- 2:30 PM

Detection of Protease Activity in Hydrolysates Additives: Effects on Monclonal Antibody Product Quality and Downstream Purification

Kimberly May, Biological and Sterile Product Development, Schering-Plough Research Institute, 1011 Morris Ave., U-1-2-2200, Union, NJ 07083

Various methods have been pursued over the years in order to increase the titer during monoclonal antibody fermentation. One such popular method in industry is the addition of soy and wheat hydrolysates to the fermentation media. Hydrolysates are commonly produced by enzyme digestion of raw material by-products from the food industry. Since the properties of the original plant source can vary from batch to batch, the hydrolysates can have significant lot-to-lot variability. Furthermore, insufficient downstream processing of the hydrolysates can result in leakage of the active digestion enzyme or other enzymes into the final product. In this work, hydrolysates from two separate companies were investigated, both soy and wheat. Batch lots were compared and preliminary results from zymograms and SDS-PAGE gels have confirmed the presence of multiple enzymes in most of the hydrolysates samples. The molecular weights vary in the range from 4 kDa to 200 kDa, depending on the batch. The preliminary results confirm that common industry standard assays are not sensitive enough to detect all possible enzyme contamination of these materials. Only reducing SDS-PAGE with hydrolysate samples containing a monoclonal antibody is truly sensitive enough to detect protease activity. Inhibition studies revealed the presence of metalloproteases, serine proteases, and cysteine proteases. Enzyme contamination of the hydrolysates coincided with insufficient downstream processing throughout common manufacturing processes.

Furthermore, the presence of these enzymes in the fermentation media may have deleterious effects on purification processes and antibody product quality. These effects were investigated for two purified monoclonal antibodies spiked into media containing varying concentrations of hydrolysates. Samples containing hydrolysates with significant proteolytic activity were shown to have much higher post-antibody peaks, increased leading shoulder, and more small clips by RP-HPLC than samples containing little to no enzyme activity. SE-HPLC also shows a post-antibody peak that may be the clipped antibody. Aside from the possible effects on antibody titer and quality, enzyme contamination could result in many purification problems downstream as well, such as the needed addition of another chromatography step to the purification process or shorter column lifetimes. These effects will also be discussed.