Proteasome-based selection systems for generation of recombinant CHOK1SV GS- KO™ cell lines with enhanced productivity

Chinese hamster ovary (CHO) cells are widely used industrially for the production of biotherapeutic proteins. In order to generate recombinant CHO cell lines expressing the target biotherapeutic gene(s) of interest, metabolic markers are used to select for those cells that have stably incorporated the gene(s) of interest. Such selection systems work very efficiently but do not directly select cells based upon secreted biotherapeutic recombinant protein productivity characteristics. When such biotherapeutic proteins are synthesised in eukaryotic cells, typically the polypeptide is co-translationally fed into the endoplasmic reticulum where it is folded, and if required, assembled with other polypeptides/domains, as in the case of antibodies. An overload of the capacity of the ER to fold and assemble recombinant proteins can result in upregulation of ER-associated degradation (ERAD) where unfolded or incorrectly folded or assembled material is retro-translocated out of the ER to the proteasome for degradation and recycling of amino acids. We have therefore investigated whether the susceptibility of cells to proteasome inhibitors during cell line construction when a recombinant load is placed upon the cell can be used to select for cells with a greater capacity for producing recombinant biotherapeutic proteins whilst maintaining or enhancing the quality of the material secreted. A number of proteasome inhibitors were therefore investigated, epoxomicin, MG-132 and bortezomib, at different concentrations to identify concentrations that would provide selection but not result in complete cell death. A range of concentrations was then added to CHO cells, along with MSX, during cell pool construction using Lonza’s CHOK1SV GS-KO™ proprietary host cell line to investigate whether this resulted in the generation of cell pools with enhanced productivity characteristics as compared to selection using MSX alone. Using this approach, and a number of different recombinant biotherapeutic molecules, we have found that CHO pools giving enhanced product concentrations can be generated (see Figure 1 for example) and validated this in ambr15 miniature bioreactor experiments. We have thus shown that stable transfectants derived from pools that had been cultured with proteasome inhibitors were more productive than pools generated without proteasome inhibitors. Further, stable transfectants generated using proteasome inhibitors retained their higher productivity characteristics even when the proteasome inhibitors were no longer added at subculture, meaning that proteasome inhibitors are only required in the initial stages of cell line construction. Please click Additional Files below to see the full abstract

Inhibition of protein degradation for improved production

Disclosed herein are methods and compositions useful for evaluating, selecting, identifying, or making a cell or cell line that has improved production capacity for generating higher yields of products and/or improved capacity to produce higher quality products. Products, as described herein, can include a polypeptide that is endogenously expressed by the cell, a recombinant polypeptide that is not endogenously expressed, or a non-naturally occurring recombinant polypeptide. The methods described herein include modulating, e.g., inhibiting, the protein degradation pathway by using a proteasome inhibitor, an ER-associated de-gradation (ERAD) inhibitor, or a ubiquitin pathway inhibitor

Cell Pool Selection of CHO Host and Recombinant Cell Pools by Inhibition of the Proteasome Results in Enhanced Product Yields and Cell Specific Productivity

Chinese hamster ovary (CHO) cells are the leading mammalian cell expression platform for biotherapeutic recombinant molecules yet some proteins remain difficult to express (DTE) in this, and other, systems. In recombinant cell lines expressing DTE proteins, cellular processes to restore proteostasis can be triggered when the folding and modification capabilities are exceeded, including the unfolded protein response and ER associated degradation (ERAD) and proteasomal degradation. We therefore investigated whether the proteasome activity of CHO cells was linked to their ability to produce recombinant proteins. We found cell lines with diverse monoclonal antibody (mAb) productivity show different susceptibilities to inhibitors of proteasome activity. Subsequently, we applied selective pressure using proteasome inhibitors on mAb producing cells to determine the impact on cell growth and recombinant protein production, and to apply proteasome selective pressure above that of a metabolic selection marker during recombinant cell pool construction. The presence of proteasome inhibitors during cell pool construction expressing two different model molecules, including a DTE Fc fusion protein, resulted in the generation of cell pools with enhanced productivity. The increased productivities, and ability to select for higher producing cells, has potential to improve clonal selection during upstream processes of DTE proteins

Methods of cell selection and modifying cell metabolism

: Described herein are compositions and methods for identifying, selecting, or culturing cells comprising a subject nucleic acid sequence of interest. Generally, a nucleic acid comprising a subject nucleic acid and a sequence encoding an enzyme molecule involved in biosynthesis of an amino acid is introduced into a cell. The cell is then grown on media lacking the amino acid, such that cells comprising the introduced nucleic acid are capable of growth. In some instances, the cell further comprises an inhibitor of the enzyme molecule to increase the stringency of the selection

Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc‐fusion protein produced in CHO cells

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins

Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production

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Data for engineering lipid metabolism of Chinese hamster ovary (CHO) cells for enhanced recombinant protein production

The data presented in this article relates to the manuscript entitled ‘Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production’, published in the Journal Metabolic Engineering [1]. In the article here, we present data examining the overexpression of the lipid metabolism modifying genes SCD1 and SREBF1 in CHO cells by densitometry of western blots and by using mass spectrometry to investigate the impact on specific lipid species. We also present immunofluorescence data at the protein level upon SCD1 and SREBF1 overexpression. The growth profile data during batch culture of control CHO cells and CHO cells engineered to overexpress SCD1 and SREBF1 during batch culture are also reported. Finally, we report data on the yields of model secretory recombinant proteins produced from control, SCD1 or SREBF1 engineered cells using a transient expression systems

Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production

Chinese hamster ovary (CHO) cell expression systems have been exquisitely developed for the production of recombinant biotherapeutics (e.g. standard monoclonal antibodies, mAbs) and are able to generate efficacious, multi-domain proteins with human-like post translational modifications at high concentration with appropriate product quality attributes. However, there remains a need for development of new CHO cell expression systems able to produce more challenging secretory recombinant biotherapeutics at higher yield with improved product quality attributes. Amazingly, the engineering of lipid metabolism to enhance such properties has not been investigated even though the biosynthesis of recombinant proteins is at least partially controlled by cellular processes that are highly dependent on lipid metabolism. Here we show that the global transcriptional activator of genes involved in lipid biosynthesis, sterol regulatory element binding factor 1 (SREBF1), and stearoyl CoA desaturase 1 (SCD1), an enzyme which catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, can be overexpressed in CHO cells to different degrees. The amount of overexpression obtained of each of these lipid metabolism modifying (LMM) genes was related to the subsequent phenotypes observed. Expression of a number of model secretory biopharmaceuticals was enhanced between 1.5-9 fold in either SREBF1 or SCD1 engineered CHO host cells as assessed under batch and fed-batch culture. The SCD1 overexpressing polyclonal pool consistently showed increased concentration of a range of products. For the SREBF1 engineered cells, the level of SREBF1 expression that gave the greatest enhancement in yield was dependent upon the model protein tested. Overexpression of both SCD1 and SREBF1 modified the lipid profile of CHO cells and the cellular structure. Mechanistically, overexpression of SCD1 and SREBF1 resulted in an expanded endoplasmic reticulum (ER) that was dependent upon the level of LMM overexpression. We conclude that manipulation of lipid metabolism in CHO cells via genetic engineering is an exciting new approach to enhance the ability of CHO cells to produce a range of different types of secretory recombinant protein products via modulation of the cellular lipid profile and expansion of the ER

A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e. g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such re-combinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline- containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super- transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression