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Developing Glycosylated Therapeutic Antibodies

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Published: Mon, 14 May 2018


The therapeutic property of an antibody relies on the extent and the form of glycosylation in that particular antibody [1]. Glyco-engineering has been developed to make the best use of this fact and thus manufacture glycosylated recombinant antibodies in a controlled manner so as to enhance its pharmacological property [1]. Mogamulizumab is the landmark in the field of glycosylation. Mogamulizumab is a glycosylated recombinant monoclonal antibody that can be used to treat patients with relapsed or refractory leukemia that is positive for CCR4, and people with lymphoma of the peripheral T-cell also or treat lymphoma of the cutaneous T-cell [2]. This makes us focus on and thus, study the glycoforms and glyco-engineering as it will open new doors to novel recombinant monoclonal antibodies [2].


Glycosylation is a post translational modification. It involves the binding of an oligosaccharide to a particular region of the fully folded protein. The pharmacokinetics of the antibody changes on glycosylation [3]. O-glycosylation and N-glycosylation are the two forms. O-glycosylation is the addition of a single monosaccharide to the polypeptide chain at the hydroxyl end of a serine or threonine residue of the fully folded protein[4]. The N-linked glycans have a major influence over the invitro therapeutic mechanisms and thus have gained a lot of interest industrially [3]. The basic structure of an antibody is the presence of two heavy and two light chains which are held together by covalent and non-covalent bonds and connected by a flexible linker [1,5]. There are two major regions on the antibody molecule, i.e the Fab region which mainly expresses specific binding sites for an antigen and the Fc region that makes reaction sites available for the ligand that are involved in clearance mechanisms. The Fc receptor for the particular ligand are found to be structurally homologous and are as follows:- FcγRI, FcγRII, FcγRIII, the C1q which is a component of complement, followed by the the neonatal Fc receptor (FcRn). The IgG-Fc portion of the antibody is a homodimer made up of the covalently bonded hinge region, the CH3 domain that is non-covalently paired. Along with it, the human igG shows to posses only one N- glycosylation site i.e the asparagine 297 located in the CH2 domain. This CH2 domain is found to be glycosylated with an oligosaccharide at its Asparagine 297 region. [1,5,6,7]The oligosaccharide might interfere with the ligand binding ability of the IgG-Fc region by causing protein conformational changes. Thus, the effector mechanisms of these receptors may get ablated.

The glycoform of the recombinant monoclonal antibodies (RMAb’s) depends on the technique used for its production and the cell conditions [1]. The currently approved and manufactured RMAb’s are usually cultured in mammalian CHO cell lines, mouse myeloma cell lines or Sp2/0 cells[1,5].

Precautions to be taken during the manufacture of glycosylated therapeutic antibodies

Small proportions of glycosylation tends to bring about tremendous changes in the properties of the RMAb’s. The extent of glycosylation must be measured and thus controlled to obtain accurate and reliable results in vivo. The glycosylated RMAb’s should not generate an immunogenic response. Validated glycoprofiling systems must ensure use of only human type glycosylation for therapeutic antibodies [8].

Process Of Glycosylation

The N-glycosylation of a molecule begins by the attachment of a precursor glycan to the protein backbone that is now present in the endoplasmic reticulum. This binding is assisted by the chaperones that also aid and monitor the proper folding of this glycoprotein. This is then transported to the golgi body to undergo processing, where, the high mannose glycans are enzymatically converted to create various glycoforms.. The variation depends on the host cell line, the conditions under which the glycosylation takes place, the enzymes used and the presence of enzyme substrates i.e the nucleotide sugar donors (NSD). The NSD are the substrates that are synthesized in the cytoplasm that then undergo processing and return to the golgi body. Thus, by manipulating the NSD processing the desired glycosylation can be brought about. Non-optimal conditions lead to production of immunogenic glycoforms from all the mentioned cell lines which are not acceptable. It has been found that the CHO cell lines are very efficient in producing IgG glycoforms that show similarity to the in vivo polyclonal IgG glycoforms. [3,5].

