Maureen Spearman

Enhanced Production of Monomeric Interferon‐β by CHO Cells through the Control of Culture Conditions

The enhancement of recombinant protein expression of a transfected cell line is essential for the development of an efficient large‐scale bioprocess. The effect of various media additives and temperature conditions were studied in an attempt to optimize protein production, stability, and protein glycosylation from a Chinese hamster ovary (CHO) cell line producing human β‐interferon (Hu‐β‐IFN). We observed a decrease in the ELISA response of the glycoprotein in the later stages of batch cultures, which was attributed to molecular aggregation. Cells were subjected to various concentrations of glycerol, dimethyl sulfoxide (DMSO), and sodium butyrate (NaBu) in a variety of culture systems and conditions. The addition of both NaBu and DMSO resulted in higher specific productivities but reduced growth rates that resulted in a net reduction of interferon produced. Glycerol appeared to stabilize the secreted β‐IFN, resulting in reduced aggregation, despite a decrease in cell growth rate. Glycosylation analysis of isolated β‐IFN showed a time‐dependent decrease in sialylation in batch culture that was ameliorated by the presence of glycerol. Low‐temperature conditions (30 °C) had the greatest effect on productivity with a significant increase in β‐IFN titer as well as a reduction in the degree of molecular aggregation.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25mM) of cultures established at high density (>10(6)ml(-1)) over 24h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation of glucose for the full 24h resulted in a 45% non-glycosylated Mab fraction. Analysis of steady state levels of intracellular lipid-linked oligosaccharides (LLOs) showed that under glucose limitation there was a reduction in the amount of full length LLO (Glc3Man9GlcNac2), with a concomitant increase in the smaller mannosyl-glycans (Man2-5GlcNAc2). Glycan microheterogeneity was quantified by galactosylation and sialylation indices (GI and SI) which showed a direct correlation to the cell specific glucose uptake. The GI increased to 0.83 following media supplementation with a cocktail of uridine, manganese and galactose. This is significantly higher than for a fully humanized antibody (DP12) produced under the similar conditions or for similar antibodies reported in the literature. The high GI of the chimeric antibody (EG2) may be due to its low molecular weight and unusual structure. These findings are important in relation to the low substrate that may occur in fed-batch cultures for Mab production.

Antisense oligodeoxyribonucleotide inhibition of TGF-β1 gene expression and alterations in the growth and malignant properties of mouse fibrosarcoma cells

Transforming growth factor (TGF-beta) is a family of multifunctional signalling molecules that play a fundamental role in both normal and malignant cell behavior. Procedures that alter mouse TGF-beta 1 gene expression provide an important approach for analyzing the complex regulatory processes associated with this member of the growth factor family. Therefore, we have designed oligodeoxyribonucleotides (oligos) in an antisense orientation, which are complementary to regions of the TGF-beta 1 message, in an attempt to obtain an oligo sequence that specifically reduces TGF-beta 1 synthesis. We observed that oligos containing a mixture of phosphorothioate and phosphodiester linkages were less toxic and more specific when compared to those only containing phosphorothioate. A non-toxic sequence was identified that markedly reduced the levels of TGF-beta 1 in oligo-treated malignant mouse fibrosarcoma cells. The invasive and metastatic properties of these fibrosarcoma cells were also significantly decreased following treatment with the antisense oligo. The results indicate an important role for altered TGF-beta 1 expression in the regulation of malignant cell proliferation, invasion and metastasis. These results also indicate that this oligo sequence is a useful tool for studies directed towards understanding the complex relationships between TGF-beta 1 and cellular regulation.

