Investigation and prediction of protein precipitation by polyethylene glycol using quantitative structure-activity relationship models.

Precipitation of proteins is considered to be an effective purification method for proteins and has proven its potential to replace costly chromatography processes. Besides salts and polyelectrolytes, polymers, such as polyethylene glycol (PEG), are commonly used for precipitation applications under mild conditions. Process development, however, for protein precipitation steps still is based mainly on heuristic approaches and high-throughput experimentation due to a lack of understanding of the underlying mechanisms. In this work we apply quantitative structure-activity relationships (QSARs) to model two parameters, the discontinuity point m* and the ß-value, that describe the complete precipitation curve of a protein under defined conditions. The generated QSAR models are sensitive to the protein type, pH, and ionic strength. It was found that the discontinuity point m* is mainly dependent on protein molecular structure properties and electrostatic surface properties, whereas the ß-value is influenced by the variance in electrostatics and hydrophobicity on the protein surface. The models for m* and the ß-value exhibit a good correlation between observed and predicted data with a coefficient of determination of R2≥0.90 and, hence, are able to accurately predict precipitation curves for proteins. The predictive capabilities were demonstrated for a set of combinations of protein type, pH, and ionic strength not included in the generation of the models and good agreement between predicted and experimental data was achieved.

High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography.

The development and manufacturing of safe and effective vaccines relies essentially on the availability of robust and precise analytical techniques. Virus-like particles (VLPs) have emerged as an important and valuable class of vaccines for the containment of infectious diseases. VLPs are produced by recombinant protein expression followed by purification procedures to minimize the levels of process- and product-related impurities. The control of these impurities is necessary during process development and manufacturing. Especially monitoring of the VLP size distribution is important for the characterization of the final vaccine product. Currently used methods require long analysis times and tailor-made assays. In this work, we present a size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) method to characterize VLPs and quantify aggregates within 3.1min per sample applying interlaced injections. Four analytical SEC columns were evaluated for the analysis of human B19 parvo-VLPs and murine polyoma-VLPs. The optimized method was successfully used for the characterization of five recombinant protein-based VLPs including human papillomavirus (HPV) VLPs, human enterovirus 71 (EV71) VLPs, and chimeric hepatitis B core antigen (HBcAg) VLPs pointing out the generic applicability of the assay. Measurements were supported by transmission electron microscopy and dynamic light scattering. It was demonstrated that the iSE-UHPLC method provides a rapid, precise and robust tool for the characterization of VLPs. Two case studies on purification tools for VLP aggregates and storage conditions of HPV VLPs highlight the relevance of the analytical method for high-throughput process development and process monitoring of virus-like particles.

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%.

High-throughput process development of an alternative platform for the production of virus-like particles in Escherichia coli.

The production of safe vaccines against untreatable or new diseases has pushed the research in the field of virus-like particles (VLPs). Currently, a large number of commercial VLP-based human vaccines and vaccine candidates are available or under development. A promising VLP production route is the controlled in vitro assembly of virus proteins into capsids. In the study reported here, a high-throughput screening (HTS) procedure was implemented for the upstream process development of a VLP platform in bacterial cell systems. Miniaturized cultivations were carried out in 48-well format in the BioLector system (m2p-Labs, Germany) using an Escherichia coli strain with a tac promoter producing the murine polyomavirus capsid protein (VP1). The screening procedure incorporated micro-scale cultivations, HTS cell disruption by sonication and HTS-compatible analytics by capillary gel electrophoresis. Cultivation temperatures, shaking speeds, induction and medium conditions were varied to optimize the product expression in E. coli. The most efficient system was selected based on an evaluation of soluble and insoluble product concentrations as well as on the percentage of product in the total soluble protein fraction. The optimized system was scaled up to cultivation 2.5L shaker flask scale and purified using an anion exchange chromatography membrane adsorber, followed by a size exclusion chromatography polishing procedure. For proof of concept, purified VP1 capsomeres were assembled under defined buffer conditions into empty capsids and characterized using transmission electron microscopy (TEM). The presented HTS procedure allowed for a fast development of an efficient production process of VLPs in E. coli. Under optimized cultivation conditions, the VP1 product totalled up to 43% of the total soluble protein fraction, yielding 1.63 mg VP1 per mL of applied cultivation medium. The developed production process strongly promotes the murine polyoma-VLP platform, moving towards an industrially feasible technology for new chimeric vaccines.

