Andrew A. Kosky

Inverse Relationship of Protein Concentration and Aggregation

AbstractPurpose. To determine the effect of protein concentration on aggregation induced through quiescent shelf-life incubation or shipping-related agitation. Methods. All aggregation was measured by size-exclusion high-performance liquid chromatography. Aggregation was induced by time-dependent incubation under stationary conditions or by agitation caused by shaking, vortexing, or vibration using simulated shipping conditions. Results. Protein aggregation is commonly a second- or higher-order process that is expected to increase with higher protein concentration. As expected, for three proteins (PEG-GCSF, PEG-MGDF, and OPG-Fc) that were examined, the aggregation increased with higher protein concentration if incubated in a quiescent shelf-life setting. However, aggregation decreased with higher protein concentration if induced by an air/water interface as a result of agitation. This unexpected result may be explained by the rate-limiting effect on aggregation of the air/water interface and the critical nature of the air/water interface to protein ratio that is greatest with decreased protein concentration. The non-ionic detergent polysorbate 20 enhanced the aggregation observed in the quiescently incubated sample but abrogated the aggregation induced by the air/water interface. Conclusions. The effect of protein concentration was opposite for aggregation that resulted from quiescent shelf-life treatment compared to induction by agitation. For motionless shelf-life incubation, increased concentration of protein resulted in more aggregation. However, exposure to agitation resulted in more aggregation with decreased protein concentration. These results highlight an unexpected complexity of protein aggregation reactions.

PEGylation prevents the N-terminal degradation of megakaryocyte growth and development factor.

AbstractPurpose. Determine the effect of PEGylation on in-vitro degradation for recombinant human Megakaryocyte Growth and Development Factor (rHuMGDF) in the neutral pH range. Methods. Degradation products were characterized by cation-exchange HPLC, N-terminal sequencing and mass spectrometry. Results. The main route of degradation was through non-enzymatic cyclization of the first two amino acids and subsequent cleavage to form a diketopiperazine and des(Ser, Pro)rHuMGDF. This reaction was prevented by alkylation of the N-terminus by polyethylene glycol (PEG). Conclusions. PEGylation of proteins is commonly performed to achieve increased in-vivo circulation half-lives. For rHuMGDF, an additional advantage of PEGylation was enhancedin-vitro shelf-life stability.

Sodium Chloride Enhances the Storage and Conformational Stability of BDNF and PEG-BDNF

AbstractPurpose. BDNF, a noncovalent homodimer, was modified by covalently attaching polyethylene glycol (PEG) with an average molecular weight of 20kDa to the N-terminal methionine. Stability of modified BDNF (PEG-BDNF) in aqueous solution was compared to BDNF after storage at elevated temperature in the presence and absence of NaCl. Methods. SDS-PAGE, Light Scattering and Size Exclusion Chromatography were used to assess conformational stability and chemical degradation. In addition, CD spectroscopy was used to follow changes in secondary and tertiary structures upon thermal stress of the protein. Results. NaCl containing formulations are more stable than NaCl-free formulations. In NaCl-free formulations, the main degradation product of BDNF and PEG-BDNF had a molecular weight of monomer that was more chemically degraded than the dimer. Additionally, the degradation of PEG-BDNF occurred at an accelerated rate compared to BDNF in NaCl-free environments. Conclusions. The addition of NaCl to formulations enhances the shelf-life and conformational stability of both BDNF and PEG-BDNF.

Optimization and applications of CDAP labeling for the assignment of cysteines.

PurposeThe aim of the study is to provide a methodology for assigning unpaired cysteine residues in proteins formulated in a variety of different conditions to identify structural heterogeneity as a potential cause for protein degradation.Methods1-Cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was employed for cyanylating free cysteines in proteins and peptides. Subsequent basic cleavage of the peptide bond at the N-terminal side of the cyanylated cysteines provided direct information about their location.ResultsCDAP was successfully employed to a wide variety of labeling conditions. CDAP was reactive between pH 2.0 and 8.0 with a maximum labeling efficiency at pH 5.0. Its reactivity was not affected by excipients, salt or denaturant. Storing CDAP in an organic solvent increased its intrinsic stability. It was demonstrated that CDAP can be employed as a thiol-directed probe to investigate structural heterogeneity of proteins by examining the accessibility of unpaired cysteine residues.ConclusionCDAP is a unique cysteine-labeling reagent because it is reactive under acidic conditions. This provides an advantage over other sulfhydryl labeling reagents as it avoids potential thiol-disulfide exchange. Optimization of the cyanylation reaction allowed the utilization of CDAP as a thiol-directed probe to investigate accessibility of sulfhydryl groups in proteins under various formulation conditions to monitor structural heterogeneity.