Large-Scale Transient Production in ExpiCHO-S™ with Enhanced N-Galactosylation-Sialylation and PEI-Based Transfection

Zhong, Xiaotian; Schwab, Ashley; Ma, Weijun; Meade, Caryl; Zhou, Jing; D’Antona, Aaron M.; Somers, Will; Lin, Laura

doi:10.1007/978-1-0716-1450-1_7

Cited by 5 publications

(2 citation statements)

References 18 publications

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“…Similarly, low fulfillment is seen in high-yield expression systems 87 necessitating, in some cases, supplementation with maturing glycoenzymes. 88–90…”

Section: Discussionmentioning

confidence: 99%

Protein structure, a genetic encoding for glycosylation

Kellman,

Sandoval,

Zaytseva

et al. 2024

Preprint

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DNA, RNA, and proteins are synthesized using template molecules, but glycosylation is not believed to be constrained by a template. However, if cellular environment is the only determinant of glycosylation, all sites should receive the same glycans on average. This template-free assertion is inconsistent with observations of microheterogeneity-wherein each site receives distinct and reproducible glycan structures. Here, we test the assumption of template-free glycan biosynthesis. Through structural analysis of site-specific glycosylation data, we find protein-sequence and structural features that predict specific glycan features. To quantify these relationships, we present a new amino acid substitution matrix that describes glycoimpact-how glycosylation varies with protein structure. High-glycoimpact amino acids co-evolve with glycosites, and glycoimpact is high when estimates of amino acid conservation and variant pathogenicity diverge. We report hundreds of disease variants near glycosites with high-glycoimpact, including several with known links to aberrant glycosylation (e.g., Oculocutaneous Albinism, Jakob-Creutzfeldt disease, Gerstmann-Straussler-Scheinker, and Gaucher's Disease). Finally, we validate glycoimpact quantification by studying oligomannose-complex glycan ratios on HIV ENV, differential sialylation on IgG3 Fc, differential glycosylation on SARS-CoV-2 Spike, and fucose-modulated function of a tuberculosis monoclonal antibody. In all, we show glycan biosynthesis is accurately guided by specific, genetically-encoded rules, and this presents a plausible refutation to the assumption of template-free glycosylation.

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“…Similarly, low fulfillment is seen in high-yield expression systems 87 necessitating, in some cases, supplementation with maturing glycoenzymes. 88–90…”

Section: Discussionmentioning

confidence: 99%

Protein structure, a genetic encoding for glycosylation

Kellman,

Sandoval,

Zaytseva

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…The CM was harvested at 120 h post-transfection. For the PEI-based ExpiCHO-S TM transfection, the procedures were previously described [ 29 ]. In essence, 7 mg/L of PEI was mixed with 2 mg/L DNAs for a 2.5-min-incubation at room temperature, prior to the mixture inoculation to the ExpiCHO-S TM culture; 16 h post-transfection, 20 mL/L of sodium butyrate (4 mM), 10 mL/L of DMSO, and 100 mL/L of CHO CD EfficientFeed TM -B medium (Thermo Fisher, CAT#A10240-01) were added.…”

Section: Methodsmentioning

confidence: 99%

Exploring Parametric and Mechanistic Differences between Expi293FTM and ExpiCHO-STM Cells for Transient Antibody Production Optimization

et al. 2023

Self Cite

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Rapidly producing drug-like antibody therapeutics for lead molecule discovery and candidate optimization is typically accomplished by large-scale transient gene expression technologies (TGE) with cultivated mammalian cells. The TGE methodologies have been extensively developed over the past three decades, yet produce significantly lower yields than the stable cell line approach, facing the technical challenge of achieving universal high expression titers for a broad range of antibodies and therapeutics modalities. In this study, we explored various parameters for antibody production in the TGE cell host Expi293FTM and ExpiCHO-STM with the transfection reagents ExpiFectamineTM and polyethylenimine. We discovered that there are significant differences between Expi293FTM and ExpiCHO-STM cells with regards to DNA complex formation time and ratio, complex formation buffers, DNA complex uptake trafficking routes, responses to dimethyl sulfoxide and cell cycle inhibitors, as well as light-chain isotype expression preferences. This investigation mechanistically dissected the TGE processes and provided a new direction for future transient antibody production optimization.

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A high‐titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics

Gonzalez‐Rivera,

Galvan,

Ryder

et al. 2024

Biotech & Bioengineering

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Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells offers a route to accelerate biologics development by delivering material weeks to months earlier than what is possible with conventional cell line development. However, low productivity, inconsistent product quality profiles, and scalability challenges have prevented its broader adoption. In this study, we develop a scalable CHO‐based TGE system achieving 1.9 g/L of monoclonal antibody in an unmodified host. We integrated continuous flow‐electroporation and alternate tangential flow (ATF) perfusion to enable an end‐to‐end closed system from N‐1 perfusion to fed‐batch 50‐L bioreactor production. Optimization of both the ATF operation for three‐in‐one application—cell growth, buffer exchange, and cell mass concentration—and the flow‐electroporation process, led to a platform for producing biotherapeutics using transiently transfected cells. We demonstrate scalability up to 50‐L bioreactor, maintaining a titer over 1 g/L. We also show comparable quality between both transiently and stably produced material, and consistency across batches. The results confirm that purity, charge variants and N‐glycan profiles are similar. Our study demonstrates the potential of CHO‐based TGE platforms to accelerate biologics process development timelines and contributes evidence supporting its feasibility for manufacturing early clinical material, aiming to strengthen endorsement for TGE's wider implementation.

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Large-Scale Transient Production in ExpiCHO-S™ with Enhanced N-Galactosylation-Sialylation and PEI-Based Transfection

Cited by 5 publications

References 18 publications

Protein structure, a genetic encoding for glycosylation

Protein structure, a genetic encoding for glycosylation

Exploring Parametric and Mechanistic Differences between Expi293FTM and ExpiCHO-STM Cells for Transient Antibody Production Optimization

A high‐titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics

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