Some potential methods for glycoengineering

  1. Manipulating the structure of the protein
  2. Use the glycosylation machinery of a cell expression system. Example, the use of a glycosyltransferase enzyme.
  3. Isolation of the fractions that have proved to be potent glycoforms[8].


  1. Glycosylation modifies the Fc effector functions

Therapeutic antibodies of IgG have two N-glycans in the CH2 domain that is present on the Fc region. Due to the increased stability of the CH2 region in the presence of the oligosaccharide, glycosylation has been proved to positively affect the ADCC (antigen dependent cytotoxicity) and the CDC (complement dependent cytotoxicty) properties. It also leads to increased endocytosis of immune cells followed by antigen presentation and then antibody mediated phagocytosis [7].The property of, antibody stability, solubility, ability to resist the action of proteases, increased binding ability of its Fc receptor are all contributed by the glycosylation that occurs on the particular site. Apart from it, the half life of the antibody and its cellular transport is controlled by the glycosylation [6]. There is a conformational change that accompanies the attachment of the oligosaccharide to the antibody. The increase in solubility and half-life in-vivo is a result of the same conformational change imposed on the glycoprotein by the appropriate oligosaccharide [5].

  1. Increase in ADCC activity mediated by de-fucosylation.

A product Herceptin from the Genentech company, produced antibodies in CHO cell lines (LEC13) that were devoid of the fucosyl group, that was originally attached at the N-acetyl glucosamine residue. Another attempt made was towards antibodies generated in rat derived cell lines YB2/0. These antibodies did not have a fucose attached to the recombinant Anti-human IL-5 receptor antibody. Both of these, showed an enhanced ADCC activity and mediator functions, nearly 40-50 times more for the former example. The ADCC activity is due to the antigen antibody interaction at the Fc region on these cells. This is due to the increased affinity of the antibody for the FcYRIII receptor. [3,2,5]. Sometimes the presence of glycoform and the absence of fucose structure leads to productive interaction due to the lack of stearic hindrance that blocks the Fc region[9].

  1. Increase in anti-inflammatory response

The addition of a galactose residue to the end terminus of the Fc region of the IgG molecule enables it to express anti-inflammatory response. Also, addition of a 2,6-linked terminating sialyl group to the IgG leads to an anti-inflammatory response[10,3].

All of these strategies can be used to make specific monoclonal antibodies, which will surely give the desired response on administration.

Glycosylated Recombinant monoclonal antibodies

Cetuximab is a recombinant monoclonal antibody that is used as a treatment for colon, head and neck cancer. Cetuximab shows a high specificity towards the epidermal growth factor. It bears an N- linked oligosaccharide at Asn-88 of the v region. Obinutuzumab is seen to have a bisecting non fucosylated complex glycosylation at the Asp-297 on the Fc region. Apart from that, Three MAb’s that are specific to alpha (1-6) dextran have been manipulated by differing the N-glycosylation site on their CDR2 region as Asn54, Asn58 and Asn60. It was found that the antigen binding ability had increased by 10-50 times when the MAb’s were glycosylated in comparison to the non glycosylated forms. Further, the antigen binding ability of the molecules that had been glycosylated at the Asn54 and Asn58 was higher than the Asn60 glysosylated form [5].


It is very evident now, that manipulating the glycoforms of the monoclonal recombinant antibodies will surely modify its the effector response. Intelligent designing will open doors to therapies for those diseases that do not have a promising treatment. Cell engineering can be a step towards knocking-out or knocking-in the appropriate gene for glycosylation. The in vitro glycosylation can be made affordable on a commercial scale. Due to the improved properties showcased by these third generation Ab’s, the future of glycosylated antibodies looks more bright as more than 15 RMAb’s are in the pipeline[1,11].

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