Allogenic and autologous nondiluted serum eye drops-validation strategy compliant with good manufacturing practice.

BACKGROUND AND AIMS: Serum eye drops alleviate ocular symptoms of diseases such as sicca syndrome, or chronic graft-versus-host disease. This study was designed for good manufacturing practice validation of our standard manufacturing, storage and transport processes for both autologous and allogenic SEDs. Specifications of quality parameters are lacking and were aimed to be defined. METHODS: Using sterile collected, coagulated whole blood, serum was separated by centrifugation and filled into single-use eye drop applicator vials. Quality control tests included visual inspection, sterility, leukocyte concentration, pH, vitamin A, TGF-ß and VEGF-A. Samples were collected after manufacture and after 24 h and 6 months of frozen storage (-20°C). Sterility testing was performed after opening the SED applicators at specified intervals. For transport validation, SEDs were packed in insulated transport bags and stored at 20-24°C and 30-32°C for 8 h. RESULTS: Vitamin A, TGF-ß and VEGF-A assays showed no difference in concentration between fresh and 24 h frozen serum. All specifications for pH (aim 7.4) and cellular contamination were met and microbiological contamination tests were negative. Shelf-life was defined as 6 months at -20°C. Once opened, the product must be used within 24 h to avoid bacterial outgrowth. Transporting frozen SEDs from the manufacturer via a local pharmacy to the patient within a maximum of 4 h was demonstrated. CONCLUSIONS: The GMP compliance of our production, storage and transport processes for autologous and allogenic SEDs was successfully validated. 100% serum eye drops in single-use applicators can be safely used for up to 24 h after opening.

Validation of an apoptosis assay for extracorporeal photopheresis.

OBJECTIVES: This validation study investigated a flow cytometric apoptosis assay according to good manufacturing practice (GMP). BACKGROUND: Extracorporeal photopheresis (ECP) is a treatment for various immunological diseases and cutaneous T-cell lymphomas. It is based on the induction of apoptosis by 8-methoxypsoralene and ultraviolet A light. The quantification of apoptosis is therefore essential for ECP improvements. However, despite numerous publications on apoptosis, validated technical details are lacking. METHODS AND MATERIALS: Mononuclear cells were collected by apheresis and treated by ECP or camptothecin. Samples taken before and after ECP were cultured for 24, 48 and 72 h and analysed for apoptosis and viability of T cells and monocytes by flow cytometry with Annexin V and 7-AAD staining. Accuracy of the assay, intra- and inter-assay precision and the pre-analytical and analytical stability of the analytes were the investigated parameters. RESULTS: Our data indicate that the median intra- and inter-assay precision coefficient of variation for T cells was 3.86% and 4.80%, respectively. Pre-analytical stability of T cells and monocytes was ensured during short-term storage for up to 2 h on ice. After staining, analytical stability was limited to 30 min, likely because of ongoing apoptosis and loss of monocytes due to plastic adhesion. CONCLUSION: The results of this validation study show that the assay is GMP-compliant and that its reliability, accuracy and precision are acceptable. While pre-analytical stability of the cells was compatible with on-site procedures, our analytical stability data indicate that this assay is not suited for batch mode analysis of ECP products.

Granulocytapheresis with modified fluid gelatin versus high-molecular-weight hydroxyethyl starch: a matched-pair analysis.

BACKGROUND: Granulocyte apheresis requires a sedimentation agent. Usually, hydroxyethyl starch (HES) is administered to donors for this purpose and, as granulocyte concentrate (GC) ingredient, also to patients. Authorities recently recommended suspending market authorizations for starch-containing products due to side effects. Therefore, we tested the efficacy of modified fluid gelatin (MFG, Gelafundin 4%) versus hetastarch (Hespan) for GC apheresis. STUDY DESIGN AND METHODS: This retrospective matched-pair analysis compared MFG- and hetastarch-derived GCs. Each group consisted of 15 unrelated male donors mobilized with dexamethasone and granulocyte-colony-stimulating factor for apheresis on 1 or 2 days with the COBE Spectra's PMN program. RESULTS: In each group, 24 GCs were collected from 15 male donors and analyzed. None of the HES-derived products, but two of the MFG-derived products (8.3%), had aggregates and could not be used. The HES-derived products had significantly higher neutrophil counts on the first day (7.7 × 1010 /unit vs. 4.0 × 1010 /unit; p = 0.00005) as well as second day of apheresis (4.0 × 1010 /unit vs. 1.1 × 1010 /unit; p = 0.0002). Median white blood cell collection efficacies were lower with MFG than with HES on Day 1 (24% vs. 43%) and Day 2 (15% vs. 37%). Twenty-one percent of the MFG-derived products had less than 1 × 1010 granulocytes. CONCLUSION: These results indicate that granulocyte apheresis is feasible with MFG as well as with hetastarch and that the latter is superior for GC production, if used in the same dosage. In addition, aggregates in GC from the COBE Spectra were observed in the MFG group but not in the hetastarch group.

A method for the quantification of 8-methoxypsoralen by mass spectrometry for offline extracorporeal photopheresis.

BACKGROUND: Extracorporeal photopheresis (ECP) is an efficient method to treat various autoimmune diseases, cutaneous T-cell lymphoma, and graft-versus-host disease. It is based on the ex vivo inactivation of lymphocytes by 8-methoxypsoralen (8-MOP)/UV light treatment. Despite the adhesive, lipophilic nature of 8-MOP, no quality control is established for the ECP procedure. METHODS: We developed a sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to monitor residual 8-MOP concentration after UVA irradiation in the whole blood supernatant after acetonitrile precipitation. RESULTS: The preanalytical stability of 8-MOP exceeded 7 days, allowing batch mode analysis. Linearity was determined with R2 above 0.99. The 8-MOP concentrations decreased exponentially after UV exposure, with decay constants of 0.0259 in plasma and 0.0528 in saline. The recovery of 8-MOP in photopheresates was about 68%, indicating binding to DNA as well as to plastic structures. UVA induced no 8-MOP fragmentation, but caused self-adducts under extreme conditions (10-fold UV dosage). CONCLUSIONS: Detection of 8-MOP proved to be feasible and demonstrated that the doses were in the pharmaceutically active range.