During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.
Bobcats (Lynx rufus) are listed in Appendix II of the Convention on International Trade in Endangered Species, which requires state agencies to affirm stability of their populations before granting exportation authority to international markets. The West Virginia Division of Natural Resources (WVDNR) has been using survival and reproductive data collected in the 1970s to guide management decisions. With current high harvest rates, it is critical to use updated demographic data. Our primary research objectives are to estimate age distribution, recruitment, and survival of West Virginia bobcats. Results will be used directly by WVDNR to update or modify the current change in population model used to determine harvest limits. Following mass public outreach, hunters and trappers across West Virginia donated 296 bobcat carcasses to the project over the 2014–2015 hunting and trapping season. Lower canines (n=296) and female reproductive organs (n=147) were collected during necropsy to estimate age at mortality and reproductive success. Age was estimated by counting cementum annuli of canines, and implantation rates were estimated by placental scar counts. Preliminary results indicate that yearling (1 - 2 years) bobcats represent 20% of the 296 bobcats collected, with juveniles (<1 year; 20%) and adults (+2 years; 60%) representing the majority of the population. Preliminary implantation rates resulted in 2.18 kittens per adult female (n=88) and 0.63 kittens per yearling female (n=30). Accurate change in population data, along with density estimates, will provide WVDNR with the necessary data to ensure sustainable harvest of bobcats in West Virginia.
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