Domingos Ng

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

Identification of IgG1 variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn: Comparison of soluble receptor-based and cell-based binding assays

Clinical response to the anti-CD20 antibody rituximab has been demonstrated to correlate with the polymorphism in the FcγRIIIa receptor where patients homozygous for the higher affinity V158 allotype showed a better response rate. This finding suggests that engineering of anti-CD20 for increased FcγRIIIa affinity could result in improved clinical outcome. To identify variants with increased affinity to FcγRIIIa, we developed quantitative assays using soluble receptors as well as engineered cell lines expressing FcγRI or FcγRIIIa on the cell surface. We assayed a set of anti-CD20 IgG(1) variants that had identical Fab regions, but alterations in the Fc regions, in both the soluble receptor-based and cell-based FcγRIIIa binding assays. We obtained similar relative binding affinity increases and assay precisions. The increase in affinity for FcγRIIIa correlated with the increase in activity in the antibody-dependent cellular cytotoxicity assay. These variants had unaltered FcγRI binding. In addition to Fcγ receptors, IgG also binds to FcRn, the receptor responsible for the long circulating half-life of IgG. The mutations in the anti-CD20 variants were previously found not to affect FcRn binding in the soluble receptor-based assays; consequently, we used anti-Her2 variants with different binding affinities to FcRn to study FcRn binding assays. We generated a cell line expressing FcRn on the cell surface to measure IgG binding and obtained similar ranking of these anti-Her2 variants in the cell-based and the soluble receptor-based FcRn binding assays. In conclusion, both the soluble receptor-based and cell-based binding assays can be used to identify IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn.

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)‐enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND‐enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies—mAb1, mAb2, and mAb3—at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND‐enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development.

An efficient route to bispecific antibody production using single-reactor mammalian co-culture

ABSTRACT Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho β that is being developed for type 2 diabetes and other obesity-linked disorders.