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June 01, 2011 (Vol. 31, No. 11)

Making Follow-On Biologics a Reality

Analytical Chemistry Methods Have a Multifaceted Role in This Complex Process

  • Protein-Characterization Methods

    Post-translational modifications of proteins encompass a wide variety of modifications, including glycosylation, oxidation, phosphorylation, sulphation, lipidation, disulphide bond formation and deamidation. Mass spectrometry (MS) has become the tool of choice for detecting and investigating these modifications.

    In some cases, nuclear magnetic resonance spectroscopy (NMR) can also be useful. Indeed, it was NMR that identified the recently publicized issue of heparin contaminated with O-linked glycans.

    The so-called higher-order structure of a protein gives it its unique 3-D shape and thus contributes to its functions. Subtle differences in such higher-order structures might explain observed biological/immunological differences between otherwise identical proteins and also serve as a basis for comparison of reference products with FOBs.

    Myriad classical biophysical techniques are used to characterize higher-order structures, including circular dichroism, fluorescence, differential scanning calorimetry, isothermal calorimetry, analytical ultracentrifugation, and size-exclusion chromatography. Detecting subtle changes requires use of additional techniques such as NMR, x-ray crystallography, and MS.

    Formation of undesirable protein aggregates represents a substantial problem for biopharmaceuticals. Aggregates can display adverse toxicological and immunological profiles, in addition to having an obvious detrimental impact on dosage. Characterizing aggregates is a complex undertaking. Among the numerous methods employed are size-exclusion chromatography, analytical ultracentrifugation, and asymmetric flow field flow fractionation. Detection of sub-visible particles present at very low concentration requires techniques such as dynamic or static light scattering.

    Sophisticated analytical protein-characterization methods will likely have an impact, not only in establishing the similarity of a FOB to its reference product, but also in accounting for potential adverse clinical events. For example, a number of FOB versions of a human erythropoietin reference product (Eprex®) have been approved by the European Medicines Agency (EMA), and while these versions were deemed by EMA to be comparable in quality, safety, and efficacy to Eprex, clear differences in structure have been documented by analytical methods.

    If any of these erythropoietin FOBs is, in the future, associated with adverse clinical events, these structural differences—and the fact that they were known in advance—may well play a role in potential litigation related to the adverse events. This, in turn, may influence similarity standards applied to FOBs by FDA and other regulatory agencies.

  • Patent Aspects

    While the scientific and clinical underpinnings of the act are undergoing scrutiny, no less significant are the act’s provisions regarding patent and data exclusivity. The act gives BLA holders 12 years of marketing exclusivity, but a FOB applicant can file its application at the FDA to start the process after just four years.

    The act lays out a byzantine process in which the FOB applicant provides the BLA holder with access to confidential information regarding the FOB application for the purpose of assessing potential patent infringement, and then the parties go back and forth trading their views regarding the BLA holder’s patents. The process culminates in lists of patent claims that are to be asserted by the BLA holder against the FOB applicant in two waves of patent litigation outlined in the act.

    In the patent context, characterizing protein structure by analytical chemistry methods will sometimes be essential to determine whether a FOB infringes a patent claim or is structurally distinct from what is claimed. These methods might also be occasionally employed by a BLA holder who seeks to develop a new, improved version of its existing product to replace the original product in the marketplace prior to loss of market share to FOBs.

    To obtain new patent protection for such an improved version, it may be useful to demonstrate just how it is structurally distinct from the original product. And regardless of patent issues, the act grants a new 12-year period of exclusivity for the BLA holder’s improved product if the improved product is shown to have a biological structure different from the original product (provided that the new structure results in a change in safety, potency, or purity of the product).

    With biosimilars due to become a reality soon, use of analytical chemistry methods to characterize proteins will take on ever-increasing importance.

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