Continuous purification of influenza A virus particles using pseudo-affinity membrane chromatography.

Robust and flexible continuous unit operations that enable the establishment of intensified bioprocesses is one of the most relevant trends in manufacturing of biopharmaceuticals, including virus-based products. Sulfated cellulose membrane adsorbers (SCMA) are one of the most promising matrices for chromatographic purification of virus particles, like influenza viruses. Here, a three 'column' periodical counter current set-up was used to continuously purify influenza A/PR/8/34 virus particles using SCMA in bind-elute mode.

Optimization of cell culture-derived influenza A virus particles purification using sulfated cellulose membrane adsorbers.

Downstream processing remains one of the biggest challenges in manufacturing of biologicals and vaccines. This work focuses on a Design of Experiments approach to understand factors influencing the performance of sulfated cellulose membrane adsorbers for the chromatographic purification of a cell culture-derived H1N1 influenza virus strain (A/Puerto Rico/8/34). Membranes with a medium ligand density together with low conductivity and a high virus titer in the feed stream resulted in optimum virus yields and low protein and DNA content in the product fraction.

Purification of Monoclonal Antibodies Using a Fiber Based Cation-Exchange Stationary Phase: Parameter Determination and Modeling.

Monoclonal antibodies (mAb) currently dominate the market for protein therapeutics. Because chromatography unit operations are critical for the purification of therapeutic proteins, the process integration of novel chromatographic stationary phases, driven by the demand for more economic process schemes, is a field of ongoing research. Within this study it was demonstrated that the description and prediction of mAb purification on a novel fiber based cation-exchange stationary phase can be achieved using a physico-chemical model.

Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol.

Preparation and characterization of high capacity, strong cation-exchange fiber based adsorbents.

Motivated by the demand for more economical capture and polishing steps in downstream processing of protein therapeutics, a novel strong cation-exchange chromatography stationary phase based on polyethylene terephthalate (PET) high surface area short-cut fibers is presented. The fiber surface is modified by grafting glycidyl methacrylate (GMA) via surface-initiated atom transfer radical polymerization (SI-ATRP) and a subsequent derivatization leading to sulfonic acid groups. The obtained cation-exchange fibers have been characterized and compared to commercially available resin and membrane based adsorbers.

Modeling and simulation of anion-exchange membrane chromatography for purification of Sf9 insect cell-derived virus-like particles.

Recombinant protein-based virus-like particles (VLPs) are steadily gaining in importance as innovative vaccines against cancer and infectious diseases. Multiple VLPs are currently evaluated in clinical phases requiring a straightforward and rational process design. To date, there is no generic platform process available for the purification of VLPs.

Rational development of two flowthrough purification strategies for adenovirus type 5 and retro virus-like particles.

We report on the rational design and implementation of flowthrough (FT) platforms for purification of virus vectors (VVs) and virus-like particles (VLPs), combining anion-exchange polyallylamine membranes (Sartobind STIC) and core-shell octylamine resins (CaptoCore 700). In one configuration, the VV bulk is concentrated and conditioned with appropriate buffer in a ultra/diafiltration (UF/DF) unit prior to injection into the STIC chromatography membrane. The FT pool and an intermediate cut of the elution pool of the STIC membrane are admixed and directed to a second UF/DF.

Impact of grafting on the design of new membrane adsorbers for adenovirus purification.

The impacts of quaternary amine ligand density and matrix structure, namely hydrogel grafted and directly grafted, on state-of-the-art chromatographic membranes operated in bind-and-elute mode were evaluated for the purification of adenovirus serotype 5. The experiments were performed on a 96-well plate membrane holder, which is a convenient high-throughput screening tool for obtaining the best operating conditions for a process yield optimization. The results show that the hydrogel-grafted membranes are more suitable for virus purification than the directly grafted ones.

Development of a novel membrane aerated hollow-fiber microbioreactor.

A new challenge in biotechnological processes is the development of flexible bioprocessing platforms, allowing strain selection, facilitating scale-up and integrating separation steps. Miniaturization of such a cultivation system allows parallel use and the saving of resources but makes the supply of oxygen to the cells difficult. In this work we present a membrane aerated hollow-fiber microbioreactor (HFMBR) which consists of an acrylic glass module equipped with two different types of membrane fibers.