Downstream processing of virus-like particles: single-stage and multi-stage aqueous two-phase extraction.

The demand for vaccines against untreated diseases has enforced the research and development of virus-like particle (VLP) based vaccine candidates in recent years. Significant progress has been made in increasing VLP titres during upstream processing in bacteria, yeast and insect cells. Considering downstream processing, the separation of host cell impurities is predominantly achieved by time-intensive ultracentrifugation processes or numerous chromatography and filtration steps. In this work, we evaluate the potential of an alternative separation technology for VLPs: aqueous two-phase extraction (ATPE). The benefits of ATPE have been demonstrated for various biomolecules, but capacity and separation efficiency were observed to be low for large biomolecules such as VLPs or viruses. Both performance parameters were examined in detail in a case study on human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A solubility-guided approach enabled the design of polyethylene (PEG) salt aqueous two-phase systems with a high capacity of up to 4.1mg/mL VLPs. Unique separation efficiencies were obtained by varying the molecular weight of PEG, the pH value and by using neutral salt additives. Further improvement of the separation of host cell impurities was achieved by multi-stage ATPE on a centrifugal partition chromatography (CPC) device in 500mL scale. While single-stage ATPE enabled a DNA clearance of 99.6%, multi-stage ATPE improved the separation of host cell proteins (HCPs). The HPLC purity ranged from 16.8% (100% VLP recovery) for the single-stage ATPE to 69.1% (40.1% VLP recovery) for the multi-stage ATPE. An alternative two-step downstream process is presented removing the ATPS forming polymer, cell debris and 99.77% DNA with a HPLC purity of 90.6% and a VLP recovery of 63.9%.

Alternative separation steps for monoclonal antibody purification: combination of centrifugal partitioning chromatography and precipitation.

Protein drugs continue to grow both in medicinal importance as in scale of their production. This furthers the interest in separation technologies that have the potential to replace chromatographic steps in a protein purification process. Two such unit operations that are employed in large scale in the chemical industry are extraction and precipitation. Their usefulness for the purification of proteins has been demonstrated, but the integration of such unit operations in a way that generate an output stream of high protein concentration and low process related impurities was missing. In this work, we employ centrifugal partitioning chromatography ('CPC') in combination with precipitation of the protein of interest to purify a cell culture supernatant of a monoclonal antibody producing cell line. Centrifugal partitioning chromatography was used as means of multi-step extraction using aqueous two-phase systems and was able to remove up to 88.2% of host cell protein ('HCP'). The following PEG driven precipitation and resolubilization of the protein of interest was use to condition the CPC output stream to suit subsequent chromatographic steps, to increase mAb concentration, remove the phase forming polymer, further improve HCP clearance, and integrate a low pH hold step for viral clearance. The entire process reduced HCP content by 99.4% while recovering 93% of the protein of interest. High throughput screening techniques were extensively employed during the development of the process.

High throughput screening based selection of phases for aqueous two-phase system-centrifugal partitioning chromatography of monoclonal antibodies.

Aqueous two-phase systems have been demonstrated to be a possible alternative to chromatographic separations during the industrial purification of proteins. While convenient high throughput screening methods were shown to drastically reduce experimental effort for the evaluations of ATPS as a unit operation, the selection of which phases to investigate is currently guided largely by prior knowledge. Correlations between protein descriptors and distribution were found, but the general applicability of such correlations especially under conditions of high protein load, is questionable, as currently no correlations take the saturation of the phases with protein into account. In this manuscript, we demonstrate how precipitation experiments using the phase forming components can guide the selection of both system type and tieline length for the purification of monoclonal antibodies. Phase selection and process development time can thus be significantly reduced, as all the necessary precipitation, binodal, and tieline experiments can be conducted within one day. Good qualitative correlations between precipitation data and both distribution and recovery of the target molecule were found. Most promising systems were selected for upscale to a 500mL CPC. Influence of operation condition on the column and on HCP clearance was investigated. An increase in HCP clearance of more than threefold compared to batch extractions was observed. The importance of load protein concentration underlined the value of using a screening approach that incorporated target protein solubility data.