Example Applications

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Some Example Applications for Protein Characterization Studies:

  1. Sedimentation velocity, light scattering, and/or native gels can be used to cross-validate SEC protocols for detection of protein aggregates. Some form of confirmation that the SEC protocol detects all relevant species is now nearly always required by the regulatory agencies. APL has now done this for dozens of products.

  2. Comparability protocols are commonly required either after changes in a manufacturing process, cell line, formulation, or manufacturing site once a product has entered clinical trials or has reached the market. They are also needed to show comparability of biosimilars to the Reference Listed Drug. It is important to demonstrate comparability of higher order structure (secondary, tertiary, and quaternary structure) as well as comparability of primary structure. (See our presentation at the FDA's State of the Art Analytical Methods meeting, June 2003, "Measuring Comparability of Conformation, Heterogeneity, and Aggregation with Circular Dichroism and Analytical Ultracentrifugation")

    APL offers a number of tools useful for comparability protocols, and has completed literally hundreds of such protocols:

  3. Pre-formulation studies of protein conformation and stability can reduce the range of conditions that need to be examined during formulation studies, saving considerable time and expense.

  4. Recombinant vaccines are often quite heterogeneous in size/MW distribution, and may use virus-like particles as carriers and adjuvants. Sedimentation velocity can be very useful for characterizing vaccines and monitoring lot-to-lot uniformity of size distributions, and circular dichroism can determine whether the recombinant antigen has a regular ordered structure.

  5. For proteins that form visible or sub-visible particulates (often leading to eventual precipitation) dynamic light scattering (DLS) is a valuable tool for detecting the early precursors (nuclei) that will eventually trigger formation of particulates. A DLS assay can often trace the damage that leads to particulate formation to a specific step in manufacturing, or give confidence that a change in formulation will truly solve the particulates issue.

  6. For characterizing PEGylated proteins and nucleic acids (and the quality of the polymers used for conjugation) we recommend

    • dynamic light scattering directly measures the increase in hydrodynamic size caused by PEGylation, a property which often correlates strongly with serum lifetime

    • on-line classical light scattering is very useful for measuring the extent of conjugation, and whether PEGylation has altered the state of association/aggregation

    • on-line classical light scattering is also very useful for verifying the true true molecular weight and homogeneity of the PEG polymer

  7. Assessing proper folding of novel proteins discovered through genomics or proteomics, for which no native protein from natural sources is available as a control, can avoid wasting time and money trying to assay biological activity or running screens using proteins that aren't properly folded. Three ways to assess proper folding are:

  1. Characterizing protein binding events can provide functional characterization of hormones, monoclonal antibodies, and small molecule drugs. Such data can be useful for:

  • selection among drug candidates

  • demonstration of comparability (e.g. after a process change)

  • quality control and process monitoring

  • development of protein mimetics

 

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