Integrated micro-scale protein a chromatography and Low pH viral inactivation unit operations on an automated platform.

High throughput process development (HTPD) is established for time- and resource- efficient chromatographic process development. However, integration with non-chromatographic operations within a monoclonal antibody (mAb) purification train is less developed. An area of importance is the development of low pH viral inactivation (VI) that follows protein A chromatography. However, the lack of pH measurement devices at the micro-scale represents a barrier to implementation, which prevents integration with the surrounding unit operations, limiting overall process knowledge. This study is based upon the design and testing of a HTPD platform for integration of the protein A and low pH VI operations. This was achieved by using a design and simulation software before execution on an automated liquid handler. The operations were successfully translated to the micro-scale, as assessed by analysis of recoveries and molecular weight content. The integrated platform was then used as a tool to assess the effect of pH on HMWC during low pH hold. The laboratory-scale and micro-scale elution pools showed comparable HMWC across the pH range 3.2-3.7. The investigative power of the platform is highlighted by evaluating the resources required to conduct a hypothetical experiment. This results in lower resource demands and increased labor efficiency relative to the laboratory-scale. For example, the experiment can be conducted in 7 h, compared to 105 h, translating to labor hours, 3 h and 28 h for the micro-scale and laboratory-scale, respectively. This presents the opportunity for further integration beyond chromatographic operations within the purification sequence, to establish a fit-to-platform assessment tool for mAb process development.

Designing of an extract production protocol for industrial application of cell-free protein synthesis technology: Building from a current best practice to a quality by design approach.

Cell-Free Protein Synthesis (CFPS) has, over the past decade, seen a substantial increase in interest from both academia and industry. Applications range from fundamental research, through high-throughput screening to niche manufacture of therapeutic products. This review/perspective focuses on Quality Control in CFPS. The importance and difficulty of measuring the Raw Material Attributes (RMAs) of whole cell extract, such as constituent protein and metabolite concentrations, and of understanding and controlling these complicated enzymatic reactions is explored, for both centralised and distributed industrial production of biotherapeutics. It is suggested that a robust cell-free extract production process should produce cell extract of consistent quality; however, demonstrating this is challenging without a full understanding of the RMAs and their interaction with reaction conditions and product. Lack of technology transfer and knowledge sharing is identified as a key limiting factor in the development of CFPS. The article draws upon the experiences of industrial process specialists, discussions within the Future Targeted Healthcare Manufacturing Hub Specialist Working Groups and evidence drawn from various sources to identify sources of process variation and to propose an initial guide towards systematisation of CFPS process development and reporting. These proposals include the development of small scale screening tools, consistent reporting of selected process parameters and analytics and application of industrial thinking and manufacturability to protocol development.

Solid-Solid Interfacial Contact of Tubing Walls Drives Therapeutic Protein Aggregation During Peristaltic Pumping.

Peristaltic pumping during bioprocessing can cause therapeutic protein loss and aggregation during use. Due to the complexity of this apparatus, root-cause mechanisms behind protein loss have been long sought. We have developed new methodologies isolating various peristaltic pump mechanisms to determine their effect on monomer loss. Closed-loops of peristaltic tubing were used to investigate the effects of peristaltic pump parameters on temperature and monomer loss, whilst two mechanism isolation methodologies are used to isolate occlusion and lateral expansion-relaxation of peristaltic tubing. Heat generated during peristaltic pumping can cause heat-induced monomer loss and the extent of heat gain is dependent on pump speed and tubing type. Peristaltic pump speed was inversely related to the rate of monomer loss whereby reducing speed 2.0-fold increased loss rates by 2.0- to 5.0-fold. Occlusion is a parameter that describes the amount of tubing compression during pumping. Varying this to start the contacting of inner tubing walls is a threshold that caused an immediate 20-30% additional monomer loss and turbidity increase. During occlusion, expansion-relaxation of solid-liquid interfaces and solid-solid interface contact of tubing walls can occur simultaneously. Using two mechanisms isolation methods, the latter mechanism was found to be most destructive and a function of solid-solid contact area, where increasing the contact area 2.0-fold increased monomer loss by 1.6-fold. We establish that a form of solid-solid contact mechanism whereby the contact solid interfaces disrupt adsorbed protein films is the root-cause behind monomer loss and protein aggregation during peristaltic pumping.

