Comprehensive Discovery and Quantitation of Protein Heterogeneity via LC-MS/MS Peptide Mapping for Clone Selection of a Therapeutic Protein.

Development of biopharmaceutical production cell lines requires efficient screening methods to select the host cell line and final production clone. This is often complicated by an incomplete understanding of the relationship between protein heterogeneity and function at early stages of product development. LC-MS/MS peptide mapping is well suited to the discovery and quantitation of protein heterogeneity; however, the intense hands-on time required to generate and analyze LC-MS/MS data typically accommodates only smaller sample sets at later stages of clone selection. Here we describe a simple approach to peptide mapping designed for large sample sets that includes higher-throughput sample preparation and automated data analysis. This approach allows for the inclusion of orthogonal protease digestions and multiple replicates of an assay control that encode an assessment of accuracy and precision into the data, significantly simplifying the identification of true-positive annotations in the LC-MS/MS results. This methodology was used to comprehensively identify and quantify glycosylation, degradation, unexpected post-translational modifications, and three types of sequence variants in a previously uncharacterized non-mAb protein therapeutic expressed in approximately 100 clones from three host cell lines. Several product quality risks were identified allowing for a more informed selection of the production clone. Moreover, the variability inherent in this unique sample set provides important structure/function information to support quality attribute identification and criticality assessments, two key components of Quality by Design.

Identification of human serum interferants in the recombinant P-selectin glycoprotein ligand-1 clinical ELISA using MALDI MS and RP-HPLC.

A colorimetric enzyme-linked immunosorbent assay (ELISA) was developed to detect circulating levels of rPSGL to permit pharmacokinetic analysis of clinical samples. The ELISA is an asymmetric sandwich utilizing a monoclonal antibody pair. Initial validation studies indicated that 57% of normal individuals scored above the limit of detection of the assay. Specificity experiments indicated that the signal was not due to circulating endogenous P-selectin glycoprotein ligand-1 (PSGL-1). Using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and sampling within the individual microplate wells, the interferant was detected in the vicinity of 6.6 kDa in lipemic and normal human sera, but not delipidized sera. These results were consistent with the ELISA data where 97.5% of known lipemic, 57% of normal, and 0% of delipidized sera scored above detectable limits in the ELISA. Preparative isolations of the 6.6 kDa species were performed using reversed-phase high performance liquid chromatography (RP-HPLC) with UV and MS detection. Edman N-terminal sequencing identified the 6.6 kDa unknown as Apolipoprotein C-I. Additional apolipoproteins were found by MALDI and RP-HPLC. Digestion of sera with liposome lipase and extraction of sera with anti-apolipoprotein C-I, C-II, and C-III antibody beads significantly reduced the ELISA interference. These experiments combined with the MALDI detection of phosphatidylcholine-type lipids from NHS eluate suggested that lipoprotein particles or remnants were causing the interference. A method combining Triton-X 100 with sonication was developed to overcome this interference without altering rPSGL recovery in the ELISA.

Purification and Characterization of an Endo-(1,3)-beta-d-Glucanase from Trichoderma longibrachiatum.

A laminarinase [endo-(1,3)-beta-d-glucanase] has been purified from Trichoderma longibrachiatum cultivated with d-glucose as the growth substrate. The enzyme was found to hydrolyze laminarin to oligosaccharides varying in size from glucose to pentaose and to lesser amounts of larger oligosaccharides. The enzyme was unable to cleave laminaribiose but hydrolyzed triose to laminaribiose and glucose. The enzyme cleaved laminaritetraose, yielding laminaritriose, laminaribiose, and glucose, and similarly cleaved laminaripentaose, yielding laminaritetraose, laminaritriose, laminaribiose, and glucose. The enzyme cleaved only glucans containing beta-1,3 linkages. The pH and temperature optima were 4.8 and 55 degrees C, respectively. Stability in the absence of a substrate was observed at temperatures up to 50 degrees C and at pH values between 4.9 and 9.3. The molecular mass was determined to be 70 kilodaltons by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, and the pI was 7.2. Enzyme activity was significantly inhibited in the presence of HgCl(2), MnCl(2), KMnO(4), and N-bromosuccinimide. The K(m) of the enzyme on laminarin was 0.0016%, and the V(max) on laminarin was 3,170 mumol of glucose equivalents per mg of the pure enzyme per min.