The role of proteomics in plasma fractionation and quality control of plasma-derived therapeutic proteins.

Human plasma is still an important row material for isolation of therapeutic proteins. Human serum albumin (HSA) was the first therapeutic protein isolated from human plasma, and plasma-derived (pd) HSA concentrates are still being used for patient treatment1, 2. Preparations of human immunoglobulins for intravenous use (IVIG) are currently the driving force for usage of human plasma in the fractionation industry1. Von Willebrand factor (VWF), protease inhibitors such as alpha-1 antitrypsin (A1-AT) and antithrombin III (AT III) and clotting factors and inhibitors are further important therapeutic proteins that are isolated form human plasma. Their concentration in this biological fluid is up to six orders of magnitude lower than HSA and IgG. Consequently, the manufacturing process for isolation of these proteins with economically sufficient yield and in form of safe and active concentrates can be a true challenge2,3. Virological safety of human plasma and human plasma-derived therapeutic products is still a primary concern, and high safety standards have been implemented to make these products virologically safe2–4. Although all plasma-derived therapeutic preparations that are on the market belong to so-called “well-characterized biologicals” 5 they still contain relatively high amount of foreign proteins. In contradiction to this name, most impurities are poorly characterized6. After a relatively slow start, proteomics has finally progressed into different branches of transfusion medicine1,7,8. As shown in figure 1, proteomic technologies can be used as an efficient tool for process development and characterization of human plasma-derived therapeutic proteins. As shown in this figure, proteomics can be used for process validation and quality control of these therapeutic proteins. It has been demonstrated that proteomics also offer fast and efficient ways for the identification of potentially harmful impurities6. After their identification, the time for process optimization towards their removal can be significantly shortened6, 9. For quality control of final products, proteomics can be used for characterization of the active component, detection of impurities and determination of batch-to-batch variations10–12. Figure 1 Application of proteomics for validation of the production process of a therapeutic protein concentrate from human plasma and for final product characterization. Human serum albumin Human serum albumin (HSA) is present in human plasma at about 35 mg/mL, and it is the most abundant protein in this biological fluid. Essentially all therapeutic pd HSA preparations are manufactured form human plasma after cryoprecipitation by ethanol fractionation2. These concentrates contain between 95 and 98% HSA, and it is well known that they contain some plasma proteins as impurities. To get highly pure HSA, e.g. for measurement of its potential biological activity, additional purification steps are necessary13. Fortis et al. 10 analyzed the 98% pure, injectable HSA solution, and after direct analysis by SDS-PAGE followed by mass spectrometry of electrophoretically separated proteins, only a single impurity, haptoglobin, was identified. However, after enrichment of trace proteins by use of hexapeptide library beads, 13 additional plasma proteins were identified as impurities in this preparation. The list of these impurities is given in table I. As shown in this table, no potentially harmful proteins such as proteases, clotting factors and inhibitors were identified, and this preparation can be considered as biologically safe. However, this work demonstrates, that proteomic investigation gives a lot of additional information about the concentrate composition, and can be very useful, especially if rare side reactions occur after use of such therapeutics. Consequently, further investigations, especially comparison between the products of different producers and batch-to-batch variations for single products are still necessary. Table I Proteins identified from injectable HSA concentrate after treatment with peptide ligand library. Modified from Reference10 with permission IVIG Clotting factor VIII used to be the driving force for human plasma usage at the end of the last century. Now, IVIG concentrates are the leading products of the plasma fractionation industry. Recent reviews about the production and quality control of these concentrates deal mostly the aspects of yield and virus safety14, 15. Quality controls that are performed for release of IVIG batches are determination of subclass composition and contents of some potentially harmful proteins that can use adverse reactions (e.g. IgA and IgM), endotoxins and chemical (e.g. residues of viral inactivation treatments) impurities15. Recently, Buchacher et al.16 investigated the anticomplementary activity (ACA) of IVIG concentrates caused by high concentration of large size polymers in some concentrates. They could demonstrate that conditions under which polymers are formed have an influence on the ACA outcome, but also other impurities related to the starting material and unforeseen changes of process condition might affect the ACA and lead to batch-to-batch variations. Further detection and characterization of these impurities, e.g. by above discussed hexapeptide library beads10 could give additional information about their identity and possible side effects. In an earlier paper, Page et al.17 demonstrate that IgG fragmentation that can cause impaired efficacy of IVIG concentrates can be caused by contamination with serum proteases such as plasmin and kallikrein. However, proteomic investigations of different IVIG preparations and batch-to-batch comparison to show potential variations of the concentrates that are on the market are still outstanding and have to be performed.