Evaluating 3D-printed bioseparation structures using multi-length scale tomography.

X-ray computed tomography was applied in imaging 3D-printed gyroids used for bioseparation in order to visualize and characterize structures from the entire geometry down to individual nanopores. Methacrylate prints were fabricated with feature sizes of 500 µm, 300 µm, and 200 µm, with the material phase exhibiting a porous substructure in all cases. Two X-ray scanners achieved pixel sizes from 5 µm to 16 nm to produce digital representations of samples across multiple length scales as the basis for geometric analysis and flow simulation. At the gyroid scale, imaged samples were visually compared to the original computed-aided designs to analyze printing fidelity across all feature sizes. An individual 500 µm feature, part of the overall gyroid structure, was compared and overlaid between design and imaged volumes, identifying individual printed layers. Internal subvolumes of all feature sizes were segmented into material and void phases for permeable flow analysis. Small pieces of 3D-printed material were optimized for nanotomographic imaging at a pixel size of 63 nm, with all three gyroid samples exhibiting similar geometric characteristics when measured. An average porosity of 45% was obtained that was within the expected design range, and a tortuosity factor of 2.52 was measured. Applying a voidage network map enabled the size, location, and connectivity of pores to be identified, obtaining an average pore size of 793 nm. Using Avizo XLAB at a bulk diffusivity of 7.00 × 10-11 m2s-1 resulted in a simulated material diffusivity of 2.17 × 10-11 m2s-1 ± 0.16 × 10-11 m2s-1.

Time-dependent sorption behavior of lentiviral vectors during anion-exchange chromatography.

Use of lentiviral vectors (LVs) in clinical Cell and Gene Therapy applications is growing. However, functional product loss during capture chromatography, typically anion-exchange (AIEX), remains a significant unresolved challenge for the design of economic processes. Despite AIEX's extensive use, variable performance and generally low recovery is reported. This poor understanding of product loss mechanisms highlights a significant gap in our knowledge of LV adsorption and other types of vector delivery systems. This work demonstrates HIV-1-LV recovery over quaternary-amine membrane adsorbents is a function of time in the adsorbed state. Kinetic data for product loss in the column bound state was generated. Fitting a second order-like rate model, we observed a rapid drop in functional recovery due to increased irreversible binding for vectors encoding two separate transgenes ( t Y 1 / 2 ${t}_{{Y}_{1/2}}$ = 12.7 and 18.7 min). Upon gradient elution, a two-peak elution profile implicating the presence of two distinct binding subpopulations is observed. Characterizing the loss kinetics of these two subpopulations showed a higher rate of vector loss in the weaker binding peak. This work highlights time spent in the adsorbed state as a critical factor impacting LV product loss and the need for consideration in LV AIEX process development workflows.

Depth filter material process interaction in the harvest of mammalian cells.

Upstream advances have led to increased mAb titers above 5 g/L in 14-day fed-batch cultures. This is accompanied by higher cell densities and process-related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture, and there is interest in utilizing these to remove process-related impurities at the harvest stage. Operation of the harvest stage has also been shown to affect the performance of the Protein A chromatography step. In addition, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. Therefore, there is an increased need for further understanding and knowledge of depth filtration. This study investigates the effect of depth filter material and loading on the Protein A resin lifetime with an industrially relevant high cell density feed material (40 million cells/ml). It focuses on the retention of process-related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in-situ. An increase in loading of the primary-synthetic filter by a third, led to earlier DNA breakthrough in the secondary filter, with DNA concentration at a throughput of 50 L/m2 being more than double. Confocal imaging of the depth filters showed that the foulant was pushed forward into the filter structure with higher loading. The additional two layers in the primary-synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Reduced filtrate clarity, as measured by OD600, was 1.6 fold lower in the final filtrate where a synthetic filter train was used. This was also associated with precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build-up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems.

