Cell line development (CLD) represents a complex but highly critical process during the development of a biological drug. To shed light on this crucial workflow, a team of BioPhorum members (authors) ...has developed and executed surveys focused on the activities and effort involved in a typical CLD campaign. An average of 27 members from different companies that participate in the BioPhorum CLD working group answered surveys covering three distinguishable stages of a standard CLD process: (1) Pre-transfection, including vector design and construction; (2) Transfection, spanning the initial introduction of vector into cells and subsequent selection and analysis of the pools; and (3) Single Cell Cloning and Lead Clone Selection, comprising methods of isolating single cells and confirming clonal origin, subsequent expansion and screening processes, and methods for identifying and banking lead clones. The surveys were very extensive, including a total of 341 questions split between antibody and complex molecule CLD processes. In this survey review, the authors interpret and highlight responses for antibody development and, where relevant, contrast complex molecule development challenges to provide a comprehensive industry perspective on the typical time and effort required to develop a CHO production cell line.
Biopharmaceuticals like therapeutic monoclonal antibodies (mAbs) and other derived proteins are popular for treating various diseases. Transient gene expression (TGE) is typically used as a fast yet ...efficient method to generate moderate amounts of material. It has been used to support early stage research and discovery processes. Introduction of a robust high yielding and predictive TGE platform in Chinese hamster ovary (CHO) is crucial. It maintains the consistency in cell lines and processes throughout the early drug discovery and downstream manufacturing processes. This helps researchers to identify the issues at an early stage for timely resolution. In this study, we have demonstrated a simple high‐titer platform for TGE in CHO based on a dilution process of seeding cells. We achieved titers ranging from 0.8 to 1.9 g/L for eight model mAbs at three scales (1, 30, 100 mL) in 10 days using our new platform. The ability to seed by dilution significantly streamlined the process and dramatically enhanced platform throughput. We observed a modest reduction in titer ranging from 11% to 28% when cells were seeded using dilution compared to when cells were seeded using medium exchange. Further studies revealed that carry over of spent medium into transfection negatively affected the DNA uptake and transcription processes, while the translation and secretion was minimally impacted. In summary, our transient CHO platform using cells prepared by dilution at high densities can achieve high titers of up to 1.9 g/L, which can be further improved by targeting the bottlenecks of transfection and transcription.
Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to ...humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest.
Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.
•Humanization of N-glycans on two recombinant therapeutic proteins.•N-glycosylation similar to human in engineered Chinese hamster ovary cells.•Knock out of ten target genes and expression of two human genes.
Perfusion cell cultures generate higher viable cell densities (VCD) and volumetric productivity compared to fed-batch cultures. However, due to the limited availability of small-scale perfusion ...models, perfusion systems are seldom used to produce licensed biotherapeutics. This study evaluated two small-scale perfusion mimic protocols to bridge the research-to-production gap. Shake flasks and the ambr250 HT were used to compare centrifugation and in situ gravity settling protocols. The centrifugation protocol achieved a peak VCD >50 million cells/mL and is well-suited to media formulation and feeding strategy comparisons. The in situ gravity settling protocol achieved ∼20 million cells/mL and is well-suited to cell productivity and stability studies. The cell retention steps resulted in temporary DO and pH changes but did not affect the overall culture's health. Further, both protocols were able to recover from an imposed long-duration DO stress, although the centrifugation protocol cultures had higher cell specific oxygen consumption rates indicative of higher culture stress. Both protocols sustained the cultures for 39 days with stable cell specific productivities (∼27 pg/cell·day). Overall, this study demonstrated the feasibility of two economic small-scale perfusion mimic protocols in a standard small-scale bioreactor system, which could be translated to other multi-unit small-scale bioreactor systems.
•Centrifugation perfusion mimic protocol enables media formulation comparisons.•Centrifugation perfusion mimic protocol assists feeding strategy comparisons.•In situ gravity perfusion mimic protocols evaluates cell productivity stability.•Cell specific oxygen uptake rates indicate increased stress for CHO cells.
