Recombinant monoclonal antibodies (rMAbs) are the predominant biotherapeutic protein under development today. FDA requires the structure characterization if rMAbs and other recombinant proteins to grant marketing approval. Characterizing such complex, inherently heterogeneous molecules is a significant analytical challenge that requires a broad array of physico-chemical tests. This article reports the use of reversed phase high-performance liquid chromatography (RP-HPLC) with on-line electrospray ionization mass spectrometry (ESI-MS) to rapidly determine the glycoform composition and the heavy chain C-terminal lysine heterogeneity of an intact rMAb. In addition, a novel multidimensional chromatographic platform was developed to investigate the two-dimensional, size exclusion chromatography (HPSEC) separation of the rMAb followed by RP-HPLC (HPSEC-RP-HPLC) with on-line ESI-MS analysis. Such analyses can characterize, identify, and confirm the structure of an intact rMAb…
Tag: <span>analysis</span>
Large scale genomics spurred the development of massively parallel methods of automated DNA purification and sequencing. These methods started with the 1962 development of a 96-well microtiter plate for miniature-scale serology studies. This simple laboratory device has since been greatly modified and extended to include numerous specialty multiwell plates contructed and/or coated with different materials for various purposes. The original 96-well (8×12 matrix) has expanded to include 384-well and higher densities. More recently, functionality and versatility have been greatly augmented by the incorporation of a filter, or thin membrane, into the bottom of the well. These multiwell microfilter plates can thus be employed in a flow-through mode, in addition to the familiar “put in” and “take out” pipetting and rinsing steps associated with traditional enzyme-linked immunosorbent assay (ELISA) microtiter plate methods…
Testing for parallelism is a fundamental requirement for assessing the validity of bioassay data used to calculate relative potency. When a test sample and the reference material are diluted and assayed, the assumption is that their dose response curves have an identical shape. The standard statistical approach to demonstrate that two curves are parallel involves testing the null hypothesis for equality. This article explains why that approach is not appropriate and presents an argument for testing the null hypothesis for a specified difference to statistically ascertain parallelism…
Glycosylation, a posttranslational modification that adds sugars to proteins, is required by many proteins to function properly. Glycosylation can modulate the biological activities of monoclonal antibodies (MAbs), including certain effector functions in the Fc region of IgG antibodies. Monoclonal antibodies produced at higher expression levels by mammalian cell culture may contain small amounts of nonglycosylated heavy chain (NGHC). Recent cell culture mini-reactor studies have shed light on the process parameters that most affect the occurrence of NGHC, and have greatly minimized NGHC levels in IgG MAb products…
The characterization of a batch cell culture process to produce a monoclonal antibody from a GS-NS0 mouse myeloma cell line is described. Productivity and cellular metabolism were monitored during scale-up to both characterize the process and aid in assessing cell culture stability. During fermentation scale-up studies, it was found that as culture generation number increased, productivity declined. In both flask and bioreactor cultures, declining production started abruptly at approximately generation 60. In this study, we assessed whether the decline in productivity was due to genetic instability of the cell line, which resulted in the generation of a non-producer sub-population, or a shift to a less productive state of cellular metabolism…