A Biological OR(XNOR) Logic Gate Couples Carbon Source and Transgene Expression Switching in a Komagataella phaffii (Pichia pastoris) Strain Co-producing Process-Enhancing Lipase and a Virus-like Particle (VLP) Vaccine.

We constructed a three-input biological logic gate: S OR (G XNOR M), where S is sorbitol, G is glycerol, and M is methanol, to optimize co-expression of two transgenes in Komagataella phaffii using batch-mode carbon source switching (CSS). K. phaffii was engineered to harbor transgenes encoding a Candida rugosa triacylglycerol lipase, which can enhance downstream processing by removing host cell lipids from homogenates, and the hepatitis B virus surface antigen (HBsAg), a protein that self-assembles into a virus-like particle (VLP) vaccine. Using the native alcohol oxidase 1 (PAOX1) and enolase 1 (PENO1) promoters to direct VLP vaccine and lipase expression, respectively, successfully provided an OR(XNOR) gate function with double-repression as the output. This logic gate functionality enabled use of CSS to ensure that approximately 80% of total VLP yield was accumulated before cells were burdened with lipase expression in 250 mL DasGip bioreactor cultivation.

Measurement of Adenovirus-Based Vector Heterogeneity.

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.

Investigating heparin affinity chromatography for extracellular vesicle purification and fractionation.

The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products. In this study, we evaluated the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process. Bind-elute chromatography, such as HAC, is an attractive method of purification because it is highly scalable, robust and can be automated. Our findings support an interaction between EVs and heparin. The recovery of EVs using HAC based on particle counts was a minimum of 68.7%. We found HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively. In addition to EV purification, HAC was used to separate EVs into three populations based on their affinity to the heparin column. Within these populations, we detected differences in the expression of the exosome-associated protein TSG101 and the tetraspanin immunophenotype. However, the significance of these observations is not clear. Overall HAC shows promise as a potential purification method to capture EVs and this study proposes a novel application of HAC for EV fractionation. Moving forward, a better understanding of the heparin-EV interaction would be required before HAC can be more widely adopted for these applications.

GFP-tagging of extracellular vesicles for rapid process development.

Extracellular vesicles (EVs) act as nano-scale molecular messengers owing to their capacity to shuttle functional macromolecular cargo between cells. This intrinsic ability to deliver bioactive cargo has sparked great interest in the use of EVs as novel therapeutic delivery vehicles; investments totaling over $2 billion in 2020 alone were reported for therapeutic EVs. One of the bottlenecks facing the production of EVs is the lack of rapid and high throughput analytics to aid process development. Here CHO cells have been designed and engineered to express GFP-tagged EVs via fusion to CD81. Moreover, this study highlights the importance of parent cell characterization to ensure lack of non-fused GFP for the effective use of this quantitative approach. The fluorescent nature of resulting vesicles allowed for rapid quantification of concentration and yield across the EV purification process. In this manner, the degree of product loss was deduced by mass balance analysis of ultrafiltration processing, reconciled up to 97% of initial feed mass. The use of GFP-tagging allowed for straightforward monitoring of vesicle elution from chromatography separations and detection via western blotting. Collectively, this work illustrates the utility of GFP-tagged EVs as a quantitative and accessible tool for accelerated process development.

Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation.

