W0283

Automated High Throughput Screening for Aggregation Prior to Crystallization. Marc N. Feiglin¹, Rick Luedke², Bingyi Yao³, and Robert P. Collins³, ¹Tecan-Biopharma, 200 Medford Ave, Boston, MA 02155, ²Tecan-US., P.O. Box 13953, Research Triangle Park, NC 27709, ³Proterion Corp., One Possumtown Rd., Piscataway, NJ 08854.

The three principle steps in the generation of a protein molecular structure are purification, crystallization, and X-ray analysis. With the integration of high speed computer processors and automation, the X-ray analysis step has become somewhat routine. The same could be said about the purification step, in that the process has been greatly simplified as a direct consequence of technological advances in protein expression and HPLC techniques. The crystallization step itself however, is still quite convoluted, and crystallographers can invest months of time following a proven crystal forming recipe, only to fail in the end for no apparent reason.

The likelihood of crystal formation is strongly dependent upon the “quality” of the stock solution from which the crystals are grown. If the solvent conditions in the stock solution are such that the protein tends toward non-specific aggregation, the likelihood of successful crystallization is low, since the aggregates tend to disrupt the lattice structure. Hence, characterization of the stock solution, prior to crystal trials, can be a time saving step.

Dynamic light scattering (DLS) is a non-invasive solution characterization technique that has been used successfully to screen stock solutions prior to crystallization trials. The scattering intensity of a sample solution is proportional to M_N², where M_N is number average molecular weight. Hence, DLS is extremely sensitive to the presence of aggregates and other large particulates that can disrupt crystal lattice formation.

This poster demonstrates the utility of integrating the Protein Solutions DynaPro Molecular Sizing Instrument into a Tecan Genesis workstation in order to automate the determination of screening conditions that may produce protein aggregation.