Photochemical Pathogen Inactivation Treatment of Human Plasma (amotosalen + UVA) Has No Major Impact on the Protein expression pattern Assessed by a 2-DIGE Proteomic Assay.
Authors: Cazenave Jean-Pierre , Ohlmann Philippe , Isola Hervé , Gachet Christian 
Published: April 2020

Background: Photochemical treatment (PCT) using amotosalen HCl and UVA light (3 J/cm2: 320 – 400 nm) inactivates pathogens and leukocytes in therapeutic single donor apheresis plasma prepared within 8 hr of collection (INTERCEPTTM [I-FFP], Cerus Europe, Amersfoort, Netherlands). Clinical trials demonstrated efficacy and safety supporting a Class III CE mark and AFSSAPS (Medicinal Products Agency of France) registration of I-FFP for primary therapeutic indications including: congenital and acquired coagulopathies and TTP. Although no evidence of immune response to neo-antigens has been detected in clinical trials (Transfusion2005; 45:1610) or in post marketing surveillance studies, it remains unknown to what extent plasma proteins may be altered by PCT. We measured the impact of PCT on plasma protein profiles using a quantitative and qualitative proteomic method (2D-DIGE) to further define the possible occurrence of protein modifications in I-FFP.

Methods: Plasma units (650 ml) from 8 donors (4 male and 4 female) were collected by apheresis (Haemonetics MCS+, Braintree, MA) with AB16 anticoagulant. Plasma from each donor was separated into two plasma units, one was treated with the INTERCEPTTM system within 8 hr after collection, and the paired untreated unit served as a Control. The proteomes of I-FFP and Control plasma were evaluated using differential two-dimensional in-gel electrophoresis (2D-DIGE). Before fluorescent cyanine (Cy) labeling and 2D-gel electrophoresis, albumin and IgG, the more abundant proteins, were removed from plasma using a depletion kit (GE Healthcare). Prior to 2D gel electrophoresis, proteins from Control and I-FFP were labeled with fluorescent Cy3 (green) and Cy5 (red), respectively, in four samples and vice versa in the other four. Fifteen μg of protein from Control and or I-FFP plasma from each donor were mixed and resolved on the same minigel (7x7 cm). An internal standard (15 μg), labeled with Cy2 (blue), composed of equal amounts of plasma from each donor, was added in all experiment as a normalizing agent to monitor spot intensity for determination of reproducible quantitative differences of statistical significance (ANOVA, p &lt; 0.05). After electrophoresis, images of 2D gels were generated using a fluorescent scanner and data were analyzed using the Samespots software (NonlinearTM).

Results: 281 common protein spots were observed in the proteome pattern of plasma from all 8 independent experiments and included in the analysis. INTERCEPTTM treatment did not change any of these 281 spots (ANOVA, p &gt; 0.05). Using the internal standard to monitor quantitative changes, more than 90% of protein spots demonstrated an expression difference of less than 10% while the highest change observed was 1.8 fold.

Conclusion: Within 8 hrs after collection, pathogen inactivation of human plasma by the INTERCEPTTM process did not show evidence of plasma protein modification either in a qualitative or a quantitative manner. Further studies would be required to assess in greater detail the impact on protein modification of the process.