Control of amino acid transport into Chinese hamster ovary cells.

Amino acid transporters (AATs) represent a key interface between the cell and its environment, critical for all cellular processes: Energy generation, redox control, and synthesis of cell and product biomass. However, very little is known about the activity of different functional classes of AATs in Chinese hamster ovary (CHO) cells, how they support cell growth and productivity, and the potential for engineering their activity and/or the composition of amino acids in growth media to improve CHO cell performance in vitro. In this study, we have comparatively characterized AAT expression in untransfected and monoclonal antibody (MAb)-producing CHO cells using transcriptome analysis by RNA-seq, and mechanistically dissected AAT function using a variety of transporter-specific chemical inhibitors, comparing their effect on cell proliferation, recombinant protein production, and amino acid transport. Of a possible 56 mammalian plasma membrane AATs, 16 AAT messenger RNAs (mRNAs) were relatively abundant across all CHO cell populations. Of these, a subset of nine AAT mRNAs were more abundant in CHO cells engineered to produce a recombinant MAb. Together, upregulated AATs provide additional supply of specific amino acids overrepresented in MAb biomass compared to CHO host cell biomass, enable transport of synthetic substrates for glutathione synthesis, facilitate transport of essential amino acids to maintain active protein synthesis, and provide amino acid substrates for coordinated antiport systems to maintain supplies of proteinogenic and essential amino acids.

Next-generation cell line selection methodology leveraging data lakes, natural language generation and advanced data analytics.

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 CLD 4, 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 (MI CL) 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 CLD 4 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 CLD 4 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. CLD 4 identified sub-optimal process conditions leading to increased levels of trisulfide bond that would not be identified through conventional cell line development methodologies. CLD 4 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.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct.

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde.

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.

Directed evolution of transketolase activity on non-phosphorylated substrates.

We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form L-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity.

Engineering the expression of an anti-interleukin-13 antibody through rational design and mutagenesis.

High levels of protein expression are key to the successful development and manufacture of a therapeutic antibody. Here, we describe two related antibodies, Ab001 and Ab008, where Ab001 shows a markedly lower level of expression relative to Ab008 when stably expressed in Chinese hamster ovary cells. We use single-gene expression vectors and structural analysis to show that the reduced titer is associated with the VL CDR2 of Ab001. We adopted two approaches to improve the expression of Ab001. First, we used mutagenesis to change single amino-acid residues in the Ab001 VL back to the equivalent Ab008 residues but this resulted in limited improvements in expression. In contrast when we used an in silico structure-based design approach to generate a set of five individual single-point variants in a discrete region of the VL, all exhibited significantly improved expression relative to Ab001. The most successful of these, D53N, exhibited a 25-fold increase in stable transfectants relative to Ab001. The functional potency of these VL-modified antibodies was unaffected. We expect that this in silico engineering strategy can be used to improve the expression of other antibodies and proteins.