Jürgen Hemberger

Tailoring recombinant protein quality by rational media design

Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C‐ & N‐terminal modifications), aggregates, low‐molecular‐weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high‐throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function.

Studies on the enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester.

The enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester, the key intermediate in the stereoselective synthesis of a statinanalogue, was studied with Hansenula anomala and Hansenula silvicola. Using whole cells of H. anomala gives complete conversion and a diastereomeric excess of 88% of the desired 3S, 4S statinanalogue. The strain contains two NADPH-dependent oxidoreductases, that can be separated by ion exchange chromatography or gelfiltration, yielding the 3S, 4S or 3R, 4S stereoisomers, respectively, with > 99% diastereomeric excess (DE). In the crude extract the 3S, 4S oxidoreductase is very unstable and could be purified with << 1% yield only. In contrast, H. silvicola, which gave poor conversions using whole cells, exhibited about 80-fold higher specific activity in the crude extract than H. anomala. The NADPH-dependent oxidoreductase was purified 317-fold in 12% yield. A single enzyme of 54 kDa reduces the substrate with 97.4% DE. Besides the statinanalogue a wide range of other compounds could be reduced, most notably diones and chinones such as isatin or campherchinone. It was demonstrated that the enzymes often discussed for the reduction of beta-ketoesters with yeast e.g. L-3-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35), the beta-ketoreductase of the fatty acid synthase complex and also the 3-hydroxy-3-methyl glutaryl-CoA dehydrogenase (EC 1.1.1.34) are separated during the purification steps from the oxidoreductase acting on N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester. The physiological role of the new enzyme is still unknown.

Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve Biosimilar product quality

Rational and high‐throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design‐of‐experiment (DoE) of CHO fed‐batch cultures in 96‐deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in‐house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D‐optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96‐deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458.

The potential of small molecules to modulate glycosylation by media design

Background A large number of recent publications demonstrate the effect of cell culture media on post-translational modifications of recombinant proteins [1]. In this study, aiming to extend the toolbox of media design beyond the commonly known media components, a large variety of cell culture compatible chemical components such as sugars in industrial relevant Chinese hamster ovary cell lines (CHO) expressing recombinant antibodies were identified and tested. One promising novel supplement is presented hereafter.

Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation

Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.