Efficient and scalable clarification of Orf virus from HEK suspension for vaccine development.

The Orf virus (ORFV) is a promising vector platform for the generation of vaccines against infectious diseases and cancer, highlighted by its progression to clinical testing phases. One of the critical steps during GMP manufacturing is the clarification of crude harvest because of the enveloped nature and large size of ORFV. This study presents the first description of ORFV clarification process from a HEK suspension batch process. We examined various filter materials, membrane pore sizes, harvest timings, and nuclease treatments. Employing the Ambr® crossflow system for high-throughput, small-volume experiments, we identified polypropylene-based Sartopure® PP3 filters as ideal. These filters, used in two consecutive stages with reducing pore sizes, significantly enhanced ORFV recovery and addressed scalability challenges. Moreover, we demonstrated that the time of harvest and the use of a nuclease play a decisive role to increase ORFV yields. With these findings, we were able to establish an efficient and scalable clarification process of ORFV derived from a suspension production process, essential for advancing ORFV vaccine manufacturing.

A Novel and Simplified Anion Exchange Flow-Through Polishing Approach for the Separation of Full From Empty Adeno-Associated Virus Capsids.

Adeno-associated viruses (AAV) are widely used viral vectors for in vivo gene therapy. The purification of AAV, particularly the separation of genome-containing from empty AAV capsids, is usually time-consuming and requires expensive equipment. In this study, we present a novel laboratory scale anion exchange flow-through polishing method designed to separate full and empty AAV. Once the appropriate conditions are defined, this method eliminates the need for a chromatography system. Determination of optimal polishing conditions using a chromatography system revealed that the divalent salt MgCl2 resulted in better separation of full and empty AAV than the monovalent salt NaCl. The efficacy of the method was demonstrated for three distinct AAV serotypes (AAV8, AAV5, and AAV2) on two different stationary phases: a membrane adsorber and a monolith, resulting in a 4- to 7.5-fold enrichment of full AAV particles. Moreover, the method was shown to preserve the AAV capsids' functional potency and structural integrity. Following the successful establishment of the flow-through polishing approach, it was adapted to a manual syringe-based system. Manual flow-through polishing using the monolith or membrane adsorber achieved 3.6- and 5.4-fold enrichment of full AAV, respectively. This study demonstrates the feasibility of separating full and empty AAV without complex linear or step gradient elution and the necessity of specialized equipment. Flow-through polishing provides a rapid and easy-to-perform platform for polishing multiple vector preparations, addressing a critical aspect in the research and development of novel gene therapies.

Scaling Up of Steric Exclusion Membrane Chromatography for Lentiviral Vector Purification.

Lentiviral vectors (LVs) are widely used in clinical trials of gene and cell therapy. Low LV stability incentivizes constant development and the improvement of gentle process steps. Steric exclusion chromatography (SXC) has gained interest in the field of virus purification but scaling up has not yet been addressed. In this study, the scaling up of lentiviral vector purification by SXC with membrane modules was approached. Visualization of the LVs captured on the membrane during SXC showed predominant usage of the upper membrane layer. Furthermore, testing of different housing geometries showed a strong influence on the uniform usage of the membrane. The main use of the first membrane layer places a completely new requirement on the scaling of the process and the membrane modules. When transferring the SXC process to smaller or larger membrane modules, it became apparent that scaling of the flow rate is a critical factor that must be related to the membrane area of the first layer. Performing SXC at different scales demonstrated that a certain critical minimum surface area-dependent flow rate is necessary to achieve reproducible LV recoveries. With the presented scaling approach, we were able to purify 980 mL LVs with a recovery of 68%.

Impact of the Membrane Structure of the Stationary Phase on Steric Exclusion Chromatography (SXC) of Lentiviral Vectors.

For steric exclusion chromatography (SXC), hydrophilic stationary phases are used to capture the target molecule in the presence of polyethylene glycol. The influence of the structure and pore size of the stationary phase on the process requirements are not yet well understood. To better understand the SXC process, membranes with different pore sizes that served as a stationary phase were compared for the purification of lentiviral vectors (LVs). A design of experiments (DoE) was performed to assess the combined impact of PEG concentration and membrane pore size on the purification performance. A visualization experiment showed that the LVs were captured on the first membrane layer for a pore size up to 2.2 µm, and for a pore size larger than 2.2 µm, LVs were also partly found on the second and third membrane layers. Moreover, we could observe that increasing membrane pore size requires a higher PEG concentration to achieve comparable LV recoveries. Using five membrane layers as a stationary phase was sufficient to achieve good performance, supporting the visualized capture results. In conclusion, we could show that each stationary phase has its optimal PEG buffer compositions for SXC, depending on the membrane structure and pore size.

Unveiling the secrets of adeno-associated virus: novel high-throughput approaches for the quantification of multiple serotypes.

Adeno-associated virus (AAV) vectors are among the most prominent viral vectors for in vivo gene therapy, and their investigation and development using high-throughput techniques have gained increasing interest. However, sample throughput remains a bottleneck in most analytical assays. In this study, we compared commonly used analytical methods for AAV genome titer, capsid titer, and transducing titer determination with advanced methods using AAV2, AAV5, and AAV8 as representative examples. For the determination of genomic titers, we evaluated the suitability of qPCR and four different digital PCR methods and assessed the respective advantages and limitations of each method. We found that both ELISA and bio-layer interferometry provide comparable capsid titers, with bio-layer interferometry reducing the workload and having a 2.8-fold higher linear measurement range. Determination of the transducing titer demonstrated that live-cell analysis required less manual effort compared with flow cytometry. Both techniques had a similar linear range of detection, and no statistically significant differences in transducing titers were observed. This study demonstrated that the use of advanced analytical methods provides faster and more robust results while simultaneously increasing sample throughput and reducing active bench work time.

