| 摘要: | Background
It is a regulatory requirement that the manufacturing process of recombinant protein products derived from mammalian cell cultures and that of human or animal plasmas products for therapeutic use should have a demonstrated capacity to effectively inactivate or remove infectious viruses potentially present in the starting material. Virus inactivation of plasma products by chemical or liquid heat treatments is typically conducted in reusable stainless steel vessels. However, re-use of equipment requires lengthy qualification and validation of the cleaning and sanitisation procedures between production batches. Single-use technology can offer operational flexibility, reduced capital infrastructure costs, and increased efficiency by minimizing time and validation requirements associated with hardware cleaning. This study determines the suitability of a single use container system for solvent/detergent (S/D) virus inactivation of human plasma and purified immunoglobulins (Ig).
Aims
The aim of the study is to validate a single-use system for S/D treatment of protein therapeutics by evaluating protein quality, rate and extent of inactivation of two model viruses, and chemical compatibility.
Materials and Methods
Plasma was collected by apheresis. To obtain Ig, plasma was cryoprecipitated at 2-4°C, and the supernatant was treated by 5% caprylic acid pH 5.5 at 25°C. Plasma and immunoglobulin test materials were S/D-treated in Mobius® or standard Polyvinyl Chloride (PVC) bags, as control, using 1% Tri (n-butyl) phosphate (TnBP) and either 1% Triton X-100 or 1% Tween-80 combinations at 31°C for 4-6 hours. Impact on protein quality was evaluated. Total protein, immunoglobulin G (IgG), IgA, IgM, SDS-PAGE, zone-electrophoresis, proteolytic activity, thrombin-like activity, Factor Xa (FXa) and plasmin were determined to assess protein quality. For plasma coagulation activity, prothrombin time (PT) and activated partial thromboplastin time (aPTT), thrombin generation assay, FV, FVIII, FIX and FXI were also tested. Volatile and non volatile organic leachables from low-density polyethylene film (Pureflex®) used in 1L scale studies after exposure to S/D in phosphate-buffered saline were identified compared to controls in glass containers. Virus spiking studies were performed with Xenotropic Murine Leukemia Virus (XMuLV) and Bovine Viral Diarrhea Virus (BVDV to determine the kinetics of virus inactivation using infectivity assays.
Results
The plasma study showed that protein parameters including total protein, albumin, IgG, IgA, IgM and protein electrophoresis profiles were unaffected by either S/D treatment nor the single-use system. Coagulation activity and thrombin generation after S/D treatment were also not altered. The process using immunoglobulin did not reveal detectable difference due to the S/D viral inactivation steps nor the single-use system. Proteolytic enzymes content and zone electrophoresis profile were unchanged before and after S/D treatment in the single-use system. Cumulative leachables levels generated after exposure to S/D were 1.5 and 1.85 ppm when using 0.3% TnBP combined with 1% Tween 80 and 1% Triton X-100, respectively. Efficient inactivation of both XMuLV and BVDV was observed, although differences in the rate of inactivation were dependent on both virus and S/D mixture.
Conclusion
Effective S/D virus inactivation using single-use technology is achievable. It does not alter plasma proteins, and induces minimal release of leachable. This development should facilitate the adoption of the single-use technology for large-scale manufacturing and support consideration for other viral-inactivation treatments such as low pH in addition to S/D treatment. |
