Biotechnology and biopharmaceuticals have emerged as a multidisciplinary area covering many aspects of biology. The emergence of rDNA based protein therapeutics is a major contribution to health care, yet today the number of small chemical entities that are in the list of prescription drugs out number protein therapeutics. On the other hand it is accepted that biologics is the future, the question that then arises is why this disparity?
A better understanding of proteins, especially when they are expressed outside their native environment will mitigate such issues to a large extent. Most of the proteins are glycosylated and different patterns of glycosylation confers different functional roles to these proteins. Expression vectors have inherent glycosylation capabilities that are different from the native pattern. This therefore brings variability in their function and immunological responses making it imperative to replicate the natural glycosylation patterns. The structural diversity of natural glycoproteins would require the construction of novel cell factories for the production of improved recombinant human therapeutics.
Polypeptide linked carbohydrates ( oligosaccharides ) play an important role as signals in many biological recognition processes : in folding, intracellular transport, regulated secretion of proteins, their in vivo half life, and the targeted transport to specific organs within the organism .In addition the oligosaccharide components of proteins and lipids on the surface of viruses and pathogens are involved in recognition within cells and cell types. Glycans participate in cell -cell interactions, in maintaining tissue integrity and adhesion phenomena of circulating cells in higher organisms.
Understanding the dynamics of glycosylation of proteins and glycolipids - programmed by the regulated expression of glycosylation machinery in the cells during embryogenesis tissue differentiation and alteration to pathological state (inflammation, tumorogenesis, and metastasis) is of importance in all biomedical research and may help answer basic biological and application related questions. Biopolymers such as nucleic acid and poly peptides are linearly connected entities and therefore simpler to elucidate their structure as compared to oligosaccharide structures that are based on glycan linkages which are based on different linkage types and on the branching of the monosaccharide building blocks.
Research on glycosylation of proteins dates back to the eighties when Interferon beta had to be produced in mammalian cells as interferon produced in E.coli was non glycosylated. All proteins expressed in E.coli will be only in the unglycosylated form as they are deposited as insoluble aggregates in the host inclusion bodies because bacteria do not have the appropriate enzyme machinery to attach oligosaccharides to polypeptides.
Interferon beta was the first recombinant human glycoprotein therapeutic for which the oligosaccharide structure was described. The carbohydrate chain is essential for proteins such as Interferon beta and EPO for their biological and medicinal properties. Today a number of analytical methodslike GC MS, ESI MS/MS, and MALDI MS are available that enable the determination ofmacromolecular structures of glycoproteins from both recombinant and natural sources.
Glycosylation patterns of a protein is tissue specific, further the clearanceand half life of glycoprotein in the serum of mammals is in part regulated by carbohydrate specific receptors that are present on the surface of hepatocytes and other cells
The biological and medicinal properties of glycoproteins are closely related to the type of oligosaccharide structural motifs for each host cell glycotransferases that are located in the golgi compartment of the cells. Each specific carbohydrate linkage chain is catalysed by a specific enzyme although overlapping substrate specificities are observed.
Two types of protein glycosylation can be distinguished:
1. N-glycosylation where complex structures consisting of 7-20 or more monosaccharide units are attached via amide nitrogen of an Aspargine side chain of polypeptide.
2. O-glycosylation of proteins which is less complex, where either the hydroxyl side chain of serine or threonine residue s are modified by small oligosaccharides with GalNAc or GlcNAc as the first monosaccharide building block .
Further chain elongation is achieved by 2-3 additonal monosaccharide units. This is achieved by action of specific glycosyltransferases using nucleotide sugar donor substrates.The typical modification for each protein is accomplished with the characteristic outer oligosaccharide structural motif for each host cell by the action of glycosidases and sequential addition of new monosaccharides units catalyzed by specific glycotransferases that are localized in the golgi compartments of the cells.
Glycoprotein preparation from natural sources contain a large number of protein glycoforms, i.e. a given polypeptide chain can bear structurally different oligosaccharide chains a phenomenon that results in microheterogenity of glycoproteins and hence suggests differences in their biological and physico-chemical properties. It is hence difficultto determine the mostbiologically active glycoproteinform within a given preparation.
