Christine Jung

LDH-C can be differentially expressed during fermentation of CHO cells.

Expression of CHO mRNA was measured with special microarrays from the Consortium for Chinese Hamster Ovary (CHO) Cell Genomics led by Prof. Wei-Shou Hu of the University of Minnesota and Prof. Miranda Yap of the Bioprocess Technology Institute of A*STAR, Singapore(http://hugroup.cems.umn.edu/CHO/cho_index.html). Cultivation experiments were performed in small scale 2L stirred tank bioreactors. During fermentation a temperature shift of -3°C was performed. This was accompanied by a reduction of the cell specific lactate production rate.The analysis of transcriptome samples before and after the temperature shift with microarrays showed several changes in the expression of available gene markers. LDH-C expression raised about 2 fold after temperature shift. LDH-A did not change. As LDH-C is known to be a specialized isoenzyme in sperm cells for consuming lactate in a lactate containing milieu, LDH-C could be proposed as a target for genetic engineering, facilitating lactate consumption in the late phase of high cell density cultures and prolonging longevity of CHO production cultures by reducing lactate and base accumulation.

High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities

Backgroundα-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This “capping” of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins.ResultsHuman α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates.ConclusionsFunctional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 – 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.

Rec. ST6Gal-I variants to control enzymatic activity in processes of in vitro glycoengineering

Background Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity. The sialyl mojety (sialic acid, 5-N-acetylneuramic acid) is usually exposed at the terminal position of N-glycosylation and therefore, a major contributor to biological recognition and ligand function, e.g. IgG featuring terminal sialic acids were shown to induce less inflammatory response and increased serum half-life. The biosynthesis of sialyl conjugates is controlled by a set of sugar-active enzymes including sialyltransferases which are classified as ST3, ST6 and ST8 based on the hydroxyl position of the glycosyl acceptor the Neu5Ac is transferred to [1]. The ST6 family consists of 2 subfamilies, ST6Gal and ST6GalNAc. ST6Gal catalyzes the transfer of Neu5Ac residues to the hydroxyl group in C6 of a terminal galactose residue of type 2 disaccharide (Galb1-4GlcNAc). To our knowledge, the access to recombinant ST6GalI for therapeutic applications is still limited due to low expression and/or poor activity in various hosts (Pichia pastoris, Spodoptera frugiperda and E. coli). The present study describes the high-yield expression of two variants of human beta-galactoside alpha-2,6 sialyltransferase 1 (ST6Gal-I, EC 2.4.99.1; data base entry P15907) by transient gene expression in HEK293 cells with yields >100 mg/L featuring distinct mono(G2 +1SA) as well as bi(G2+2SA) sialylation activity. Materials and methods Two N-terminally truncated fragments of human ST6Gal-I (delta89, residues 89-406, and delta108, residues 109-406) were designed for transient gene expression (TGE): Instead of the natural leader sequence and N-terminal residues, both ST6Gal-I coding regions harbor the Erythropoietin (EPO) signal sequence in order to ensure correct processing of the polypeptides by the secretion machinery. Following cloning into pM1MT, expression of the ST6Gal-I coding sequences is under control of a hCMV promoter followed by an intron A. Sialyltransferase assays: 1. Asialofetuin was used as acceptor and CMP-9F-NANA as donor substrate. Enzymatic activity was determined by measuring the transfer of 9F-NANA to asialofetuin. 2. Recombinant humanized IgG1 and IgG4 monoclonal antibodies (mabs), characterized as G2+0SA, as well as desialylated EPO were used as targets in sialylation experiments (30 μg enzyme/300 μg target protein). Both enzyme variants of ST6Gal-I (delta89 and delta108) were used under identical reaction conditions and the sialylation status was analyzed by mass spectrometry.

Two N-terminally truncated variants of human β-galactoside α2,6 sialyltransferase I with distinct properties for in vitro protein glycosylation.

Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Km for N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.

Discerning key parameters influencing high productivity and quality through recognition of patterns in process data.

Background The adoption of Quality by Design (QbD) approach to biologics manufacturing requires fundamental understanding of complex relationship between the quality of the product, especially critical quality attributes (CQAs), and various parameters of the manufacturing process [1]. This can be approached through multivariate analysis of historical cell culture bioprocess data [2]. In this study, process parameters and raw materials data obtained from 51 runs with final titer varying from 0.8 to 2.0 units and Gal0 glycan ranging from 47.5 to 67.5% was investigated. The aim was to discover prominent patterns which may cause the spread of final process outcome.

Non-fucose level as a function of glycoenzyme transcription in a glycoengineered CHO cell line

Background and novelty Antibody-dependent cellular cytotoxicity (ADCC) is one important mode of action for therapeutic mAbs in the field of oncology. It is strongly dependent on the glycan pattern of the Fc N-glycan: low core-fucose levels (= high non-fucose levels) typically result in an increased ADCC. There are several options to reach high non-fucose levels like the selection of the right host that expresses the desired pattern, in vitro glycosylation, use of glycosylation inhibitors, alteration of process parameters and generating glycosylation mutants with modified glycan synthesis activities by cell line engineering. Within Roche Pharma we are working with CHO cell lines designed to produce therapeutic antibodies based on the GlycArt system. These cell lines contain in addition to the recombinant gene for the therapeutic monoclonal antibody (mAb), also recombinant genes for two glycosyltransferases, N-acetylglucosaminyltransferase-III (GntIII) and mannosidase-II (ManII). As a result the CHO cells produce antibodies with a modified glycosylation structure characterized by a low-fucose Fc part. The selection system for the two glycosylation enzymes is based on the use of puromycin whereas for the mAB MSX is used.