A scalable, integrated downstream process for production of a recombinant measles virus-vectored vaccine.

Purification of very large and complex, enveloped viruses, such as measles virus is very challenging, it must be performed in a closed system because the final product cannot be sterile filtered and often loss of virus titer and poor product purity has been observed. We developed a purification process where the clarified and endonuclease treated culture supernatant is loaded on a restricted access chromatography medium where small impurities are bound and the virus is collected in the flow-through, which is then concentrated, and buffer exchanged by ultra/diafiltration. Up to 98.5% of host cell proteins could be captured by direct loading of clarified and endonuclease treated cell culture supernatant. Reproducible process performance and scalability of the chromatography step were demonstrated from small to pilot scale, including loading volumes from 50 mL up to 9 L. A 10-fold virus concentration was achieved by the ultrafiltration using a 100 kDa flat-sheet membrane. The order of individual process steps had a large impact on the virus infectivity and total process yields. The developed process maintained virus infectivity and is twice as fast as the traditional process train, where concentration is performed before loading on the chromatography column. Capturing impurities by the restricted access medium makes it a platform purification process with a high flexibility, which can be easily and quickly adapted to other vectors based on the measles virus vector platform.

Fractal dimension of antibody-PEG precipitate: Light microscopy for the reconstruction of 3D precipitate structures.

Protein and in particular antibody precipitation by PEG is a cost-effective alternative for the first capture step. The 3D structure of precipitates has a large impact on the process parameters for the recovery and dissolution, but current technologies for determination of precipitate structures are either very time consuming (cryo-TEM) or only generate an average fractal dimension (light scattering). We developed a light microscopy based reconstruction of 3D structures of individual particles with a resolution of 0.1-0.2 µm and used this method to characterize particle populations generated by batch as well as continuous precipitation in different shear stress environments. The resulting precipitate structures show a broad distribution in terms of fractal dimension. While the average fractal dimension is significantly different for batch and continuous precipitation, the distribution is broad and samples overlap significantly. The precipitate flocs were monofractal from micro- to nanoscale showing a random but consistent nature of precipitate formation. We showed that the fractal dimension and 3D reconstruction is a valuable tool for characterization of protein precipitate processes. The current switch from batch to continuous manufacturing has to take the 3D structure and population of different protein precipitates into account in their design, engineering, and scale up.

Mid-manufacturing storage: Antibody stability after chromatography and precipitation based capture steps.

Antibodies of the IgG2 subclass were captured from the clarified cell culture fluid either by protein A chromatography or by polyethylene glycol precipitation. The captured intermediates were stored as neutralized eluates (protein A chromatography) or in solid form as polyethylene glycol precipitates over a period of 13 months at three temperatures, -20°C, 5°C, and room temperature to compare the capture technologies in regard of the resulting product storability. Monomer content, high molecular mass impurities product loss and changes in the composition of the charge variants were determined at six time points during the storage. At the beginning and end of the study, samples were additionally tested by differential scanning calorimetry, differential scanning fluorimetry, and circular dichroism to determine structural alterations occurring during storage. Protein A purified material was highly stable at all tested temperatures in regard of monomer content and product losses. A transient, acidic isoform was formed during the chromatography step which re-converted to the main charged variant upon storage within a matter of days. Precipitated antibodies could be stored at -20 or 5°C for 3 months without product losses but afterwards recovery yields dropped to 65%. At room temperature, the precipitated antibody was not stable and degraded within 3 months.

Continuous integrated antibody precipitation with two-stage tangential flow microfiltration enables constant mass flow.

Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two-stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two-stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre-existing high-molecular-weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter-current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow-through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.

Continuous cell flocculation for recombinant antibody harvesting.

BACKGROUND: Integrated continuous production technology is of great interest in biopharmaceutical industry. Efficient, flexible and cost effective methods for continuous cell removal have to be developed, before a fully continuous and integrated product train can be realized. The paper describes the development and testing of such an integrated continuous and disposable set-up for cell separation by flocculation combined with depth filtration. RESULTS: Screening of multiple flocculation agents, depth filters, and conditions demonstrated that the best performance was obtained with 0.0375% polydiallyldimethylammonium chloride (pDADMAC; a polycationic flocculation agent) in combination with Clarisolve® depth filters. Using this set-up, a 4-fold decrease of filtration area was achieved relative to standard filtration without flocculation, with yields of ≥97% and DNA depletion of up to 99%. Continuous operation was accomplished using a simple tubular reactor design with parallelization of the filtration. The reactor length was selected to allow a 13.2-min residence time, which was sufficient to complete flocculation in batch experiments. Continuous flocculation performance was monitored on-line using focused beam reflectance measurement. Filter switch cycles based on upstream pressure were controlled by in-line pressure sensors, and were stable from one filter to the next. CONCLUSION: It was demonstrated that stable and efficient continuous flocculation associated with depth filtration can be easily accomplished using tubular reactors and parallelization. Continuous cell separation is essential for the development of fully continuous integrated process trains. This cost-efficient disposable design run in continuous mode significantly reduces facility foot print, process costs and enables great flexbility. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis.

The rapid quantification of enveloped virus-like particles (VLPs) requires orthogonal methods to obtain reliable results. Three methods-nanoparticle tracking analysis (NTA), size-exclusion HPLC (SE-HPLC) with UV detection, and detection with multi-angle light scattering (MALS)-for quantification of enveloped VLPs have been compared, and the lower and upper limits of detection and quantification have been evaluated. NTA directly counts the enveloped VLPs, and a particle number is obtained with a lower limit of detection (LLOD) of 1.7×107part/mL and lower limit of quantification (LLOQ) of 3.4×108part/mL. SE-HPLC with UV detection was calibrated with standards characterized by NTA, and a LLOD of 6.9×109part/mL and LLOQ of 2.1×1010part/mL were found. SE-HPLC with MALS does not require a pre-calibrated sample because with a spherical model based on the Rayleigh-Gans-Debye approximation, the particle concentration can be directly deduced from the scattered light. A LLOD of 4.8×108part/mL and LLOQ of 2.1×109part/mL were measured and substantially lower compared to the UV method. The absolute particle concentration measured by SE-HPLC-MALS is one order of magnitude lower compared to measurement by NTA, which is explained by the wide size distribution of an enveloped VLP suspension. The model used for evaluation of light scattering data assumes monodisperse, homogeneous, and spherical particles.

Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.

Purification of HIV-1 gag virus-like particles and separation of other extracellular particles.

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×10(9) particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

BACKGROUND: Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. RESULTS: Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. CONCLUSIONS: P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.