Glycosylation patterns of a protein is tissue specific, further the clearanceand half life of glycoprotein in the serum of mammals is in part regulated by carbohydrate specific receptors that are present on the surface of hepatocytes and other cells. A central role in the clearance of circulating glycoproteins can be assigned in particular to the asialoglycoproteins receptor.At least five oligosaccharide receptors of different monosaccharide specificity have been described on the surface of liver cells. Body fluids such as blood, cerebrospinal fluid lymph etc contain a large number of proteins/glycoproteins components and represent dynamic systems whose composition in healthy organisms must be precisely regulated under different physiological conditions.
The regulation of the concentration of each specific glycoprotein form in the body fluids is subject to complex mechanisms which are so far only partially understood.
The in vivo active forms of medically used recombinant glycoprotein therapeutic s should be "human identical "or at least should have structures as similar as possible to natu-rally occurring glycoforms. Because natural glycoproteinseg those isolated from natural sources are characterized by a significant microheterogenity of their oligosaccharide structures and have structures which depend on differentiation phenomenon of the cells in which they are synthesized. It is often difficult to define the correct/ optimal glycoforms and subsequently to produce them by recombinant techniques for clinical application.
A challenge for the biotechnological production of medicinal glycoprotein in recombinant expression systems is the peripheral modification of the heterologous polypeptide with carbohydrate structural motifs. This occurs in a host cell specific manner. This means that a host cell chosen for production will decorate the outer antennae of oligosaccharides with monosaccharide building blocks in the linkage type that results from specific terminal glycosyltransferases activity that are expressed by the given host .therefore in many cases recombinant glycoprotein can differ significantly from those isolated from natural sources or that might be optimal for in vivo application.
Unlike DNA, RNA and polypeptide there are no known templates encoding the structural variety of carbohydrates, thus their molecular structural elucidation would require sensitive methodologies
The serum half life, the antigenicproperties, stability, and the interaction with specific receptors of glycoprotein's is significantly influenced by peripheral glycosylation motifs present. It is therefore necessary to carefully investigate these parameters for each recombinant product to guarantee its optimal efficacy. in human therapeutic applications, and to optimize manufacturing processes if necessary.
Insect cell lines, using baculovirus vectors and higher fungi cannot be used as hosts if the recombinant production of glycoproteins if the carbohydrate structure is important for its biological function, although these expression systems have advantages over mammalian systems. Their higher productivity and cost of production is compromised as the carbohydrate structures from these products are not compatible with the human clearance mechanism. They have antigenic properties that are different .complex type oligosaccharide structural motifs which contain important biological information in mammalian systems are not synthesized in these hosts.
The posttranslational modification properties of the most frequently used host cells for the production of human therapeutics can give rise to products with oligosaccharide structural motifs that are different from their natural counter parts. The therapeutic properties and quality of a recombinant glycoprotein is therefore in part predetermined by the expression of glycotransferases of the host cells used.
The construction of new cell lines with specific novel glycosylation properties via the expression of specific human glycotransferases together with secretory glycoproteins represents a promising alternative for producing polypeptides with a desired glycosylation pattern and should eventually improve their properties when to be used in patients. In general, the design of the host cells withmodified glycosylation properties by transfectionwith foreign glycotransferases genes should take into consideration possible competition between the newly introduced transferases and the endogenous enzymes for the same acceptor oligosaccharide motifs in glycoprotein substrates.
So far the significance of the structural variety of carbohydrates has been poorly understood. Unlike DNA, RNA and polypeptide there are no known templates encoding the structural variety of carbohydrates, thus their molecular structural elucidation would require sensitive methodologies that can describe diversity under different physiological states of cell in normal and diseased states. An interdisciplinary approach is required to identify the relevant structural functional relationships. Micro-analytical methods to be developed and combined with suitable biological and immunological methods would lead to a new era and dimension in the safe use of recombinant protein therapeutics.
(The author is former director and chief scientific officer at Magene Life Sciences)