Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure.

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.1 In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and FcγR. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.

Identification and Characterization of a Monoclonal Antibody Variant Species with a Clipping in the Complementarity Determining Region Isolated by Size Exclusion Chromatography Under Native Conditions.

The content of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Peptide bonds in the hinge region of mAbs are susceptible to hydrolysis, generating Fc-Fab fragments, which are associated with lower efficacy than the intact antibody. Fc-Fab fragments can be separated from intact antibody molecules under native conditions by size exclusion chromatography (SEC). Although several fragments generated by a clip in the complementarity determining region (CDR) have been reported, their efficacies have not been analyzed. This is because these fragments could not be separated from intact antibodies under native conditions owing to their similar molecular sizes. Here, we report that bevacizumab variant with clipping in the CDR, with the resulting fragments remaining intact in the variant, can be isolated under native conditions by selecting an adequate SEC column.

C. elegans AMPKs promote survival and arrest germline development during nutrient stress.

Mechanisms controlling development, growth, and metabolism are coordinated in response to changes in environmental conditions, enhancing the likelihood of survival to reproductive maturity. Much remains to be learned about the molecular basis underlying environmental influences on these processes. C. elegans larvae enter a developmentally dormant state called L1 diapause when hatched into nutrient-poor conditions. The nematode pten homologue daf-18 is essential for maintenance of survival and germline stem cell quiescence during this period (Fukuyama et al., 2006; Sigmond et al., 2008), but the details of the signaling network(s) in which it functions remain to be elucidated. Here, we report that animals lacking both aak-1 and aak-2, which encode the two catalytic α subunits of AMP-activated protein kinase (AMPK), show reduced viability and failure to maintain mitotic quiescence in germline stem cells during L1 diapause. Furthermore, failure to arrest germline proliferation has a long term consequence; aak double mutants that have experienced L1 diapause develop into sterile adults when returned to food, whereas their continuously fed siblings are fertile. Both aak and daf-18 appear to maintain germline quiescence by inhibiting activity of the common downstream target, TORC1 (TOR Complex 1). In contrast, rescue of the lethality phenotype indicates that aak-2 acts not only in the intestine, as does daf-18, but also in neurons, likely promoting survival by preventing energy deprivation during L1 diapause. These results not only provide evidence that AMPK contributes to survival during L1 diapause in a manner distinct from that by which it controls dauer diapause, but they also suggest that AMPK suppresses TORC1 activity to maintain stem cell quiescence.