A scalable adenovirus production process, from cell culture to purified bulk

Adenovirus (AdV) vectors are commonly used in cancer gene therapy trials, evaluated in gene therapy and used as vaccines for various diseases. AdV vectors are well studied and are suitable as vaccine vectors due to their ability to infect different cell types, remain episomal and produce stable high titer material. Manufacturing of safe and efficacious clinical-grade virus relies on a scalable and cost-effective production process. In this study, we have combined experimental work and process economy calculations, from AdV production in cell culture to purified bulk product up to 10L scale. An efficient and scalable process for AdV production was developed by evaluation of each process step. The most studied vector is serotype 5, making this a suitable system for process development of AdV vectors. Human AdV5 expressing the green fluorescent protein (GFP) was used for process development. First, suspension HEK 293 cells adapted to serum-free cell culture medium were optimized for AdV production and evaluated in different single use bioreactor systems. Tween 20 was used for cell lysis as a replacement for the traditionally used Triton X-100 (now on the Authorization list (Annex XIV) of REACH, the regulation on Registration, Evaluation, Authorization and restriction of Chemicals). A residual Tween 20 assay with low detection limit was set-up. Filters and conditions for clarification, concentration and buffer exchange by tangential flow filtration were optimized. Anion exchange based capture step alternatives were compared, including different chromatography resins and membrane formats. Finally, core bead technology was evaluated as an alternative to size exclusion chromatography for the polishing step before the final formulation. Analytical methods for virus titer are challenging and depend on purity and quality of the sample. For total virus titer, qPCR and HPLC methods were used. Furthermore, a method based on surface plasmon resonance (Biacore) was developed for analysis of adenovirus titer. For infectious virus titer, we have used a cell based assay with automatic image analysis. Based on analytical data different downstream process alternatives were compared regarding load capacity, recovery and purity and we propose a robust and scalable process with a favorable process economy. Please click Additional Files below to see the full abstract

Effects of a person-centred telephone-support in patients with chronic obstructive pulmonary disease and/or chronic heart failure - A randomized controlled trial.

PURPOSE:To evaluate the effects of person-centred support via telephone in two chronically ill patient groups, chronic obstructive pulmonary disease (COPD) and/or chronic heart failure (CHF). METHOD:221 patients ≥ 50 years with COPD and/or CHF were randomized to usual care vs. usual care plus a person-centred telephone-support intervention and followed for six months. Patients in the intervention group were telephoned by a registered nurse initially to co-create a person-centred health plan with the patient and subsequently to discuss and evaluate the plan. The primary outcome measure was a composite score comprising General Self-Efficacy (GSE), re-hospitalization and death. Patients were classified as deteriorated if GSE had decreased by ≥ 5 points, or if they had been re-admitted to hospital for unscheduled reasons related to COPD and/or CHF or if they had died. RESULTS:At six-month follow-up no difference in the composite score was found between the two study groups (57.6%, n = 68 vs. 46.6%, n = 48; OR = 1.6, 95% CI: 0.9-2.7; P = 0.102) in the intention-to-treat analysis (n = 221); however, significantly more patients in the control group showed a clinically important decrease in GSE (≥ 5 units) (22.9%, n = 27 vs. 9.7%, n = 10; OR = 2.8, 95% CI: 1.3-6.0; P = 0.011). There were 49 clinical events (14 deaths, 35 re-admissions) in the control group and 41 in the intervention group (9 deaths, 32 re-admissions). Per-protocol analysis (n = 202) of the composite score showed that more patients deteriorated in the control group than in the intervention group (57.6%, n = 68 vs. 42.9%, n = 36; OR = 1.8, 95% CI 1.0-3.2; P = 0.039). CONCLUSION:Person-centred support via telephone mitigates worsening self-efficacy without increasing the risk of clinical events in chronically ill patients with CHF and/or COPD. This indicates that a patient-healthcare professional partnership may be established without the need for face-to-face consultations, even in vulnerable patient groups. TRIAL REGISTRATION:ISRCTN.com ISRCTN55562827