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. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 105 U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

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. It was possible to recover 67.4% of the HA-activity and to remove 67.4% and 99.8% of the total protein and DNA, respectively. The performance of the continuous process operated over a total of 10 loops, was slightly inferior to was obtained in a comparable batch process. Nevertheless, it was possible to increase the effective usage of binding capacity to 80%, resulting on a productivity of 22.8 kHAU mlmemb-1 min-1. As a proof-of-principle, SCMA were successfully used as matrix for purification of cell-derived influenza virus particles, in continuous mode.

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. Fibers of polyethersulfone and polyvinyldifluoride were used for substrate and oxygen supply, respectively. Cultivation of E. coli as model organism and production of His-tagged GFP were carried out in the extracapillary space of the membrane aerated HFMBR and compared with cultivations in shaking flask which are commonly used for screening experiments. The measurement of the oxygen transfer capacity and the online monitoring of the dissolved oxygen during the cultivation were performed using a fiber optic oxygen sensor. Online measurement of the optical density was also integrated to the bioreactor. Due to efficient oxygen transfer, a better cell growth than in the shaking flask experiments was achieved, while no negative influence on the GFP productivity was observed in the membrane aerated bioreactor. Thus the feasibility of a future integrated downstreaming could also be demonstrated.

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. Flow rate and salt concentration in the buffer used for elution were of secondary importance while membrane permeability had no significant impact on separation performance. A virus loss of 2.1% in the flow through, a yield of 57.4% together with a contamination level of 5.1 pgDNA HAU-1 and 1.2 ngprot HAU-1 were experimentally confirmed for the optimal operating point predicted. The critical process parameters identified and their optimal settings should support the optimization of sulfated cellulose membrane adsorbers based purification trains for other influenza virus strains, streamlining cell culture-derived vaccine manufacturing.

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. High volumetric static binding capacities for lysozyme (90mg/mL) and polyclonal human IgG (hIgG, 92mg/mL) were found, suggesting an efficient multi-layer binding within the grafted hydrogel layer. A packed bed of randomly orientated fibers has been tested for packing efficiency, permeability and chromatographic performance. High dynamic binding capacities for lysozyme (50mg/mL) and hIgG (54mg/mL) were found nearly independent of the bed-residence time, revealing a fast mass-transport mechanism. Height equivalent to a theoretical plate (HETP) values in the order of 0.1 cm and a peak asymmetry factor (AF) of 1.8 have been determined by tracer experiments. Additionally inverse size-exclusion chromatography (iSEC) revealed a bimodal structure within the fiber bed, consisting of larger transport channels, formed by the voidage between the fibers, and a hydrogel layer with porous properties.

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. All relevant mass-transport phenomena during a bind and elute chromatographic cycle, namely convection, axial dispersion, boundary layer mass-transfer, and the salt dependent binding behavior in the fiber bed were described. This work highlights the combination of model adaption, simulation, and experimental parameter determination through separate measurements, correlations, or geometric considerations, independent from the chromatographic cycle. The salt dependent binding behavior of a purified mAb was determined by the measurement of adsorption isotherms using batch adsorption experiments. Utilizing a combination of size exclusion and protein A chromatography as analytic techniques, this approach can be extended to a cell culture broth, describing the salt dependent binding behavior of multiple components. Model testing and validation was performed with experimental bind and elute cycles using purified mAb as well as a clarified cell culture broth. A comparison between model calculations and experimental data showed a good agreement. The influence of the model parameters is discussed in detail.

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. In order to accelerate and simplify VLP downstream processing, there is a demand for novel development approaches, technologies, and purification tools. Membrane adsorbers have been identified as promising stationary phases for the processing of bionanoparticles due to their large pore sizes. In this work, we present the potential of two strategies for designing VLP processes following the basic tenet of 'quality by design': High-throughput experimentation and process modeling of an anion-exchange membrane capture step. Automated membrane screenings allowed the identification of optimal VLP binding conditions yielding a dynamic binding capacity of 5.7 mg/mL for human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A mechanistic approach was implemented for radial ion-exchange membrane chromatography using the lumped-rate model and stoichiometric displacement model for the in silico optimization of a VLP capture step. For the first time, process modeling enabled the in silico design of a selective, robust and scalable process with minimal experimental effort for a complex VLP feedstock. The optimized anion-exchange membrane chromatography process resulted in a protein purity of 81.5%, a DNA clearance of 99.2%, and a VLP recovery of 59%.

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. Finally, the retentate is injected into a CC700 packed bed adsorber where the purified VVs are collected in the FT pool, whereas the residual amount of DNA and host cell protein (HCP) are discarded in the eluate. The experimental recovery achieved with this downstream processing (DSP) platform is close to 100%, the DNA clearance is roughly a 4-log reduction, and the HCP level is reduced by 5 logs. The platform developed for VLP purification is simpler than the previous one, as the STIC membrane adsorber and CC700 bed are connected in series with no UF/DF unit in between. Experimentally, the FT scheme for VLP purification gave a recovery yield of 45% in the chromatography train; the experimental log reduction of DNA and HCP were 2.0 and 3.5, respectively. These results are in line with other purification strategies in the specific field of enveloped VLPs. Both DSP platforms were successfully developed from an initial design space of the binding of the major contaminant (DNA) to the two ligands, determined by surface plasmon resonance, which was subsequently scaled up and confirmed experimentally.

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. By reducing the number of grafted ligands to low (1.7µmol/cm(2)) or medium (2.4µmol/cm(2)) density, it is possible to increase the recovery of purified virus by 60% compared to a highly charged membrane (3.3µmol/cm(2)) that yielded a recovery rate lower than 30%. In the reported experiments, Sartobind(®) Q, chosen as benchmark comparison, provides a better compromise between high recovery and large dynamic binding capacity. Overall, this work contributes to the understanding and development of new membrane adsorbers specifically designed for virus purification.