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Biopharmaceutical is constantly growing depending on the uniqueness of treating a disease. Now, Biopharmaceutical industries are concentrating on therapeutic proteins i.e. majorly glycoproteins . Various proteins undergo different types of modifications during the process of translation or later. The latter modifications are referred to as 'Post-translational modifications'. These are held responsible for the changes in their structure and therapeutic properties . One of the PTM's is Glycosylation which utilizes a specific enzyme or target a particular pathway .
An enzymatic pathway in which the oligosaccharide side chains/sugar moieties are attached covalently to either the side of asparagine (N-linked) or serine/threonine (O-linked) of a protein is called glycosylation . Today there is a need for glycosylation in the clinical field in order to treat a disease efficiently as it plays a role in understanding the activity, immunogenicity, bioavailability and kinetics of the active compound . Thus, glycosylation is a pre-requisite for medical applications .
Antibodies have been proved to be very efficient as a specific protein based binding component . They are soluble serum glycoproteins .They recognize foreign bodies and eliminate them. There are five classes of human antibodies namely; IgG, IgM, IgA, IgD and IgE. These differ in their structures & function . Due to this variability they are widely used in therapeutics . Immunoglobulin G antibody is a tetrameric glycoprotein 150kDa, which is a very important class composed of two heavy and two light chains . The light chain consists of one variable chain (VL) and one constant domain (CL). The heavy chain consists of 3 constant domains (CH1, CH2, CH3) and one variable domain (VH) .There is one disulfide bond between the heavy and light chains and two or three disulfide bond depending on the type of the heavy chain. 3-D structure is conserved by the non-covalent interactions . The structure of antibody is divided into two i.e. antigen binding fragment (Fabs) and constant (Fc) region. These are interconnected by a flexible hinge region . The domains are responsible for the immunoglobulin fold. The variable region consists of 3 sections which have hypervariable sequence answerable to the loop formation. These loops are the prime section for antigen recognition and are referred as Complementarity determining region (CDRs) . The remaining amino acids in the V domain help in supporting the loop and are referred as framework residues (FR) . The natural and recombinant antibodies contain a particular consensus sequence i.e. Asn-X-Ser/Thr (where X is any amino acid except proline) . This sequence is utilized for the N-glycosylation in the constant domain (CH2) . The Asn present on 297 residue plays a role in the stability and in the functioning of the constant region . N-glycan is attached covalently to the conserved Asn residue . These N-linked sugar moiety chains of IgG are heterogeneous in nature commonly referred as microheterogeneity. The glycan complex consists of core fucose .
Figure 1: Human IgG structure shows a core fucose attached to Gn. (where, Asn = asparagine; Gn = N-acetylglucosamine; Fuc = fucose; M = mannose; Gal = galactose; NANA = N-acetyl neuraminic acid.) 
PROCESS OF N-GLYCOSYLATION
The N-glycosylation first starts at the endoplasmic reticulum (ER) where it transfers the pre-existing lipid-linked oligosaccharide onto a nascent protein backbone (Asn residue) .The glycoprotein is then transported along the secretory pathway, where it will mature. The maturation steps involve the removal of glucose and mannose residues by the action of glucosidase I/ II and mannosidase . The Golgi apparatus will then finally form the N-glycan complex . The oligosaccharide to be attached also follows a biosynthetic pathway which is conserved in animals, plants and fungi. The precursor oligosaccharide is also produced in the ER with the help of an enzyme glycosyltransferase . The misfolded glycoproteins will undergo protein degradation .The attachment will enhance properties such as self-association, solubility and protein sorting within the cell. It modifies the immunogenicity, clearance rate, ligand binding and its stability .
ANTIBODIES PRODUCED IN DIFFERENT SYSTEMS
Recombinant antibodies are produced to treat the dreadful diseases which do not have cure and most of these rIgG molecules are manufactured by in vitro cell culture methods. But due to increasing demand to produce antibodies new different methods are explored . Every expression system has certain variation, which will be discussed below.
