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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.

In this paper a generic process model for the simulation of various downstream processes for protein purification is presented. It consists of detailed, dynamic models for ion-exchange membrane adsorbers, ion-exchange and size-exclusion chromatography and ultra-/diafiltration. For the implemented ion-exchange membrane adsorber model, the steric mass action model has been applied. The isotherm parameters have been determined experimentally for bovine serum albumin (BSA) and immunoglobulin G (IgG). The comparison between simulation results of the ion-exchange membrane and experiments for two protein mixtures containing BSA and IgG shows satisfactory agreement. Two case studies based on the purification process of human serum albumin are performed to illustrate the influence of structural changes in the process flowsheet on the process performance. The simulation results show that the replacement of an anion-exchange chromatography column by an anion-exchange membrane adsorber leads to a significant reduction in overall process time at constant yield and purity. The case studies demonstrate the applicability of the generic process model for detailed process analysis.

Human and equine influenza A virus in cell culture supernatant (serum-free and serum-containing cultivation) was directly adsorbed to Sartobind Q and D MA75 anion-exchangers. A light scattering detector was used to trace virus particles online. The intensity of scattered light was shown to correlate with viral HA activity. Dynamic capacities of Sartobind Q (operated at a flow rate of 264 cm h −1 ) were consistent but depended on the cell culture medium (3.12 kHAU cm −2 for serum-free and 5.19 kHAU cm −2 for serum-containing medium). Elution of adsorbed virus from Sartobind Q by displacement with sodium chloride (up to 1.5 M, pH 7.0) resulted in average yields of 86% (based on HA activity). Elution from Sartobind D resulted in yields of only 38%. Lowering the pH (down to 4.2) or combining a low pH with salt displacement failed to elute virions. The pH of loaded supernatant was important with respect to recovery but not capacity. Preparative runs with Sartobind Q resulted in about five-fold enrichment of HA activity and 77% reduction in total protein. Host-cell DNA, in contrast, was recovered completely in the product fraction. The average virus yield was 72%. A productivity of 67 l of culture broth m −2 h −1 was achieved. Due to their high productivity, ease of operation and acceptable yields Sartobind Q anion-exchangers can be considered promising candidates for the large-scale purification of cell culture derived influenza virus.

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.

Spodoptera frugiperda-9 (Sf-9) cells used in conjunction with the baculovirus expression vector system (BEVS) represent a promising platform for the rapid development and manufacture of protein complexes and virus-like particle (VLP) products. Several studies have described the superiority of single-use wave-mixed bioreactors although reusable stirred and, more recently, single-use stirred bioreactors have also been successfully applied. Due to their bioengineering characteristics (more homogeneous energy dissipation, reduced foam formation), wave-mixed systems are often preferred. However, a direct comparison of the influence of single-use wave-mixed and single-use stirred bioreactors on cell growth and protein expression in Sf-9/BEVS-based production processes was still lacking. We investigated Sf-9 cell growth and expression of a recombinant secreted alkaline phosphatase (rSEAP) in the wave-mixed BIOSTAT® RM as well as the stirred UniVessel® SU and a serum-free culture medium. Irrespective of the bioreactor system, comparable growth, substrate, and metabolite courses as well as peak cell densities (>1.2 × 107 cells mL−1) were observed in Sf-9 cell expansions performed in batch mode. Additionally, identical rSEAP quality and maximum rSEAP activities were found in biphasic productions in both bioreactor systems. Concluding, comparability of single-use wave-mixed and stirred bioreactors for insect cell culture processes was demonstrated for the first time.

Background: To improve cultivation conditions for human bone-marrow-derived mesenchymal stem cells, we redesigned the commercially available UniVessel® SU bioreactor using results obtained from computational fluid dynamics. The goal was to produce ≥1 × 109 cells and to achieve expansion factors ≥30. Screening studies suggested that microcarrier solid fractions of at least 0.3% are required to reach the appropriate cell densities. Results: The fluid flow pattern found in the most promising modification (#2) was altered by increasing the impeller blade angle and lowering the off-bottom clearance. As a result, the maximum required specific power input was reduced by a factor of 2.2–4.6, depending on the microcarrier concentration, and peak cell densities were 3.4-times higher than in the standard version. Conclusion: The peak cell number of nearly 1.1 × 109 cells (expansion factor = 35), which was achieved in our low-serum cultivations, indicates an improvement in the redesigned UniVessel® SU configuration f...

For the production of recombinant proteins for clinical use animal cell cultivation is used. Only these cells are able to perform correct folding and glycosylation of the desired protein. As the production process is expensive and long, cheaper and/or swifter cultivation routines are required. Chinese hamster ovary (CHO) cells were used to examine the expansion of cells and the production of recombinant human growth hormone in different cell culture systems which are supposed to achieve higher cell densities and product concentrations compared to conventional cell culture systems. The CHO cells were grown in suspension in serum-free, low-protein medium. Five different culture systems were used for batch-cultivation: Biostat B, BelloCell 500, spinner flask, RCCS-D and miniPERM. The systems differed in oxygen supply and medium agitation. While cells are agitated by stirrers in Biostat B and spinner flask, the whole medium is revolved in BelloCell 500, RCCS-D and miniPERM. Unlike the other systems the BelloCell 500 retains the CHO cells on a matrix. The aim was to maximize cell growth and productivity, which was achieved best in BelloCell and RCCS-D. In a second step the influence of temperature on growth and product formation was examined.

Tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots by converting plasminogen in its active form, plasmin, dissolving the major component of blood clots, fibrin. t-PA and plasminogen possess a high affinity binding site for fibrin, but also some synthetic polymers can provide the stimulating effects of plasminogen activation. High performance monolithic disk chromatography (HPMDC) is a very fast, efficient and suitable tool for the isolation of biological active compounds. A step by step modelling of possible affinity pairs and different oligo/polymer forms of linear and branched lysine derivatives using HPMDC is shown. The results of the evaluation of these affinity interactions were compared to natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The results enable a practical choice of affinity systems to be used for the fast and efficient analytical and preparative methods for the down stream processing of recombinant production of t-PA.

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.

The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-L-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.