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Jul 1, 2014 (Vol. 34, No. 13)

Glycosylation Affirmed as Quality Metric

  • Producers of biopharmaceuticals are finding that the assessment of critical quality attributes is increasingly a matter of evaluating post-translational modifications (PTMs), especially glycosylation.

    One such producer, the contract manufacturer Fujifilm Diosynth Biotechnologies, treats PTMs as indicators of process and product consistency. In fact, Min Zhang, Ph.D., the company’s principal scientist for upstream process development, uses the terms “quality” and “PTM” nearly interchangeably.

    Bioprocessors and contractors such as Fujifilm have a wealth of experience with PTMs, at least in certain contexts—late in development and through manufacturing. Data, however, is scarce in other contexts—preclinical and early clinical trials.

    “In many cases, we are limited to using knowledge of PTMs from a generic antibody as the standard,” admits Dr. Zhang. One example consists of high-mannose glycans, which are associated with very short clearance. As experience accumulates, desirable molecular attributes are based more on the specific molecule and its performance in studies.

    The focus on PTMs throughout development underscores the iterative nature
    of process development. Developers know which PTMs, particularly glycosylations, are associated with desirable and undesirable attributes, and appropriately try to overexpress or eliminate them.

    Dr. Zhang counts protein folding/misfolding and aggregation as independently significant PTMs, even though process conditions and not the usual cell-derived post-translational factors may be responsible. Immunogenicity caused by aggregation is one of the major factors influencing late clinical-stage failures.

    Upstream processing, including culture conditions, media, and feed, are known to influence PTMs, but no formula exists for dialing in quality attributes. Process interventions are, as Dr. Zhang puts it, “the million dollar question,” as almost any upstream factor affects quality.

    “That includes molecule design, cell line, and process conditions.” Even such pedestrian values as dissolved oxygen and pH are relevant. There is no single way to guarantee quality attributes within spec, but Dr. Zhang assures that “we have many different tools available.”

  • The Glycan Is the Process

    As a testing lab, Eurofins Lancaster Laboratories analyzes therapeutic proteins for such PTMs as deamidation, methionine oxidation, glycosylation, and to a lesser degree phosphorylation. Eurofins obtains glycan profiles by fluorescently labeling them and analyzing by high-performance liquid chromatography using a hydrophilic interaction liquid chromatography (HILIC) column under an organic-to-aqueous gradient, and confirming identity by mass spectrometry. “Fluorescence detection provides high sensitivity,” asserts John Snyder, Ph.D., who works at the company as principal scientist and group leader, proteomics.

    Fluorescence labeling is required because sugars lack an ultraviolet chromophore, but tagging adds a step to the analysis. Label-free analysis is possible as well. For example, Agilent’s mAb Glycochip analysis system consists of a microfluidic chip incorporating sample introduction, cleavage of N-glycans by the enzyme glycopeptide N-glycosidase, microchannel ion exchange separation, and direct injection of fractions into a time-of-flight mass spectrometer. Glycans are identified based on matches to a built-in database.

    The strength of methods based on initial glycan cleavage is rapid determination of glycan composition and comparison with a reference glycosylation pattern, which is useful for quality testing. The drawback is lack of information on glycan location. For that type of analysis, investigators first subject the protein to protease digestion, followed by mass spectrometry analysis of the fragments for mass additions typical of glycans. Analysis is simplified by knowing what glycans are present to begin with, and what fragments result from proteolytic digestion.

    Despite the intermolecular variability in glycosylation, even within a batch, aggregate glycan analysis is a powerful measure of quality assessment. Out-of-specification batches suggests that something has gone wrong with the process, usually upstream.

  • Mass Spectrometry Approaches

    Glycan analysis may be approached from the top down (peptide/amino acid plus glycan) or the bottom up (free glycans). Softly ionizing mass spectrometry techniques are useful in both cases. LAESI (laser ablation electrospray ionization), a softly ionizing mass spectrometry technique, is useful for both.

    LAESI involves two steps. First, an infrared laser tuned to the absorption band of water pulses and vaporizes the sample. Second, the sample is carried to a chamber for electrospray ionization. LAESI is an ambient ionization technique, meaning it does not require high vacuum and in many cases samples may be introduced with little or no sample preparation. As with conventional electrospray, samples may be multiply charged. The method was discovered in 2007 at George Washington University and commercialized by Protea Biosciences.

    For the bottom-up method, glycans are first released enzymatically in 18O-labeled water. This has the effect of labeling glycosylation sites with that isotope, to distinguish those from deamidation sites that do not take up 18O.

    IgGs, the most common therapeutic antibodies today, contain 3–5 kDa of carbohydrate consisting of 3–10 isoforms that predominantly use four or five sugars. Glycoforms differ in terms of number and spacing of sugar residues, sometimes often near the ends of branched chain glycans.

    “Today’s high-resolution accurate mass spectrometers can easily distinguish among intact antibody glycoforms, which may differ in molecular weight by as little as 162 daltons,” says Greg W. Kilby, Ph.D., director of molecular imaging and bioanalytical services at Protea Biosciences. That molecular weight difference signifies an extra hexose, for example.

    LAESI can also assist in sequencing glycans, similarly to picking apart a peptide or protein. “But this requires a three-stage mass spectrometry analysis, or MS3, usually with an ion trap or Orbitrap,” Dr. Kilby advises. Sequence is often less important than sugar composition, a quality attribute of great interest in biosimilars. This value is accessible through tandem mass spectrometry (MS/MS, or MS2) methods.

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