Studies on the effect of glycoprotein processing inhibitors on fusion of L6 myoblast cell lines

The effect of oligosaccharide processing inhibitors on the fusion of L6 myoblasts was studied. The glucosidase inhibitors, castanospermine, 1-deoxynojirimycin and N-methyl-deoxynojirimycin were potent inhibitors of myoblast fusion, as was the mannosidase II inhibitor, swainsonine. Inhibition of fusion was reversed when inhibitors were removed. However, the mannosidase I inhibitor, 1-deoxymannojirimycin did not inhibit fusion. Changes in cell membrane oligosaccharide structure were followed by monitoring the binding of concanavalin A (conA) and wheat germ agglutinin (WGA) to cell surface membranes in cells treated with processing inhibitors. All the processing inhibitors resulted in increased binding of conA and decreased binding of WGA; this is consistent with the known mechanisms of inhibition of the inhibitors used in the study. Inhibition of fusion by the processing inhibitors also resulted in reduced activities of creatine phosphokinase, an enzyme used as a marker for biochemical differentiation during fusion. Treatment of a non-differentiating conA-resistant cell line with processing inhibitors did not induce fusion, but the cells did show altered lectin-binding properties. The main conclusion drawn from these studies is that cell surface glycoproteins probably containing the mannose (Man)9 structure are important for the fusion reaction.

High productivity of human recombinant beta-interferon from a low-temperature perfusion culture.

Recombinant human interferon-beta (β-IFN), used in the therapeutic treatment of multiple sclerosis (MS), can be produced on a large-scale from genetically engineered Chinese hamster ovary (CHO) cells. However, its hydrophobicity causes non-reversible, molecular aggregation in culture. The parameters affecting aggregation were determined to be concentration, culture residence time, temperature and glycosylation. Although the protein can be produced in Escherichia coli in a non-glycosylated form, the addition of glycans confers a reduced rate of aggregation as well as a 10-fold higher bioactivity. We report on the application of a low temperature perfusion culture designed to control the parameters that cause aggregation. In this three-phase culture system there is a transition to a low temperature (32°C) in a batch mode prior to implementing perfusion at 1 volume/day using an acoustic cell separator. Perfusion at the low temperature resulted in a 3.5-fold increase in specific productivity and a 7-fold increase in volumetric productivity compared to the batch culture at 37°C. The percentage aggregation of β-IFN was reduced from a maximum of 43% in batch culture to a minimum of 5% toward the end of the perfusion phase. The glycosylation profile of all samples showed predominantly sialylated biantennary fucosylated structures. The extent of sialylation, which is important for bioactivity, was enhanced significantly in the perfusion culture, compared to the batch culture.

Production and Glycosylation of Recombinant β‐Interferon in Suspension and Cytopore Microcarrier Cultures of CHO Cells

Microcarriers are suitable for high‐density cultures of cells requiring surface attachment and also offer the advantage of easy media removal for product recovery. We have used the macroporous microcarriers Cytopore 1 and 2 for the growth of CHO cells producing recombinant human β‐interferon (β‐IFN) in stirred batch cultures. Although these cells may grow in suspension, in the presence of Cytopore microcarriers they become entrapped in the inner bead matrix where they can be maintained at high densities. Cell growth rates were reduced in microcarrier cultures compared to suspension cultures. However, the β‐IFN yield was up to 3‐fold greater as a result of an almost 5‐fold higher specific productivity. Maximum productivity was found in cultures containing 1.0 mg/mL of Cytopore 1 or 0.5 mg/mL of Cytopore 2 with a cell/bead ratio of 1029 and 822, respectively. β‐IFN molecules aggregated in the later stages of all cultures, causing a decrease in response by ELISA. However, the degree of aggregation was significantly less in the microcarrier cultures. The N‐linked glycans from β‐IFN were isolated and analyzed by normal phase HPLC. There was no apparent difference in the profile of glycans obtained from each of the suspension and Cytopore culture systems. This suggests that Cytopore microcarriers may be useful in bioprocess development for enhanced recombinant glycoprotein production without affecting the glycosylation profile of the protein.

Production of α2,6-sialylated IgG1 in CHO cells

The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgGs effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fcs amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.

