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Apr 15, 2012 (Vol. 32, No. 8)

Cracking the PTM Code

  • Post-translational modifications contribute to the structure, function, and efficacy of the therapeutic antibody. The extent to which the antibody is modified and the location of the modifications all contribute to the overall effectiveness of the biotherapeutic. What we are learning in the selection and, later, the production of these therapeutic agents is that it is critical to monitor and control the level of modifications that are found in the antibody. Any changes in the pattern of modification can have a significant effect on the antibody as a therapeutic.

    Monoclonal antibodies are the fastest growing class of human pharmaceuticals, with more than 30 mAbs approved for various therapeutic indications including oncology, inflammatory disease, and viral infection. With the guidance from immunologists like emeritus professor Roy Jefferis, Ph.D., from the University of Birmingham, the drug discovery and development of biotherapeutics is coming of age. This is because we better understand the significance of post-translational modifications in the establishment of antibody structure and function.

    Most of the therapeutic antibodies that are being developed today are produced as recombinant glycoproteins in eukaryotic cells. The key to production of a successful therapeutic is to make sure that the therapeutic antibody has the same structural characteristics as an antibody produced in the normal human immune response. During the normal human immune response, glycosylation of the Fab portion is selected for via somatic mutation as the patient shifts from primary antibody response, production of IgM class of antibody, to a secondary response, with the production of IgG class of antibody.

    Therapeutic antibodies are of the IgG class and so need to contain the proper level of glycosylation, which, in turn, sets the appropriate level of therapeutic efficacy. The type of glycosylation is also important; introduction of a fucose moiety can knock out efficacy and so needs to be avoided. Recent advances including the isolation of a CHO cell line that has been engineered to eliminate fucose transferase has eliminated this concern.

    Glycosylation patterns in the Fab portion of the therapeutic antibody are also linked to the intrinsic solubility of the antibody. “By maintaining the proper level of glycosylation in the Fab portion during production, we can assure that there will be minimal aggregation of the protein.”

    Professor Jefferis explained, “This guarantees that the antibody can be made in high concentration (>120 mg/mL), which is essential to keep the patient within therapeutic range over the course of multiple treatments for cancer therapy. This is less critical for the treatment of rheumatoid arthritis (RA), for example, where antibody concentrations can be maintained at lower levels over the course of the therapeutic regime.”

    Interestingly, glycosylation at the Fc portion of the antibody, though heterogeneous, can’t be changed by the culture conditions. Specifically, the biantennary modification in the Fc portion of the antibody is hardwired into the structure and serves the critical role of triggering downstream immunological responses, for example, when the therapeutic is bound to cancer cells. These responses include the attraction of NK cells, initiating a ADCC response and/or activation of complement. These responses ultimately effect lysis of antibody-bound cancer cells.

    In addition to the discussion about the impact of post-translational modifications on the structure and function of the biotherapeutic, Professor Jefferis added that we can’t forget about the threat that biotherapeutics, when injected in patients, might be immunogenic, inducing an allergic response and thereby precluding them from the benefit of the therapy.

    Such an allergic response can be induced if any of the modifications are of nonhuman origin. Hence the effort over the years to humanize monoclonal antibodies. But at the same time, you can’t eliminate all glycosylations without negatively impacting both the efficacy and solubility of the antibody. So it becomes a balancing act and requires the need for constant monitoring of biotherapeutics.

    At Merck, the effort to develop viable antibody-based biotherapeutics is led by Shara Dellatore, Ph.D. Dr. Dellatore is focused on the amino acid sequence of the variable domain of an antigen receptor, specifically the complementarity determining region (CDR) sequences of therapeutic monoclonal antibodies to make sure the engineering effort leaves the right sequence intact to confer target specificity while minimizing the susceptibility to degradation via oxidation (removal of methionine residues), deamindation (removal of asparagine residues), or isomerization.

    It’s a balancing act; the team employs directed mutagenesis to make changes in the primary amino acid sequence in defined hot-spots that can reduce the impact of degradation of the monoclonal without changing target specificity. Dr. Dellatore pulls from a toolkit of greater than 30 analytical and characterization assays to assess whether primary, secondary, tertiary, and quarternary structures are maintained without loss of function and specificity during the entire developmental process.

    For early-stage developmental assessments, the assays are selected based on the target properties of interest for a particular therapeutic protein, including characteristics that can impact mechanism of action such as glycans, modeling information, the type of immunoglobulin, and limited forced degradation to delineate degradation pathways. Typical assays include ion-exchange and size-exclusion chromatography, peptide mapping, capillary electrophoresis, and binding ELISA.

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