Predicting Antibody Developability Profiles Through Early Stage Discovery Screening.

Monoclonal antibodies play an increasingly important role for the development of new drugs across multiple therapy areas. The term 'developability' encompasses the feasibility of molecules to successfully progress from discovery to development via evaluation of their physicochemical properties. These properties include the tendency for self-interaction and aggregation, thermal stability, colloidal stability, and optimization of their properties through sequence engineering. Selection of the best antibody molecule based on biological function, efficacy, safety, and developability allows for a streamlined and successful CMC phase. An efficient and practical high-throughput developability workflow (100 s-1,000 s of molecules) implemented during early antibody generation and screening is crucial to select the best lead candidates. This involves careful assessment of critical developability parameters, combined with binding affinity and biological properties evaluation using small amounts of purified material (<1 mg), as well as an efficient data management and database system. Herein, a panel of 152 various human or humanized monoclonal antibodies was analyzed in biophysical property assays. Correlations between assays for different sets of properties were established. We demonstrated in two case studies that physicochemical properties and key assay endpoints correlate with key downstream process parameters. The workflow allows the elimination of antibodies with suboptimal properties and a rank ordering of molecules for further evaluation early in the candidate selection process. This enables any further engineering for problematic sequence attributes without affecting program timelines.

Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays.

The reliable use of purified Cry1Ab protein standards is a prerequisite for ecological studies and resistance monitoring programs of Cry1Ab-expressing transgenic corn. In this study the stability and activity of different Cry1Ab protein batches expressed in and purified from Escherichia coli were determined during two-year storage at different temperature conditions (4 degrees C, -20 degrees C, and -80 degrees C). SDS-Polyacrylamide gel electrophoresis showed degradation of the protein stored at 4 degrees C over four months, whereas no difference in the band intensity of the Cry1Ab proteins stored at -20 degrees C and -80 degrees C was observed. Bioassays with neonate larvae of Ostrinia nubilalis indicated that the biological activity of Cry1Ab varied from batch to batch, depending on the production process. Cry1Ab protein stored at 4 degrees C for four months showed a significantly decreasing activity measured as median lethal concentration (LC(50)), whereas the protein activity declined less than 11-fold after two years storage at -20 degrees C. When stored at -80 degrees C the toxin activity remained relatively stable for at least 30 months, as indicated by low LC(50) values of 7-10 ng Cry1Ab per cm(2) diet. These experiments demonstrate that appropriate long-term storage conditions of Cry1Ab protein standards are crucial for resistance monitoring programs of Bt corn, and storage at -80 degrees C is recommended.

Expression of Cry3Bb1 in transgenic corn MON88017.

To evaluate the effects of transgenic expression of Coleopteran-specific Bt protein Cry3Bb1 on target and nontarget insects in fields with Bt crops, it is necessary to quantify the Cry3Bb1 contents in the plants. Here, we describe the optimization and validation of the quantitative detection of Cry3Bb1 by adapting the commercially available qualitative PathoScreen double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) for quantitative measurements. The optimized method had an average accuracy of 84-109% and was used to quantify the Cry3Bb1 contents of different tissues of Bt corn MON88017 at four developmental stages during three years (2005-2007) in a field trial in Germany. The Cry3Bb1 contents were determined based on both dry weight and fresh weight. Cry3Bb1 expression was highest in young leaves (228.4 microg/g dw and 35.5 microg/g fw) and lowest in pollen (3.8 microg/g fw). In root tissues, the Cry3Bb1 content declined during the growing season from 130 to 40 microg/g dw. A significant decline of Cry3Bb1 contents was also observed during the growing season in other plant tissues. The Cry3Bb1 contents of different plant tissues strongly correlated to each other. On the basis of the total corn biomass produced on 1 hectare, it was estimated that up to 905 g of Cry3Bb1 is produced per hectare Bt corn MON88017.