Monoclonal antibodies (mAbs) represent a significant segment of biopharmaceuticals, with the market for mAb therapeutics expected to reach $200 billion in 2021. Chinese Hamster Ovary (CHO) cells are ...the industry standard for large-scale mAb production owing to their adaptability and genetic engineering capabilities. However, maintaining consistent product quality is challenging, primarily because of the inherent genetic instability of CHO cells. In this study, we address the need for advanced technologies for quality monitoring of host cells in biopharmaceuticals. We highlight the limitations of traditional cell assessment techniques such as flow cytometry and propose a noninvasive, label-free image-based analysis method. By utilizing advanced image processing and machine learning, this technique aims to non-invasively and quantitatively evaluate subtle quality changes in suspension cells. The research aims to investigate the use of morphological analysis for identifying subtle alterations in mAb productivity of CHO cells, employing cells stimulated by compounds as a model for this study. Our results show that the mAb productivity of CHO cells (day 8) can be predicted only from their early morphological profile (day 3). Our study also discusses the importance of strategic methods for forecasting host cell mAb productivity using morphological profiles, as inferred from our machine learning models specialized in predictive score prediction and anomaly prediction.
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•Stable image-based analysis for suspension cells was established.•Early morphological profile explains the host cell productivity, but total profile reflects their viability and growth.•Using time-course morphological profile, antibody productivity can be predicted.•Using time-point morphological profile at day 3, productivity at day 8 can be predicted.
► We sequenced the CHO cell line transcriptome with pyrosequencing technology from 454 Life Sciences generating sequence reads of ≈400
bp. ► These long reads enabled a
de novo assembly of the ...generated transcriptome data with regard to the exon-intron structure of eukaryotic transcripts. ► We assembled more than 29,000 transcripts and are able to distinguish between different isoforms of genes. ► Finally, we publish assembled data to enable further research to other scientific groups.
The pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences with the Newbler Assembler were used to achieve a major step forward to unravel the transcriptome of Chinese hamster ovary (CHO) cells. Normalized cDNA libraries originating from several cell lines and diverse culture conditions were sequenced and the resulting 1.84 million reads were assembled into 32,801 contiguous sequences, 29,184 isotigs, and 24,576 isogroups. A taxonomic classification of the isotigs showed that more than 70% of the assembled data is most similar to the transcriptome of
Mus musculus, with most of the remaining isotigs being homologous to DNA sequences from
Rattus norvegicus. Mapping of the CHO cell line contigs to the mouse transcriptome demonstrated that 9124 mouse transcripts, representing 6701 genes, are covered by more than 95% of their sequence length. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein
N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the
N-glycosylation pathway of CHO cells were detected. The present manuscript represents a data set of assembled and annotated genes for CHO cells that can now be used for a detailed analysis of the molecular functioning of CHO cell lines.
•A novel small-scale perfusion bioreactor system for animal/human cells with working volumes <250 mL was developed.•Densities maintained at ≥80 × 106HEK293 cells/mL at 10pL/cell/day cell specific ...perfusion rate using enriched feed medium.•HEK293 cell expressing Erythropoietin cultured in high cell density perfusion process more than 75 days.
Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines.
In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 × 106 cells/mL) and high cell densities (80 × 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes.
•Mechanistic model developed for acoustic wave separation using population balance model.•Acoustic agglomeration kernel predicts CHO cell size distribution as a function of time.•Cell trajectory has ...been analysed numerically based on the inertial and acoustic forces.•Significant effect of flowrate and acoustic power on cell dynamics in the acoustic zone.•A generalized correlation is developed using Buckingham Pi theorem to predict agglomeration efficiency.
Acoustic wave separation (AWS) is gradually emerging as an effective method for continuous clarification of Chinese hamster ovary (CHO) cells. In this paper, we present a mechanistic model for AWS based on population balance model (PBM) along with acoustic agglomeration kernel to predict cell size distribution as a function of time. Results show that more than 90% CSE was achieved for flowrates <5 mL min−1 and acoustic power >12.5 W. Experimental results confirm that the mechanistic model offers an accurate prediction of the CHO agglomeration process. Furthermore, a generalized correlation has been developed using the Buckingham Pi theorem between the dimensionless numbers composed of relevant process and geometrical variables to predict the agglomeration efficiency. To our knowledge, this is the first such study to model the agglomeration dynamics of CHO cells in acoustic standing waves.
The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of ...biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host‐ or biopharmaceutical‐specific product quality obstacles.
In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools. We focus on genes for knockout/knockdown and overexpression for meaningful approaches to improve biopharmaceutical PTMs and discuss their applicability as well as future trends in the field.
The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality.
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