Stability studies for the identification of critical process parameters for a pharmaceutical production of the Orf virus.

A promising new vaccine platform is based on the Orf virus, a viral vector of the genus Parapoxvirus, which is currently being tested in phase I clinical trials. The application as a vaccine platform mandates a well-characterised, robust, and efficient production process. To identify critical process parameters in the production process affecting the virus' infectivity, the Orf virus was subjected to forced degradation studies, including thermal, pH, chemical, and mechanical stress conditions. The tests indicated a robust virus infectivity within a pH range of 5-7.4 and in the presence of the tested buffering substances (TRIS, HEPES, PBS). The ionic strength up to 0.5 M had no influence on the Orf virus' infectivity stability for NaCl and MgCl2, while NH4Cl destabilized significantly. Furthermore, short-term thermal stress of 2d up to 37 °C and repeated freeze-thaw cycles (20cycles) did not affect the virus' infectivity. The addition of recombinant human serum albumin was found to reduce virus inactivation. Last, the Orf virus showed a low shear sensitivity induced by peristaltic pumps and mixing, but was sensitive to ultrasonication. The isoelectric point of the applied Orf virus genotype D1707-V was determined at pH3.5. The broad picture of the Orf virus' infectivity stability against environmental parameters is an important contribution for the identification of critical process parameters for the production process, and supports the development of a stable pharmaceutical formulation. The work is specifically relevant for enveloped (large DNA) viruses, like the Orf virus and like most vectored vaccine approaches.

Steric exclusion chromatography of lentiviral vectors using hydrophilic cellulose membranes.

Enveloped viral vectors like lentiviral vectors pose purification challenges due to their low stability. A gentle purification method is considered one of the major bottlenecks for lentiviral vector bioprocessing. To overcome these challenges, a promising method is steric exclusion chromatography which has been used to purify a variety of target molecules. In this study, we successfully identified optimal process parameters for steric exclusion chromatography to purify lentiviral vectors. Lentiviral vector particle recoveries and infectious recoveries of 86% and 88%, respectively, were achieved. The process parameters optimal for steric exclusion chromatography were determined as follows: polyethylene glycol with a molecular weight of 4000 Da, a polyethylene glycol concentration of 12.5%, and a flow rate of 7 mL⋅min-1 using 5 layers of stabilized cellulose membranes as a stationary phase. High protein and dsDNA removal of approximately 80% were obtained. The remaining polyethylene glycol concentration in the eluate was determined. We defined the maximum loading capacity as 7.5 × 1012 lentiviral particles for the lab device used and provide deeper insights into loading strategies. Furthermore, we determined critical process parameters like pressure. We demonstrated in our experiments that steric exclusion chromatography is a gentle purification method with high potential for fragile enveloped viral vectors as it yields high recoveries while efficiently removing impurities.

A new simplified clarification approach for lentiviral vectors using diatomaceous earth improves throughput and safe handling.

Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream processing of these vectors is however still a great challenge, particularly because of the low stability of the virus. Harvesting and clarification are critical and until now insufficiently characterized steps for lentivirus processing. To address this bottleneck, we analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method with the filter aid diatomaceous earth (DE) and bioburden reducing membrane filters achieving high lentivirus recoveries. Using a design of experiment approach we found that higher DE concentrations are advantageous for a higher turbidity reduction and shorter filtration times, but at the same time LV titer decreases with increasing DE concentration. A DE concentration of 9 g/L was identified with a DoE as a robust set-point. Clarification with DE was compared with for lab-scale traditionally employed centrifugation and subsequent bioburden reduction filtration of viral vectors. The use of DE allows to perform a harvest and clarification process, which does not only facilitate faster and safer virus handling, but enables a lower material consumption due to the extremely increased filter capacity, thus representing an efficient and robust lab-scale clarification process.

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach.

The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity.

Continuous microbiological air monitoring for aseptic filling lines.

BACKGROUND: Pharmaceutical aseptic filling lines are used to fill sterile biotechnology products without affecting their quality by a terminal sterilization step. Standard grade A filling environments are required to have < 1 colony-forming unit (CFU) per cubic meter (m3) of air. Aseptic filling is the production cycle with one of the highest contamination risks. A typical method of contamination monitoring is to actively draw air and filter it through special gelatin filters. OBJECTIVE: The study aims to establish whether continuous sampling provides effective monitoring for the entire production process by determining whether trapped organisms can withstand long-term drying stress with unaltered recovery. METHODS: In two experimental phases, the study examined microbial recovery in long-term air-stressed membranes as well as the viability of microorganisms on gelatin filters during 8-hour runs of filtration with high-efficiency particulate air-filtered air from a laminar flow hood using the MD 8 Airscan system. Stressed and unstressed filters were compared with parallel-run reference filters as controls. The CFUs were counted and the genus of the identified microorganism populations determined to examine any changes in microbiological flora occurring during continuous long-term sampling. RESULTS: Compared to the unstressed reference filters, neither total recovery nor recovered bacterial diversity changed. No statistically significant differences in CFUs were found between test filters and reference filters, nor were any statistically significant differences found in the microbiological flora between test filters and reference filters. CFU populations were comparable in both experiments. CONCLUSION: Eight hours of continuous air sampling on gelatin filters with the MD 8 Airscan system did not affect total recovery or change the diversity of recovered microorganisms compared to the controls.