Use of proteomics for validation of the isolation process of clotting factor IX from human plasma

Abstract

The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documentedOur methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.

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

“…Residual impurities that were still present in the F IX fraction, such as IaIp and vitronectin, were removed by nanofiltration, as documented by use of iTRAQ quantitation methods. The final product was pure F IX, with a specific activity greater than 200 IU/ mg protein [3,12]. These two papers [3,12] show that use of proteomic techniques can significantly extend the knowledge of this industrial process.…”

“…These two papers [3,12] show that use of proteomic techniques can significantly extend the knowledge of this industrial process. In addition to the identification of impurities, the use of iTRAQ methods in the MS analysis also provides quantitative information about the degree of their removal [12,106].…”

“…3). It was demonstrated that some serine proteases such as mannan-binding lectin serine protease (MBLSP) and HBP2 were not completely removed in anion-exchange chromatography steps [12]. Interestingly, HBP2 was also detected as impurity in some pd F VIII concentrates ( [106], see above).…”

“…In order to detect potentially harmful impurities and to prevent undesired changes of the target molecule, thorough proteomic investigation has been recommended for the early phases of product development [7,12]. After detection and identification of impurities and undesired changes of the therapeutically active molecule, the time for further process optimization (towards impurity removal and keeping the target protein molecule intact) can be significantly shortened [13,14].…”

Proteomic applications in blood transfusion: working the jigsaw puzzle

“…The analysis of prothrombin complex concentrates (PCCs) was used as a model to evaluate to what extent these technologies can detect differences in blood‐derived therapeutics beyond that of standard quality control tests [16]. Additional studies have reported on fractionated blood products including FVIII and FVIII/von Willebrand factor concentrates [17] and FIX concentrates [18]. These studies showed a surprising degree of difference between products from different manufacturers but revealed low batch to batch variability of single products.…”