Glycosylation can be a critical quality attribute in biologic manufacturing. In particular, it has implications on the half-life, immunogenicity, and pharmacokinetics of therapeutic monoclonal antibodies (mAbs), and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering, to yield more predictably glycosylated mAbs and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. A more robust approach would be a conjoined upstream-downstream processing strategy. This could include implementing novel downstream technologies, such as the use of Fc γ-based affinity ligands for the separation of mAb glycovariants. This review highlights the importance of controlling therapeutic antibody glycosylation patterns, the challenges faced in terms of glycosylation during mAb biosimilar development, current efforts both upstream and downstream to control glycosylation and their limitations, and the need for research in the downstream space to establish holistic and consistent manufacturing processes for the production of antibody therapies.

Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy.

Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%-50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.

Identification and classification of host cell proteins during biopharmaceutical process development.

As significant improvements in volumetric antibody productivity have been achieved by advances in upstream processing over the last decade, and harvest material has become progressively more difficult to recover with these intensified upstream operations, the segregation of upstream and downstream processing has remained largely unchanged. By integrating upstream and downstream process development, product purification issues are given consideration during the optimization of upstream operating conditions, which mitigates the need for extensive and expensive clearance strategies downstream. To investigate the impact of cell culture duration on critical quality attributes, CHO-expressed IgG1 was cultivated in two 2 L bioreactors with samples taken on days 8, 10, 13, 15, and 17. The material was centrifuged, filtered and protein A purified on a 1 ml HiTrap column. Host cell protein (HCP) identification by mass spectrometry (MS) was applied to this system to provide insights into cellular behavior and HCP carryover during protein A purification. It was shown that as cultivation progressed from day 8 to 17 and antibody titer increased, product quality declined due to an increase in post-protein A HCPs (from 72 to 475 peptides detected by MS) and a decrease in product monomer percentage (from 98% to 95.5%). Additionally, the MS data revealed an increase in the abundance of several classes of post-protein A HCPs (e.g., stress response proteins and indicators of cell age), particularly on days 15 and 17 of culture, which were associated with significant increases in total overall HCP levels. This provides new insight into the specific types of HCPs that are retained during mAb purification and may be used to aid process development strategies.

Liposome Sterile Filtration Characterization via X-ray Computed Tomography and Confocal Microscopy.

Two high resolution, 3D imaging techniques were applied to visualize and characterize sterilizing grade dual-layer filtration of liposomes, enabling membrane structure to be related with function and performance. Two polyethersulfone membranes with nominal retention ratings of 650 nm and 200 nm were used to filter liposomes of an average diameter of 143 nm and a polydispersity index of 0.1. Operating conditions including differential pressure were evaluated. X-ray computed tomography at a pixel size of 63 nm was capable of resolving the internal geometry of each membrane. The respective asymmetry and symmetry of the upstream and downstream membranes could be measured, with pore network modeling used to identify pore sizes as a function of distance through the imaged volume. Reconstructed 3D digital datasets were the basis of tortuous flow simulation through each porous structure. Confocal microscopy visualized liposome retention within each membrane using fluorescent dyes, with bacterial challenges also performed. It was found that increasing pressure drop from 0.07 MPa to 0.21 MPa resulted in differing fluorescent retention profiles in the upstream membrane. These results highlighted the capability for complementary imaging approaches to deepen understanding of liposome sterilizing grade filtration.

Analytics of host cell proteins (HCPs): lessons from biopharmaceutical mAb analysis for Gene therapy products.

Analytics for host cell protein (HCP) analysis of therapeutic monoclonal antibody preparations have developed enormously. We consider how learnings from this can inform HCP analysis of gene therapy viral vector products. The application of mass spectrometry (MS) approaches for analysis of HCPs in viral vector preparations is being established, although such information remains limited and is yet to be widely applied into process or host cell line development to reduce HCP amounts or risk. As these MS approaches, and the data from them, are applied and become available, the process understanding created will speed process development activity. We describe technologies that have been, or can be, applied to viral vector HCP analysis to aid process development, reduce HCP amounts, identify critical HCPs and thus inform risk assessment and management based on a knowledge of specific HCPs, ultimately delivering safe and efficacious gene therapy products to the clinic.

