Engineering Perspectives of Glycoconjugate Fermentation

There are many factors affecting the biosynthesis of carbohydrates of glycoconjugates such as the cell types, medium composition, and environmental conditions. Although comprehensive studies regarding the research area have not been reported, numerous significant findings of glycoproteins have been achieved as follows.

Choose of Appropriate Hosts and Media

Eukaryotic expression systems are required for complex recombinant proteins. Among the eukaryotic hosts, yeasts including Saccharomyes and Pichia can perform only mannosylated glycosylation, and plants do not have the enzymes to process N-linked glycosylation of oligosaccharide chains terminating with p-1, 4 galactose and /or sialic acid residues (30, 31). Thus, animal cell or incest cell cultures have opened the door for the availability of a multitude of therapeutic proteins that required complex post-modification for their biological functions.

Paradoxically, the authenticity of these recombinant proteins is still a major problem by using the biosynthetic route in the in vitro cell culture cultivation. Glycosylation is a common post-modification process influencing the authenticity of recombinant glycoproteins. In other words, natural glycoproteins usually exist as a spectrum of glycosylated forms (glycoforms), where each protein might associated with an array of oligosaccharide structures. There are several engineering approaches developed in improving or monitoring the glycosylation patterns of therapeutic glycoproteins.

Choice of complex or defined medium for recombinant protein proteins is based not only on yields of proteins but also on the quality of proteins and the cost of downstream processing. Serum was used as a growth-promoting component in the animal cell culture medium because it was a source of nutrients, hormones, growth factors and protease inhibitors. However, serum has several disadvantages. It is a mixture with respect to its chemical composition and many undesired components are present. For example, the bovine serum can be contaminated with viruses causing transmissible spongiform encephalopathy (TSE) and bovine spongiform encephalophathy (BSE) in human. Besides, high protein content of serum-containing culture media will result high cost and difficulty in downstream processes sing. Thus, adding meat hydrolysates has developed serum-free culture medium. However, these serum replacements still suffer from similar problems to those of serum (32). As a result, a chemically defined, protein-free, culture medium, has been developed to avoid the potential risk of introducing foreign agents or contamination derived from raw materials. The main challenge in designing a protein-free medium composition is to find the replacements of protein used for conventional culture media. In addition to the nutritional roles of proteins might play in the medium, the iron-carrier transferrin is replaced by EDTA or citrate (33), and the cell protection property of proteins from shear damage is replaced by the surfactant Pluronic F68 (34).

Medium Optimization

Besides the choice of medium, the chemical compositions have substantial influence on glycosylation and cell metabolism. For example, the glycosylation of the recombinant glycoprotein of NSO and CHO cells might be manipulated by controlling their intracellular nucleotide-sugar contents through medium supplementation with the nucleotide sugar precursors glucosamine and N-acetylmannosamine (35, 36). Precursor studies indicate that the aromatic rings of the aglycone of macrolides are composed of two unusual amino acids that are metabolically derived from tyrosine and acetate, respectively (37). Thus, sufficient precursor supply by adding nitrogen sources such as peptone and amino acids into the medium might improve specific macrolide production such as vancomycin, but phosphate plays a negative control on macrolide and aminoglycoside antibiotics (38- 40).

Another fermentation strategy of medium optimization is to add inhibitors to block the biosynthetic pathways of undesired by-products. For instance, amphotericin B high producing strains of Streptomyces nodosus usually produce substantial more amphotericin A than amphotericin B as a result of costly downstream processing. A parallel biosynthetic pathway in the formation of amphotericin A and B is suggested upon biosynthetic studies, and a patented fermentation process is proposed to add chloramphenicol, a protein synthesis inhibitor, into the media to prevent the formation of amphotericin A, whereas amphotericin B production is slightly affected (12).

