yield recombinant human Erythropoietin biosimilar

Nag¹,

Islam²,

Khan³

et al. 2023

Preprint

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“…The titer of a specific clone is not determined by gene copy number alone in a TI platform. One possible explanation is that epigenetic changes induced by subcloning lead to the isolation of higher titer cell lines 17,55 …”

Section: Resultsmentioning

confidence: 99%

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies

Barnard,

Zhou,

Shen

et al. 2023

Biotechnology Progress

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Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID‐19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore “pandemic mAb” timelines need to be shortened. One acceleration tool is “deferred cloning” and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single‐cell cloning for mAb1 and mAb2, identifying three high‐expressing clones from each pool. CHO pools and clones were inoculated side‐by‐side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3‐month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future “pandemic mAb.”

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“…9 The CpB treatment protocol was also applied to improve the main peak by removing C-terminal lysines for mAb-A and mAb-B production in this study. In addition to three widely used in vivo CHO cell culture improvement strategies, including cell line engineering, [36][37][38] media development, [39][40][41][42][43] and process parameter optimization, 32,44,45 in vitro enzymatic treatments may represent useful options for titer improvement and control of critical quality attributes. Although cell culture media and process parameters are environmental factors, the reason why we defined as in vivo above is because in vivo metabolisms within cells play a major role, while the CpB treatment is an in vitro reaction regardless of cells present or not.…”

Section: Cell Culture Performance and Critical Quality Attributes Bef...mentioning

confidence: 99%

Antibody charge variant modulation by in vitro enzymatic treatment in different Chinese hamster ovary cell cultures

2022

Biotechnology Progress

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Charge variants represent a critical quality attribute that must be controlled during the development and manufacturing of monoclonal antibodies (mAb). Previously, we reported the development of a cost‐effective enzymatic treatment capable of removing the C‐terminal lysine from a mAb produced by a Chinese hamster ovary (CHO) GS cell line. This treatment resulted in a significant decrease in basic charge variants and a corresponding improvement in the main peak, enabling a longer cell culture production duration for titer improvement. Here, we describe this enzymatic treatment protocol in detail and demonstrate its applicability to two additional mAbs produced by distinct industrial cell lines. The simple addition of carboxypeptidase B (CpB) at a ratio of 1:10,000 (w/w) to whole cell cultures significantly improved the main peaks for both mAbs without affecting other critical quality attributes, including size exclusion chromatography impurities and N‐glycans. Our results demonstrate that this in vitro CpB treatment protocol can be used as a platform strategy to improve main peak for mAbs that exhibit high levels of basic variants attributable to C‐terminal lysines. An in vitro enzymatic treatment in general may be another good addition to existing in vivo CHO cell culture strategies for titer improvement and control of critical quality attributes.

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Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning

Cited by 4 publications

References 37 publications

Development and qualification of a high-yield recombinant human Erythropoietin biosimilar

Development and qualification of a high-yield recombinant human Erythropoietin biosimilar

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies

Antibody charge variant modulation by in vitro enzymatic treatment in different Chinese hamster ovary cell cultures

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