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September 15, 2015 (Vol. 35, No. 16)

An Ear for Proteins’ Bells and Whistles

Appropriate Post-Translational Modification Techniques Must Be Critically Employed

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    SAFC’s recent innovations in media development have enabled clients to identify critical raw materials at early stages of cell culture development. Besides significantly expanding process knowledge, this next-generation media development approach has made robust manufacturing processes more predictable.

    Therapeutic proteins are subject to deamidation, oxidation, glycosylation and other post-translational modifications (PTMs). These finishing touches are not merely ornamental, they can alter essential factors such as structure (including higher-order structure), binding, stability, and activity—factors that can influence a biotherapeutic drug’s clinical effectiveness and safety.

    Accordingly, “PTMs become critical quality attributes,” advises Asish Chakraborty, Ph.D., business development manager at Waters. “Developers need to determine which modifications are present and their implications.”

    Because of their sensitivity and selectivity, liquid chromatography–mass spectrometry (LC-MS) methods constitute the foundation of modern PTM analysis. LC-MS identifies PTMs, specifies their locations, and determines their relative abundances. This kind of information helps define product quality. Low-level occurrence at a critical location—say, the complementarity-determining region of a monoclonal antibody—may be more critical than higher preponderance at a less-critical site.

    Data handling becomes the bottleneck in LC-MS identification and quantitation of PTMs. Making sense of PTM occurrence and trends involves comparing data from many batches and large numbers of samples over time. “Data mining, generation, interpretation, and communication are what slow the process down,” Dr. Chakraborty explains.

    Waters has developed an analytical platform solution that provides the critical tools needed for biotherapeutic drug development. It is called the Biopharmaceutical Platform Solution with UNIFI® (BPS/UNIFI).

    UNIFI software enables the deployment of high-resolution analytics, such as LC/MS, across a biopharmaceutical organization, with workflows for specific PTMs. For analyzing deamidation and oxidation, the package applies a peptide mapping workflow that generates precursor and fragment ions in a single run to identify, quantify, and compare the PTMs from different lots and batches. This workflow approach, which is called an LC/MSE, uses simultaneous acquisition of exact mass at high and low collision energy.

    BPS/UNIFI integrates robust ultra-performance LC/MS characterization with comprehensive software suitable for applications in bioseparations, intact protein mass analysis, peptide mapping, and released glycan analysis. “UNIFI is the first commercial software combining LC and MS data in a single package that supports data acquisition, data processing, visualization, reporting, and compliance,” asserts Dr. Chakraborty.

  • MS—The Way to Go

    Since PTMs result in proteins that are chemically nearly identical with the original molecules, analytical methods must deliver maximum resolution and sensitivity. “Multidimensional chromatographic and MS/MS platforms enable researchers to break down complex molecules to look for specific PTMs,” says Scott Kuzdzal, Ph.D., life science business unit manager at Shimadzu Scientific Instruments. “Ion traps are especially powerful as they enable MSn detection by holding analytes in a trap and sequentially ejecting the fragmented ions.” (The n in MSn refers to the number of product ion stages in multistage MS experiments.)

    For example, researchers employ ion traps to characterize glycan composition and sequencing information without labeling or glycosidase treatment.

    Phosphoproteomics and glycomics continue to be the principal methods for analyzing PTMs. “Nearly half of all proteins are glycosylated,” Dr. Kuzdzal notes. Phosphoproteomics remains a significant challenge because of issues such as the low stoichiometry of phosphorylation, lowered ionization efficiencies of phosphopeptides, reduced digestion efficiencies, and phosphopeptide losses during sample chromatographic separations.

    For glycan work with highly branched structures, MSn analysis is the way to go. An alternative is collision-induced dissociation, which provides valuable information such as simultaneous sequencing of the peptide and determination of glycan structures. Obtaining such information at lower collision energies is not possible.

    Current PTM detection has limitations. Whereas modifications occur once per position, many different PTM types may occur simultaneously. “Biological systems are not restrained to single—or even a handful—of modifications,” Dr. Kuzdzal explains. “No single technology available can interrogate all potential modifications simultaneously, and we have a long way to go technically and computationally to even begin to look at the synergistic effects of multiple, tandem modifications.”

  • Multi-Attribute Assays

    Much has been written on the significance of PTMs occurring in therapeutic proteins. According to St. John Skilton, Ph.D., global market manager at Sciex, PTMs remain topical for issues related to patient benefits, bioproduction, and meeting quality control requirements.

    “Deeply understanding PTMs strengthens manufacturers’ ability to correlate them with safety and efficacy,” Dr. Skilton says. “Furthermore, monitoring PTMs as efficiently as other bioprocess parameters will boost manufacturing efficiency.”

    Fully understanding PTMs helps biomanufacturers meet internal quality goals, monitor stability, and convince regulators of batch-to-batch comparability because the variations can be clearly understood.

    These benefits are not lost on Sciex, whose MS instruments form the cornerstone of the company’s PTM analysis platforms. “The more information we can provide in terms of hard analytical, statistical information, with measures of abundance, the better off our customers will be,” Dr. Skilton remarks.

    There is a massive trend toward multi-attribute PTM assays, which Sciex offers by combining traditional separations like capillary electrophoresis or liquid chromatography with mass spectrometry: CESI (a type of electrospray MS ionization with a capillary electrophoresis front end is suitable for peptide mapping, intact mass analysis, and glycan analysis) plus MS, MS/MS, and Sciex’s BioPharmaView™ software. The company also offers its own SelexION™ differential ion mobility instrument as an additional separation mode.“This set of capabilities enables us to construct multi-attribute assays from one analysis,” asserts Dr. Skilton. “From one peptide map, I can look at all the PTMs flagged as important, quantify them, and determine their variability across a batch. Having separations that are complementary and orthogonal makes this comprehensive.”

    These analysis platforms enable scientists to probe deeply into PTM structure so that they can determine, for example, the degree of branching of specific sugar residues in glycans. This is one strength of capillary electrophoresis coupled with MS. “We can get to crazy levels of finding very low abundance glycosylations that people were not aware of,” Dr. Skilton explains. This will be a boon to developers of biosimilars who must demonstrate both physicochemical and therapeutic similarity.

    Dr. Skilton likens the ability to exploit deep, broad glycan analysis to an arms race: “Innovator companies can use this information to create market entry barriers, but biosimilars firms can also use it to demonstrate similarity with analytical tools and measure the heterogeneity.”

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