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

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.

Knowing that the specific glycoform modifications are essential for function puts the burden on the production side of biotherapeutics to insure that the specific glycoforms are maintained from lot to lot. Bruker designed its Mass Spec Toolbox to help production scientists monitor modifications during the course of the production process. The Mass Spec Toolbox incorporates instrumentation and software solutions and facilitates complete characterization of biopharmaceutical products and potential product impurities that are essential for product development, regulatory approval, and production.

Among other challenging tasks, the analysis of post-translational modifications including glycosylation, phosphorylation, oxidation, and deamidation can be addressed using these tools. According to Catherine Evans, business development manager at Bruker’s European Biopharma division, Bruker recognizes glycopeptide and glycan profiling to be among the most difficult characterization challenges for production of biopharmaceuticals due to the microheterogeneity of the sample.

Given that multiple different glycans can exist on a single amino acid, the interpretation of the glycan MS-MS spectra has traditionally required the input of a glyco expert. But now, with GlycoQuest, Bruker has developed a new software tool that will enable nonexpert users to monitor subtle changes in biotherapeutics during the manufacturing processes and so avoid altered drug efficacy.

With these new tools, the identification and relative quantitation of glycans in the active sites can be easily compared following production runs in the development of biosimilar/biobetter drugs.

“We have focused on glycosylation at this time as it is one of the most challenging biopharmaceutical characterization tasks which traditionally required expert knowledge to interpret the data produced. We believe that GlycoQuest will enable nonexpert users to perform this critical characterization task, enabling them to identify both the location of the sugar attachment as well as the sugar composition when characterizing glycopeptide composition,” Evans explained.

At Millennium, The Takeda Oncology Company, Niclas Tan, Ph.D., scientist in analytical development, biologics, has optimized the use of a novel integrated microfluidic-based LC-MS chip developed by Agilent. The Agilent mAb-Glyco chip enables rapid online cleavage, purification, separation, identification, and quantitation of label-free N-linked glycans from monoclonal antibodies.

The intact antibody in deglycosylation buffer is directly loaded onto the chip containing immobilized PNGase F enzyme and two porous graphitized carbon (PGC) columns for enrichment/desalting and separation, respectively. After incubation with the enzyme, the released glycans are enriched on the column, washed, separated by reversed-phase gradient, and then analyzed on an Agilent 6540 Q-TOF mass spec. The chip not only provides a significant time savings—the glycan data readout is available in 20 minutes, as compared to the traditional HPLC-based glycan assay, which took three to four days—but also is done label-free, which has allowed for detection of more glycan species than before.

“The mAb-Glyco LC-MS chip from Agilent is amenable to the analysis of N-linked glycosylated therapeutic proteins other than monoclonal antibodies,” shared Dr. Tan. “This is a true manifestation of the lab-on-a-chip concept where the sample is processed and analyzed within the chip. The current glyco chip method allows label-free detection and identification of released glycans using accurate mass. Other post-translational modifications are readily detectable by label-free mass spec via peptide mapping on other LC-MS chip formats.”

Open communication between immunologists and the companies focused on the production of viable biotherapeutics is critical throughout the entire discovery and development process. Meetings like CHI’s “Biotherapeutics Analytical Summit” will help to insure that the quality of viable therapeutic proteins will continue to improve.

The Glycoquest module in ProteinScape handles complex LC-MALDI-MS/MS data from an enzymatic digestion of an antibody enabling classification and identification of peptides and glycopeptides, according to Bruker.

For a review of a paper in Science that points to the relative abundance of PTMs in human cells, click here.

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