Proteomic analysis for process development and control of therapeutic protein separation from human plasma

Abstract

The use of proteomics technology during the development of a new process for plasma protein separation was demonstrated. In a two-step process, the two most abundant proteins, human serum albumin and IgG, were removed in a first step of anion-exchange chromatography using a gel with very high capacity. Subsequently, two fractions containing medium and low abundance proteins were re-chromatographed on a smaller column with the same type of gel. Collected fractions were separated by SDS-PAGE and 2D electrophoresis, and excised proteins were digested with trypsin and identified by LC-ESI-MS/MS. This proteomic analysis proved to be a useful method for detection of low abundance therapeutic proteins and potential harmful contaminants during process development. Based on this method low abundance therapeutic proteins, such as vitamin K dependent clotting factors and inhibitors, could be identified as present in target fractions after chromatographic separation. In addition, the tracking of potentially dangerous impurities and designing proper steps for their removal are important outcomes when developing, refining or controlling a new fractionation schema. For the purpose of in-process control, in-solution digestion of complete fractions followed by protein identification with LC-ESI-MS/MS was demonstrated as a rapid and simple alternative to the entire analysis including 1D or 2D-electrophoretic steps.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen and Angiogenin Interact with Common Host Proteins, Including Annexin A2, Which Is Essential for Survival of Latently Infected Cells

“…Protein bands visible by Coomassie staining that were unique to LANA-1 and ANG lanes (Fig. 1E, lanes 2 and 3) were excised for proteomic analysis using LC-ESI-MS methods at the Midwest Proteome Center, RFUMS (116). Repetition of MS analysis four times demonstrated that only about 30% of the proteins were consistently immunoprecipitated, which corroborated earlier reports (5).…”

Mass spectrometry of peptides and proteins from human blood

“…Serum itself may be dried and subsequently completely re-dissolved in water; however, treatment with organic solvents and heating can affect the subsequent solubility of blood polypeptides. MS analysis might be used to monitor sample handling and quality procedures (Zheng et al, 2006c;Yi, Kim, & Gelfand, 2007;Gast et al, 2009a;Sun et al, 2009a;Yang et al, 2009a). It may also be possible to store blood samples dried on filter paper (Kulik et al, 2008) or PVDF (Gharahdaghi et al, 1996).…”

Therapeutic plasma proteins – application of proteomics in process optimization, validation, and analysis of the final product

“…We recently analyzed a highly enriched laboratory IgG concentrate (prepared by cation-exchange chromatography) by use of electrophoretic methods followed by LC-MS/MS, and transferrin, hemopectin, and a-2-macroglobulin were identified as impurities. However, no traces of potentially harmful components were detected [64]. Further detection and characterization of trace proteins can be achieved by use of the above-mentioned techniques for high-throughput analysis [33] and hexapeptide library beads [54], combined with LC-MS/MS.…”