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

Cracking the PTM Code

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    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.

    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.

  • 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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