Infections with pestiviruses occur in cattle, sheep, pigs and also in numerous other ungulate species. In the present study, pestiviruses from goat, buffalo, deer and giraffe were analysed at the molecular level ; unusual strains from cattle and pigs were also included. A phylogenetic analysis of the respective pestiviruses was undertaken on the basis of a fragment from the 5h noncoding region as well as the gene encoding autoprotease N pro . Statistical analyses of the respective phylogenetic trees based on the 5h NCR revealed low confidence levels for most of the branches, while the structure of the tree based on the N pro gene was supported by high bootstrap values. Accordingly, the isolates from goat, buffalo and deer can be grouped together
Two border disease virus (BDV) pairs each consisting of cytopathogenic (cp) and non-cp viruses have been analyzed at the molecular level. Within the NS2-3 (p125) encoding region of both cp viruses, insertions of cellular sequences were identified which were absent in the corresponding non-cp isolates. A comparative sequence analysis revealed that within each pair the cp and non-cp viruses are almost identical. This strongly suggests that the cp BDV isolates developed from the non-cp viruses by RNA recombination between the viral genome and cellular sequences. Nonstructural protein NS3 (p80) was demonstrated after infection with both cp BDV strains. In addition, fusion proteins composed of cellular and viral sequences were identified. In contrast, expression of NS3 and the fusion proteins was not found after infection with the respective non-cp counterparts.
Combination therapy is a fast-growing strategy to maximize therapeutic benefits to patients. Coformulation of two or more therapeutic proteins has advantages over the administration of multiple medications, including reduced medication errors and convenience for patients. Characterization of coformulated biologics can be challenging due to the high degree of similarity in the physicochemical properties of co-formulated proteins, especially at different concentrations of individual components. We present the results of a deamidation study of one monoclonal antibody component (mAb-B) in coformulated combination antibodies (referred to as COMBO) that contain various ratios of mAb-A and mAb-B. A single deamidation site in the complementarity-determining region of mAb-B was identified as a critical quality attribute (CQA) due to its impact on biological activity. A conventional charge-based method of monitoring mAb-B deamidation presented specificity and robustness challenges, especially when mAb-B was a minor component in the COMBO, making it unsuitable for lot release and stability testing. We developed and qualified a new, quality-control-friendly, single quadrupole Dalton mass detector (QDa)-based method to monitor site-specific deamidation. Our approach can be also used as a multi-attribute method for monitoring other quality attributes in COMBO. This analytical paradigm is applicable to the identification of CQAs in combination therapeutic molecules, and to the subsequent development of a highly specific, highly sensitive, and sufficiently robust method for routine monitoring CQAs for lot release test and during stability studies.
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