Q5D Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products

Plavsic, Mark

doi:10.1002/9781118971147.ch13

Cited by 15 publications

(9 citation statements)

References 17 publications

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“…The HEK-293 cell line was selected because it stably expresses human FSHR, allowing for the evaluation of hormone receptor binding affinity and activation, and has been shown to produce cAMP in response to FSH stimulation [34,35]. HEK-293 is qualified for such use within a defined working window; to ensure method suitability, test acceptance criteria are maintained, and each cell bank is tested for functionality at the beginning, middle and end of the defined working window [7,36].…”

Section: Determination Of Fsh Potency 451 In Vitro Bioassay To Assess...mentioning

confidence: 99%

Biological Assay to Determine Gonadotropin Potency: From In Vivo to In Vitro Sustainable Method

Nevelli¹,

Palmese

Gleixner³

et al. 2023

IJMS

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Various preparations of follicle-stimulating hormone (FSH) are commercially available; however, they differ in glycoforms composition and purity owing to their respective sources. Additional chemical/physical changes can also be introduced during manufacturing and can impact their biological activity (biopotency), which is routinely assessed using an in vivo bioassay (Steelman–Pohley). This study aimed to determine whether an in vitro bioassay could assess biopotency by distinguishing between r-hFSH chemical/physical variants with similar ability to the in vivo bioassay. The specific activity (units of biological activity per mg of product) of variants of r-hFSH generated through enrichment (acidic/basic), stress (oxidative/acidic pH) and enzymatic treatment (desialylation and desialylation/degalactosylation) was compared using the in vivo and in vitro bioassays. The in vitro bioassay reliably detected potential chemical/physical modifications in r-hFSH variants that may impact biopotency. Overall, the methods demonstrated a comparable ability to detect changes in specific activities due to chemical/physical differences in r-hFSH variants. These data indicate that the in vitro bioassay is suitable to replace the in vivo bioassay.

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Section: Determination Of Fsh Potency 451 In Vitro Bioassay To Assess...mentioning

confidence: 99%

Biological Assay to Determine Gonadotropin Potency: From In Vivo to In Vitro Sustainable Method

Nevelli¹,

Palmese

Gleixner³

et al. 2023

IJMS

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“…On the other hand, to ensure the quality, consistency and safety of these biologics, regulatory authorities require that new cell lines stem from a single-cell progenitor. 3 Upon submission of a New drug application (NDA) or Investigational new drug (IND), organizations must provide evidence that their cell lines are clonally derived. The combination of these two factors creates a continuous need to improve CLD workflows for faster clone generation while providing assurance of clonal derivation.…”

Section: Introductionmentioning

confidence: 99%

An optimized and validated workflow for developing stable producer cell lines with >99.99% assurance of clonality and high clone recovery

Scherzinger

Türk

Aprile-Garcia

2022

Preprint

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There is a constant pressure to reduce timelines in mammalian cell line development (CLD) for biotherapeutic protein production. Demonstration of clonal derivation of the generated cell lines is key for health authorities' approval. To meet these regulatory and process-oriented demands, single-cell dispensers have become vital instruments for single-cell cloning. We conducted validation experiments with the UP.SIGHT (CYTENA GmbH) to determine this instrument's single-cell dispensing efficiency (SCDE) and probability of clonal derivation (p(clonal)). Process optimization to maximize clone recovery with several cell lines was also performed, focusing on cloning media and plate type. With a SCDE >97%, p(clonal) >99.99% and clone recovery values of up to 80%, the data reported here support the notion that the UP.SIGHT covers all steps in the single-cell dispensing process with assurance of clonality and colony tracking, leading to faster and more efficient CLD workflows. This work also serves as a guideline for instrument validation and guidance towards process optimization.

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“…In a standard CLD workflow, host cells are transfected with the genes of interest, selection pressure is introduced to enrich for positively transfected cells, and limiting dilution or fluorescence‐activated cell sorting (FACS) is applied to sort the fully recovered population into single cells. Single‐cell cloning (SCC) represents a critical step in the CLD workflow as regulatory guidance requires production cell lines to be derived from “a single cell progenitor.” 1 These clonally‐derived cultures must then be individually propagated and assessed for growth, productivity, and product quality. As a result, achieving a highly productive cell line is a time‐consuming and resource‐intensive process that involves the selection of cell pools that retain the desired genes of interest, scale‐up of the pools to confirm desired expression and product quality, SCC and scale up from a single cell, and screening of hundreds to thousands of clones.…”

Section: Introductionmentioning

confidence: 99%

Microfluidic chip‐based single‐cell cloning to accelerate biologic production timelines

Diep

Lê

et al. 2021

Biotechnology Progress

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Cell line development (CLD) represents a critical, yet time-consuming, step in the biomanufacturing process as significant resources are devoted to the scale-up and screening of several hundreds to thousands of single-cell clones. Typically, transfected pools are fully recovered from selection and characterized for growth, productivity, and product quality to identify the best pools suitable for single-cell cloning (SCC) using limiting dilution or fluorescence-activated cell sorting (FACS). Here we report the application of the Berkeley Lights Beacon Instrument (BLI) in an early SCC process to accelerate the CLD timeline. Transfected pools were single-cell cloned when viabilities reached greater than 85% or during selection when viabilities were less than 30%. Clones isolated from these accelerated processes exhibited comparable growth, productivity, and product quality to those derived from a standard CLD process and fit into an existing manufacturing platform. With these approaches, up to a 30% reduction in the overall CLD timeline was achieved. Furthermore, early process-derived clones demonstrated equivalent long-term stability compared with standard process-derived clones over 50 population doubling levels (PDLs). Taken together, the data supported early SCC on the BLI as an attractive approach to reducing the standard CLD timeline while still identifying clones with acceptable manufacturability.

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Q5D Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products

Cited by 15 publications

References 17 publications

Biological Assay to Determine Gonadotropin Potency: From In Vivo to In Vitro Sustainable Method

Biological Assay to Determine Gonadotropin Potency: From In Vivo to In Vitro Sustainable Method

An optimized and validated workflow for developing stable producer cell lines with >99.99% assurance of clonality and high clone recovery

Microfluidic chip‐based single‐cell cloning to accelerate biologic production timelines

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