Process Optimization

Environmental factors including dissolved oxygen concentration, osmotic pressure, and carbon dioxide concentration have significant effects on the biosynthesis of oligosaccharides and glycoproteins. Some macrolide antibiotics including erythromycin B (41) and doxorubicin (15, 42) are considered to be secondary metabolites, and the limitation of the dissolved oxygen during cultivation of various microbial strains can decrease the activity of cytochrome P-450 monooxygenases required for the processing of pathway intermediates into their final forms. It is believed that the increase in DO concentration will affect the changes in energy metabolism with a higher proportion of glucose utilized at higher oxygen concentrations. However, some results from batch fermentation contradicted each other as shown below. Sialyltransferase activity, sialic acid content, and specific productivity of a recombinant glycoprotein, human follicle stimulating hormone, in CHO cells all increased as DO increased (43). In a well-controlled bioreactor, the glycan processing of the recombinant human secreted alkaline phosphatase in insect cell-baculovirus system was inhibited at both low and high dissolved oxygen concentrations (44). In contrast, another study indicated that the N-glycosylation of an interleukin-2 variant from BHK cells did not changed under low oxygen concentration (45). In a continuous culture study, alternations of the steady state DO concentration in the serum-free hybridoma culture dramatically affected the galactosylation of the monoclonal antibody (46). Their research results indicated that a significant decrease in galactosylation of at 10% DO as compared to those under 50 and 100%.

Carbon dioxide is a metabolic byproduct of mammalian cell culture that can accumulate in a poorly ventilated culture. High carbon dioxide partial pressure is likely to affect intracellular pH even though the medium pH is controlled. High intracellular pH might alter die endoplasmic reticulum and the Golgi apparatus. Changes in the pH of these organelles could alter the protein processing including glycosylation (47). Under elevated carbon dioxide concentration, the proportion of sialic acids comprising N-glycolylneuraminic acid of recombinant tissue plasminogen activator decreased from 2.3-4% under 36 mmHg C02 to 1.5-2.2% under 250 mrnHg C02 (48).

Considering the production of oligosaccharides such as acarbose by fermentation, the process and product yields should be significantly optimized and improved in order to be economic competitive. The osmolality of the fermentation broth has been found to have a very considerable effect on the final yield of die acarbose fermentation (//). Optimal range of osmolality for acarbose production is between 200 to 600 mosmol/kg. Under osmolarity-controlled fermentation, it was estimated to have 31 % increases in final yield.

Challenge and Solutions to Maintaining Homogeneous Glycoforms of Glycoproteins

Heterogeneous glycoforms of recombinant proteins in mammalian cell cultures remains a common and complicated problem faced in current pharmaceutical. A simplied diagram of the synthesis of N-linked glycan is shown in Fig. 4 (49).

Heterogeneity of glycoforms of recombinant carbohydrate-based therapeutics has affected not only the efficacy of product but also the production cost There are several engineering approaches developed in improving or monitoring the glycosylation patterns of therapeutic glycoproteins. Recombinant human erythropoietin (rhEPO), manufactured by Amgen, is discarded 80% for years because of its inadequate glycosylation, which causes rapid clearance from the blood (22). The pharmacokinetics of ihEPOs can be improved by adding two extra N-linked oligosaccharide chains to rhEPO to from a super-sialated erythropoietic protein, darbepoetin alpha (Aranesp). Aranesp has been proven with threefold greater circulating half-life than rhEPO (50,57).

Incomplete synthesis of the N-linked carbohydrate structures on the recombinant glycoproteins is considered to the caused of heterogeneous glycoforms. Thus, the availability of sugars, the relative amounts of glycoprocessing enzymes, and die local protein structure around the glycosylation site in die cells will determine die glycoforms. Although several approaches including medium optimization, pathway engineering and process monitoring have been investigated to control the glycoforms, relatively little success in maintaining a homogeneous glycoform has been accomplished by using only fermentation techniques.

Lumen ER

Synthesis of Gtycan W lipid-linked transfer v precursor

Cytosol

Trimming and processing

Golgi

v Goigi

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