Effects of nutrient levels and average culture pH on the glycosylation pattern of camelid-humanized monoclonal antibody.

The impact of operating conditions on the glycosylation pattern of humanized camelid monoclonal antibody, EG2-hFc produced by Chinese hamster ovary (CHO) cells has been evaluated by a combination of experiments and modeling. Cells were cultivated under different levels of glucose and glutamine concentrations with the goal of investigating the effect of nutrient depletion levels and ammonia build up on the cell growth and the glycoprofiles of the monoclonal antibody (Mab). The effect of average pH reduction on glycosylation level during the entire culture time or during a specific time span was also investigated. The relative abundance of glycan structures was quantified by hydrophilic interaction liquid chromatography (HILIC) and the galactosylation index (GI) and the sialylation index (SI) were determined. Lower initial concentrations of glutamine resulted in lower glucose consumption and lower cell yield but increased GI and SI levels when compared to cultures started with higher initial glutamine levels. Similarly, reducing the average pH of culture resulted in lower growth but higher SI and GI levels. These findings indicate that there is a tradeoff between cell growth, resulting Mab productivity and the achievement of desirable higher glycosylation levels. A dynamic model, based on a metabolic flux analysis (MFA), is proposed to describe the metabolism of nutrients, cell growth and Mab productivity. Finally, existing software (GLYCOVIS) that describes the glycosylation pathways was used to illustrate the impact of extracellular species on the glycoprofiles.

The inhibition of platelet aggregation of metastatic H-ras-transformed 10T12 fibroblasts with castanospermine, an N-linked glycoprotein processing inhibitor

A series of T24-H-ras-transformed 10T1/2 fibroblasts with varying metastatic potential was tested for the ability to aggregate platelets. Results indicate that although platelet activation was always detected in the highly metastatic cells, some non-metastatic cells also have the ability to cause platelet aggregation, suggesting that this is a necessary but not sufficient characteristic of the metastatic phenotype. Apyrase, an ADP scavenger, effectively inhibited platelet aggregation by metastatic cells, however, there was no significant increase in ADP secretion or relation to the ability of the tumor cells to activate platelets. Hirudin, a thrombin inhibitor, did not affect aggregation, suggesting that the pathway of activation is thrombin-independent. The glycoprotein processing inhibitor, castanospermine, which reduces glycosidase I activity and metastatic capability, inhibited the ability of metastatic cells to cause platelet aggregation. However, another inhibitor of oligosaccharide processing, swainsonine, which inhibits mannosidase II activity and does not reduce metastasis, had no effect on platelet aggregation. These results show that the integrity of N-linked oligosaccharide structure of glycoproteins is an important feature of the ability of ras-transformed fibroblasts to activate platelets.

Tuning a MAb glycan profile in cell culture: Supplementing N-acetylglucosamine to favour G0 glycans without compromising productivity and cell growth.

Glycosylation is a critical quality attribute of many therapeutic proteins, particularly monoclonal antibodies (MAbs). Nucleotide-sugar precursors supplemented to growth medium to affect the substrate supply chain of glycosylation has yielded promising but varied results for affecting glycosylation. Glucosamine (GlcN), a precursor for N-acetylglucosamine (GlcNAc), is a major component of mammalian glycans. The supplementation of GlcN to CHO cells stably-expressing a chimeric heavy-chain monoclonal antibody, EG2-hFc, reduces the complexity of glycans to favour G0 glycoforms, while also negatively impacting cell growth. Although several researchers have examined the supplementation of glucosamine, no clear explanation of its impact on cell growth has been forthcoming. In this work, the glucosamine metabolism is examined. We identified the acetylation of GlcN to produce GlcNAc to be the most likely cause for the negative impact on growth due to the depletion of intracellular acetyl-CoA pools in the cytosol. By supplementing GlcNAc in lieu of GlcN to CHO cells producing EG2-hFc, we achieve the same shift in glycan complexity with marginal impacts on the cell growth and protein production.