Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ

The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for 2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat 2,6-ST was expressed in a recombinant CHO cell line making interferon-, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon- being linked in the 2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of 2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN- productivity or other aspects of IFN- glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.Abbreviations AT-III antithrombin-III - CHO Chinese hamster ovary - dhfr dihydrofolate reductase - EPO erythropoietin - IFN- human interferon- - NEO neomycin - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - ST sialyltransferase - tPA tissue plasminogen activator     

Rapid protein production using CHO stable transfection pools

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post‐transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence‐activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three‐fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin‐opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level

The molecular heterogeneity of recombinant humanerythropoietin (EPO) increased during the course of abatch culture of transfected Chinese hamster ovary(CHO) cells grown in serum-free medium. This wasshown by both an increased molecular weight and pIrange of the isolated EPO at the end of the culture. However, analysis of the N-glycan structures of themolecule by fluorophore-assisted carbohydrateelectrophoresis (FACE) and HPLC anion exchangechromatography indicated a consistent pattern ofglycosylation. Seven glycoforms were identified, thepredominant structure being a fully sialylatedtetra-antennary glycan. The degree of sialylationwas maintained throughout the culture. Analysis ofthe secreted EPO indicated a time-dependent increasein the molecular weight band width of the peptideconsistent with proteolytic degradation. A highglutamine concentration (16–20 mM) in the culturedecreased the apparent degradation of the EPO.     

The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures

To investigate the effect of dextran sulfate (DS), a widely used anti‐aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM‐0.025 and CS13‐1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13‐1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13‐1.00 cells, and showed the highest maximum mAb concentration. For SM‐0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N‐glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13‐1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3–1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113–1122, 2016     

Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ibα

A fragment (residues His1-Val289) of the chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, and inhibitor of N-linked glycosylation. The recombinant GP Ib fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 g of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ib fragment.Abbreviations ABTS 2.2-azino-di-(3-ethylbenzthiazoline sulphonate) - CHO Chinese hamster ovary - dhfr dihydrofolate reductase - GP Ib-IX glycoprotein Ib-IX complex - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - IEF isoelectric focusing - Ig immunoglobulin - mAb monoclonal antibody - MEM minimum essential medium - PMSF phenyl-methylsulfonyl fluoride - SDS sodium dodecyl sulfate - vWF von Willebrand factor