Search

ABSTRACTIntrons are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to

Cell line development is an essential stage in biopharmaceutical development that often lies on the critical path. Failure to fully characterise the lead clone during initial screening can lead to lengthy project delays during scale-up, which can potentially compromise commercial manufacturing success. In this study, we propose a novel cell line development methodology, referenced as CLD4, which involves four steps enabling autonomous data-driven selection of the lead clone. The first step involves the digitalisation of the process and storage of all available information within a structured data lake. The second step calculates a new metric referenced as the cell line manufacturability index (MICL) quantifying the performance of each clone by considering the selection criteria relevant to productivity, growth and product quality. The third step implements machine learning (ML) to identify any potential risks associated with process operation and relevant critical quality attributes (CQAs). The final step of CLD4 takes into account the available metadata and summaries all relevant statistics generated in steps 1–3 in an automated report utilising a natural language generation (NLG) algorithm. The CLD4 methodology was implemented to select the lead clone of a recombinant Chinese hamster ovary (CHO) cell line producing high levels of an antibody-peptide fusion with a known product quality issue related to end-point trisulfide bond (TSB) concentration. CLD4 identified sub-optimal process conditions leading to increased levels of trisulfide bond that would not be identified through conventional cell line development methodologies. CLD4 embodies the core principles of Industry 4.0 and demonstrates the benefits of increased digitalisation, data lake integration, predictive analytics and autonomous report generation to enable more informed decision making.

With the aim of increasing protein productivity of Chinese hamster ovary (CHO) cells, we sought to generate new CHO hosts with favorable biomanufacturing phenotypes and improved functionality. Here, we present an innovative approach of enriching the CHO host cells with a high mitochondrial membrane potential (MMP). Stable transfectant pools and clonal cell lines expressing difficult-to-express bispecific molecules generated from the MMP-enriched host outperformed the parental host by displaying (1) improved fed-batch productivity; (2) enhanced long-term cell viability of pools; (3) more favorable lactate metabolism; and (4) improved cell cloning efficiency during monoclonal cell line generation. Proteomic analysis together with Western blot validation were used to investigate the underlying mechanisms by which high MMP influenced production performance. The MMP-enriched host exhibited multifaceted protection against mitochondrial dysfunction and endoplasmic reticulum stress. Our findings indicate that the MMP-enriched host achieved an overall “fitter” phenotype that contributes to the significant improvement in biomanufacturing capability.

Summary: Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.

Summary: Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.