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.

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. 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:

   	CD (equivalent secondary and tertiary structure; equivalent conformational stability)
   	sedimentation velocity and native gels (equivalent solution conformation; equivalent aggregate content)
   	sedimentation equilibrium (equivalent quaternary structure and/or binding of ligands)
   	on-line classical light scattering and/or dynamic light scattering (equivalent aggregate content)

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. 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.

5. 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

6. 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:

	near-UV CD (is there any regular tertiary structure?)
	thermal unfolding by far-UV CD (is there a cooperative unfolding transition?)
	hydrodynamic shape analysis by sedimentation velocity or dynamic light scattering (is it highly extended or a compact folded structure?)

	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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