CHO cell line
Chinese hamsters used in the laboratories as specimen showed low chromosome number and hence were ideal then in 1919 to use for tissue culture. Later, an ovary of female hamster was isolated and used in culture plates. This technique leads to faster generation times . Mouse derived cell lines such as hybridomas and NS0s are used host cells. There is an analogy seen in the glycosylation mechanism between CHO cell line and humans . The difference spotted is the absence of acetyleglucosaminyl transferase. This enzyme is the transporter for GlcNAc to complex glycan. But this variation does not affect the quality and activity significantly .Another modification is the nature of sialic acid linkages. Human IgG comprise of the Î± 2, 6-linked and CHO derived antibodies involve the Î± 2, 3- linked sialic acid. But this also doesn't affect the activity of the antibodies. In the structure of the antibody, the core fucose complex assembled on the N-acetylglucosamine consists of termination 0, 1 or 2 Gal residues denoted as G0, G1 or G2 respectively. Depending on the proportions of these residues the variations will differ from culture to culture .
Avian system are used in producing antibodies because of several advantages such as high protein production in eggs, short span to reproduce and the similarity in the machinery . But there is no data published available in literature. There might be shortcomings faced during the trials and hence further research must be done in this field .
Plants have a conserved secretory pathway similar to the mammals. The production speed and yield of the resultant product is also greater due to which it is highly preferred .It is also advantageous because it produces the similar N-glycan complex as that of mammals. Variation is seen after the addition of the precursor molecule onto the polypeptide chain. There is an addition of xylose in the Î² position of the inner most mannose and the in fucose (Î±-1,3 position) of the core . This modification in plants is undesirable due the procedure is re-considered during research .
Pichia pastoris and Saccharomyces cervisiae are widely used yeast in the homologous recombination and are successfully used in glycosylation of antibodies .By eliminating the yeast specific glycosylation the antibody can be glycosylated avoiding the hypermannosylation by the knockout of yeast mannosyltransferase. This technique was successful in producing mAb rituximab used for cancer .
ANALYSIS OF GLYCOSYLATION
Past years researchers have selected mass spectrometric method for structural analysis of the biomolecules. This method is considered to be very sensitive. The most commonly used comprise of electrospray mass spectrometry (ES-MS), matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) and gas chromatography mass spectrometry (GC-MS). [ptm biopharma] . Electrophoresis gives the glycan profile to analyse the carbohydrate portion of the molecule. ES-MS detects the ionisable molecules and can detect upto 150kDa. MALDI measures the mass of large molecules (500kDa) . Capillary electrophoresis - SDS gives a quantitative analysis and help in replacement of the gels to identify the glycan profile .
Antibody- dependent cellular cytotoxicity (ADCC) and complement- dependent cytotoxicity (CDC) are important in treating oncology. The N- acetylglucosamine present on the oligosaccharide in the conserved residue increases ADCC in vitro 100 fold times . The Fc region attaching to effector cells and leads to destruction (exocytosis) [cancer sci]. Major anti-tumor antibodies such as rituximab, trastuzuman and alemtuzumab are anti-CD53 mAb. The variations in affinity of FcÎ³RIIIa lead to a distinguishing study in patients with follicular non-hodgkin's lymphoma . CDC depends on the complement systems i.e. the classical pathway. This mechanism is functionalized when the binding of C1q to Fc domain takes place on the cell surface. Different types of antibodies are raised against HLA-II, Carcinoembryonic antigen (CEA) etc induce CDC. It is seen that rituximab attacks the tumor cells via complement dependent pathway. 
Cetuximab is an anti-tumor antibody used against the epidermal growth factor receptor. This is used for treating colorectal cancer and squamous- cell carcinoma of neck and head . Gemtuzumab and ozagamicin play a very important role in the hematogical malignancies .
mAbs are large weighing about 150 kDa. This needs machinery to keep it in their active form and produce more. Hence eukaryotic organisms are selected. Due to the alarming issue of protein degradation in a particular system large amount of antibodies are injected . This will increase the cost of the production and maintainance of the cell lines or the eukaryotic organism .
Out of the all the produced mAbs only twenty-four of them are approved by USA Food and drug administration for clinical use . Therapeutic antibodies are safe due to high specificity . A new era to these "magic bullets" will be attained by further research and development and overcome their technical difficulties the future will shine bright and reach at the top in medical fields.