Enriching leukapheresis improves T cell activation and transduction efficiency during CAR T processing.

The majority of CD19-directed CAR T cell products are manufactured using an autologous process. Although using a patient's leukapheresis reduces the risks of rejection, it introduces variability in starting material composition and the presence of cell populations that might negatively affect production of chimeric antigen receptor (CAR) T cells, such as myeloid cells. In this work, the effect of monocytes (CD14) on the level of activation, growth, and transduction efficiency was monitored across well plate and culture bag platforms using healthy donor leukapheresis. Removal of monocytes from leukapheresis improved the level of activation 2-fold, achieving the same level of activation as when initiating the process with a purified T cell starting material. Two activation reagents were tested in well plate cultures, revealing differing sensitivities to starting material composition. Monocyte depletion in culture bag systems had a significant effect on transduction efficiency, improving consistency and increasing the level of CAR expression by up to 64% compared to unsorted leukapheresis. Cytotoxicity assays revealed that CAR T cell products produced from donor material depleted of monocytes and isolated T cells consistently outperformed those made from unsorted leukapheresis. Analysis of memory phenotypes and gene expression indicated that CAR T cells produced using depleted starting material displayed a more rested and naive state.

Escherichia Coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles.

Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an Escherichia coli-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 µg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs.

Advanced control strategies for bioprocess chromatography: Challenges and opportunities for intensified processes and next generation products.

Recent advances in process analytical technologies and modelling techniques present opportunities to improve industrial chromatography control strategies to enhance process robustness, increase productivity and move towards real-time release testing. This paper provides a critical overview of batch and continuous industrial chromatography control systems for therapeutic protein purification. Firstly, the limitations of conventional industrial fractionation control strategies using in-line UV spectroscopy and on-line HPLC are outlined. Following this, an evaluation of monitoring and control techniques showing promise within research, process development and manufacturing is provided. These novel control strategies combine rapid in-line data capture (e.g. NIR, MALS and variable pathlength UV) with enhanced process understanding obtained from mechanistic and empirical modelling techniques. Finally, a summary of the future states of industrial chromatography control systems is proposed, including strategies to control buffer formulation, product fractionation, column switching and column fouling. The implementation of these control systems improves process capabilities to fulfil product quality criteria as processes are scaled, transferred and operated, thus fast tracking the delivery of new medicines to market.

High-Throughput Process Development for the Chromatographic Purification of Viral Antigens.

Chromatography is a widely used method in the biotechnology industry and functions to separate the desired product from process and product related impurities. There is a multitude of resins available based on different modalities (such as charge, hydrophobicity, and affinity) to provide a spectrum of approaches to meet the separation challenges of the diverse products. The challenge of developing viral antigen purification processes is addressed in this method. A unique feature of this product class is that in order to protect against more than one strain of an antigen, vaccines are often multivalent. This entails multiple production processes for each antigen, all of which will require separate development and validation. Ideally, a universal purification method is sought, but differences in the protein subunits (frequently used as the antigens) make this challenging and often-bespoke purification steps are required. This means process development for the chromatographic stages of these products can be particularly challenging and labour intensive. With the numerous choices available, making critical process decisions that are usually unique to each product, process, and strain, can be costly and time-consuming. To address this, scale down purification at <1.0 mL column volume and automation approaches are increasingly applied to increase throughput. In this work, a method is described wherein a Tecan Freedom EVO® automated liquid handler is deployed for the evaluation of different resin chemistries and buffer conditions to find a suitable purification strategy. This method allows for the rapid evaluation of the separation viral antigens where limited information on chromatography behavior is known at the early stages of process development. Here, we demonstrate the methodology firstly by explaining the automated purification script and secondly by applying the script for an efficient purification development for different serotypes of rotavirus antigens.

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions.

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.