Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated     

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     

Growth and interferon-γ production in batch culture of CHO cells

The relationship between growth and interferon- (IFN-) production in the recombinant cell line CHO 320 was studied by varying the foetal calf serum (FCS) concentration. The specific growth rate varied with the initial FCS concentration in a manner which could be well fitted by the Monod model. TheK _s and _max values were found to be 0.771% (v/v) serum and 0.031 h^–1 respectively. The average specific IFN- production rates during the exponential phase increased with increasing FCS concentration. A good correlation between specific production rate and specific growth rate was found in all phases of the culture except the lag phase and it was clearly demonstrated that IFN- production was growth associated. Specific glucose and glutamine utilisation rates were inversely related to specific growth rates.     

CHO cell growth and recombinant interferon-γ production: Effects of BSA, Pluronic and lipids

The role of bovine serum albumin in mammalian cell cultures and the possibility of its substitution by other components in a serum-free medium has been investigated. In this study, BSA was shown to be important for growth and product formation in CHO cells expressing recombinant human interferon-. There were indications that its stimulating growth effect was dependent on the source of BSA used and probably was related to the purification procedure used for the production of the desired albumin fraction. Cell growth did not occur in the absence of BSA but at low concentration (1 mg ml^–1) it was stimulated by the addition of a combination of a commercial lipid mixture plus Pluronic F68. However, under the latter conditions IFN- production was adversely effected. The importance of individual lipid components was investigated using a statistical approach based on a Plackett-Burman design. Linoleic acid was identified as a positive variable for cell growth while cholesterol was identified as a negative variable for both cell growth and IFN- production. When a combination of linoleic acid plus Pluronic F68 was included in the formulation of low BSA medium, cell growth was similar to that at high BSA concentration (5 mg ml^–1) but the IFN- concentration was significantly reduced (ca. 45%).Abbreviations IFN- interferon- - CHO Chinese Hamster Ovary cells - BSA bovine serum albumin - FAF-BSA fatty acid-free bovine serum albumin     

Genetic engineering of CHO cells producing human interferon-γ by transfection of sialyltransferases

Natural human interferon- (hIFN-) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN- in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: 1,6-D-mannoside 1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN- having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN- having GlcNAc1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Gal1-4GlcNAc1-3)n was observed. In the present study, we introduced mouse Gal1-3/4GlcNAc-R 2,3-sialyltransferase (ST3Gal IV) and/or rat Gal1-4GlcNAc-R 2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of 2,3-ST and/or 2,6-ST. The extent of sialylation of hIFN- from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of 2,3- and 2,6-sialic acid corresponded to the activity ratio of 2,3-ST to 2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells.     

Use of cell cycle analysis to characterise growth and interferon-γ production in perfusion culture of CHO cells

The importance of cell cycle analysis in cell culture development has been widely recognised. Whether such analysis is useful in indicating future performance of high cell density culture is uncertain. Using flow cytometric approach to address this question, we utilised the fraction of cells in the S phase to control specific growth rate and productivity in spin filter perfusion cultures and found a significant increase in the accumulated interferon-γ over that obtained from the nutrient-based controlled fed culture. While a general decrease with time exists in both percentage of S phase cells and specific growth rate, a clear oscillatory behaviour of both parameters is found in perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.     

Industrial production of α-amylase by genetically engineered Bacillus

A genetically engineered Bacillus subtilis strain (ALKO 84) has been introduced for industrial production of α-amylase. This strain carries the α-amylase gene from a traditionally developed production strain B. amyloliquefaciens (ALKO 89) on the multicopy plasmid pUB110.⁸At laboratory scale the recombinant strain ALKO 84 produced in industrial medium about twice as much α-amylase as the traditional strain ALKO 89. The process for production of the enzyme was scaled-up to 60m³. At this scale B. subtilis ALKO 84 retained its relative superiority to B. amyloliquefaciens ALKO 89, producing about 85% of the activity obtained at laboratory scale. Stability of the recombinant plasmid was found acceptable during the large-scale cultivations with over 90% of cells retaining plasmid-encoded characteristics throughout.     

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 IgG''s 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 Fc''s amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.     

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 IgG's 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 Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.     

Optimization of α-amylase and glucoamylase production by recombinant strains ofSaccharomyces cerevisiae

Summary Replacement of the regulatory sequence of theBacillus amyloliquefaciens α-amylase gene (AMY1) by the yeast alcohol dehydrogenase gene promoter (ADC1 _p) resulted in increased levels of extracellular α-amylase production inSaccharomyces cerevisiae. Negative regulation of glucoamylase synthesis by theSTA10-encoded repressor was alleviated by replacing the nativeSTA2 gene promoter fromS. cerevisiae var.diastaticus withADC1 _p. Enhanced degradation of starch was achieved when the modified versions of theAMY1 andSTA2 genes were introduced jointly intoS. cerevisiae.     

Continuous production of α-peptide using immobilized recombinant yeast cells: A model for continuous production of foreign peptide by recombinant yeast

α-Peptide, a portion of Escherichia coli β-galactosidase, was cloned downstream of the yeast α-factor promoter and the signal peptide by one of the authors. In this study, we utilized recombinant yeast cells, transformed the α-peptide secretion vector and attempted continuous production of α-peptide as a model of foreign peptide production. The continuous production of α-peptide was performed by using immobilized recombinant yeast cells on a column reactor, after characterizing the secretion, using minimal and complex medium. Utilizing minimal medium, with a productivity of 100 000 U h⁻¹ l⁻¹, α-peptide was continuously produced for more than 200 h. We then attempted to improve the productivity of α-peptide by alternating minimal and complex medium. Utilizing this medium changing method, 1.4 times higher α-peptide was produced during 150 h of operation compared with that achieved only by feeding minimal medium.     

Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-β in serum-free suspension culture of CHO cells

The Bombyx mori 30Kc gene is known to have anti-apoptotic activity and can enhance the cell growth and expression of recombinant proteins in anchorage-dependent CHO cell cultures. In this study, an interferon-β (IFN-β)-producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed to investigate the effect of 30Kc6 expression on the production of IFN-β in serum-free suspension culture. The 30Kc6 expressing cell line showed lower apoptotic activity and prolonged cell viability under apoptotic conditions induced by the addition of sodium butyrate, staurosporine, or the removal of serum. The 30Kc6 expressing cell line also suppressed the loss of mitochondrial membrane potential induced under these conditions. It was observed that viability, and production of IFN-β were also enhanced by 30Kc6 expression in serum-free suspension cultures. These results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.     

Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coli using size exclusion chromatography

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×10⁷ IU/mg.     

Monitoring proteolysis of recombinant human interferon-γ during batch culture of Chinese hamster ovary cells

Proteolytic cleavage of recombinant human interferon- (IFN-) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN- was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN- polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN- polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln¹³³-Met¹³⁴. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln¹³³ occurred during the later stages of the culture resulting in a heterogeneous IFN- polypeptide population with 'ragged' C-termini.     

Enhanced production of α-ketoglutarate by fed-batch culture in the metabolically engineered strains of Corynebacterium glutamicum

The fed-batch culture system was employed to enhance production of α-ketoglutarate (α-KG) by the strainsof Corynebacterium glutamicum, whose genes encoding the key enzymes responsible for the biosynthesis of L-glutamate from α-KG were deleted. In a shake flask fermentation, C. glutamicum JH110 in which the 3 genes, gdh (encoding glutamate dehydrogenase), gltB (encoding glutamate synthase), and aceA (encoding isocitrate lyase) were disrupted showed the highest production of α-KG (12.4 g/L) compared to the strains JH102 (gdh mutant), JH103 (gltB mutant), and JH107 (gdh gltB double mutant). In the fed-batch cultures using a 5 L-jar fermenter, the strain JH107 produced more α-KG (19.5 g/L), but less glutamic acid (23.3 g/L) than those produced by the parent strain HH109, as well as JH102. The production of α-KG was significantly enhanced and the accumulation of glutamicacid was minimized by the ammonium-limited fed-batch cultures employing C. glutamicum JH107. Further improvement of α-KG production by the strain JH107 was achieved through the ammonium-limited fed-batch culture with the feeding of molasses, and the levels of α-KG and glutamic acid produced were 51.1 and 0.01 g/L, respectively.     

Stability of recombinant bovine interferon-γ antiviral activity in the absence of stabilizing additives

The stability of recombinant bovine interferon-γ (rbIFN-γ) produced by a baculovirus expression system was investigated under different storage conditions: freezing-thawing and storage for 30 days at temperatures of -80, 4, 25, and 37°C. Antiviral activity was not significantly decreased by freeze-thawing at least five times. Furthermore, although not statistically different, antiviral activity gradually decreased as temperature increased. These findings suggest that rbIFN-γ possesses high thermal and freeze-thaw stability.     

Surface α2-3- and α2-6-sialylation of human monocytes and derived dendritic cells and its influence on endocytosis

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that α2-3-sialylated O-glycans and α2-6- and α2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented α2-6-sialylated N-glycans; ST3Gal1 contributes for the α2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased α2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.     

Identification of cytosine-phosphorothioate-guanine oligodeoxynucleotide sequences that induce interferon-γ production in feline immune cells

Unmethylated CpG-ODN are known to enhance Th1-type immune response. However, optimal sequences of CpG-ODN for activating Th1-type immune cells vary among species. It is necessary to identify the effective CpG-ODN sequences in each species. In the present study, in order to identify the sequences of CpG-ODN that produce fIFN-γ in cats, 14 kinds of ODN were synthesized and examined regarding their ability to induce fIFN-γ in feline PBMC and splenocytes. It was shown that some CpG-ODN significantly induced fIFN-γ production in splenocytes, but not in PBMC. We found that three kinds of CpG-ODN (no. 2, 5'-ggTGCATCGATGCAGggggG-3'; no. 5, 5'-ggTGCGTCGACGCAGggggG-3'; no. 10, 5'-ggTGCTACGTAGCAGggggG-3') specifically and significantly induced fIFN-γ production in feline splenocytes. The reverse sequences, GpC-ODN, do not cause significant fIFN-γ production. The fIFN-γ production inductivity of a mixture of CpG-ODN nos. 2, 5 and 10 was higher than those of individual CpG-ODN. When the CpG-ODN mixture was encapsulated in an MCL and administrated to cats, the number of fIFN-γ(+) cells in PBMC significantly increased. CpG-ODN nos. 2, 5 and 10 should be useful to elicit a Th1-type immune response as a vaccine adjuvant in cats.