Process intensification for Peste des Petites Ruminants Virus vaccine production.

Process intensification for Peste des Petites Ruminants Virus (PPRV) vaccine production in anchorage dependent Vero cells is challenging, involving substantial amount of bioprocess development. In this study, we describe the implementation of a new, scalable bioprocess for PPRV vaccine production in Vero cells using serum-free medium (SFM), microcarrier technology in stirred-tank bioreactors (STB), in-situ cell detachment from microcarriers and perfusion. Vero cells were successfully adapted to ProVero™-1 SFM, reaching growth rates similar to serum-containing cultures (0.030 1/h vs 0.026 1/h, respectively). An in-situ cell detachment method was successfully implemented, with efficiencies above 85%. Up to 2.5-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining perfusion with the in-situ cell detachment method enabled the scale-up to 20 L STB directly from a 2 L STB, surpassing the need for a mid-scale platform (i.e. 5 L STB) and thus reducing seed train duration. Head-to-head comparison of cell growth and PPRV production in the 2 L and 20 L STB was performed, and no significant differences could be observed. Estimated infectious PPRV titers in Tissue Culture Infection Dose (TCID50) (TCID50/mL = 5 × 106 and TCID50/cell = 5) are within the log-range reported in literature for PPRV production in STB and SFM by Silva et al. (2008), thus confirming the feasibility and scalability of the seed train designed [1]. The novel and scalable vaccine production process herein proposed has the potential to assist the upcoming Peste des Petites Ruminants (PPR) Global Eradication Program (targeted by FAAO for 2030) by providing African local and/or regional manufacturers with a platform capable of generating over 25,000 doses of Nigeria 75/1 strain in just 19 days using a 20 L STB.

Fast and efficient protein purification using membrane adsorber systems.

The purification of proteins from complex cell culture samples is an essential step in proteomic research. Traditional chromatographic methods often require several steps resulting in time consuming and costly procedures. In contrast, protein purification via membrane adsorbers offers the advantage of fast and gentle but still effective isolation. In this work, we present a new method for purification of proteins from crude cell extracts via membrane adsorber based devices. This isolation procedure utilises the membranes favourable pore structure allowing high flow rates without causing high back pressure. Therefore, shear stress to fragile structures is avoided. In addition, mass transfer takes place through convection rather than diffusion, thus allowing very rapid separation processes. Based on this membrane adsorber technology the separation of two model proteins, human serum albumin (HSA) and immungluboline G (IgG) is shown. The isolation of human growth hormone (hGH) from chinese hamster ovary (CHO) cell culture supernatant was performed using a cation exchange membrane. The isolation of the enzyme penicillin acylase from the crude Escherichia coli supernatant was achieved using an anion exchange spin column within one step at a considerable purity. In summary, the membrane adsorber devices have proven to be suitable tools for the purification of proteins from different complex cell culture samples.

Monolithic peptidyl sorbents for comparison of affinity properties of plasminogen activators.

Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein's denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA-EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.