Integrated autolysis, DNA hydrolysis and precipitation enables an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral and clinical-stage anti-COVID-19 candidate.

Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic. Here we report an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral currently in clinical trials for COVID-19. Q-Griffithsin is produced at high titer in E. coli and purified to anticipated clinical grade without conventional chromatography or the need for any fixed downstream equipment. The process is thus both low-cost and highly flexible, facilitating low sales prices and agile modifications of production capacity, two key features for pandemic response. The simplicity of this process is enabled by a novel unit operation that integrates cellular autolysis, autohydrolysis of nucleic acids, and contaminant precipitation, giving essentially complete removal of host cell DNA as well as reducing host cell proteins and endotoxin by 3.6 and 2.4 log10 units, respectively. This unit operation can be performed rapidly and in the fermentation vessel, such that Q-GRFT is obtained with 100% yield and > 99.9% purity immediately after fermentation and requires only a flow-through membrane chromatography step for further contaminant removal. Using this operation or variations of it may enable improved bioprocesses for a range of other high-value proteins in E. coli.

Rapid optimization of processes for the integrated purification of biopharmaceuticals.

Straight-through chromatography, wherein the eluate from one column passes directly onto another column without adjustment, is one strategy to integrate and intensify manufacturing processes for biologics. Development and optimization of such straight-through chromatographic processes is a challenge, however. Conventional high-throughput screening methods optimize each chromatographic step independently, with limited consideration for the connectivity of steps. Here, we demonstrate a method for the development and optimization of fully integrated, multi-column processes for straight-through purification. Selection of resins was performed using an in silico tool for the prediction of processes for straight-through purification based on a one-time characterization of host-cell proteins combined with the chromatographic behavior of the product. A two-step optimization was then conducted to determine the buffer conditions that maximized yield while minimizing process- and product-related impurities. This optimization of buffer conditions included a series of range-finding experiments on each individual column, similar to conventional screening, followed by the development of a statistical model for the fully integrated, multi-column process using design of experiments. We used this methodology to develop and optimize integrated purification processes for a single-domain antibody and a cytokine, obtaining yields of 88% and 86%, respectively, with process- and product-related variants reduced to phase-appropriate levels for nonclinical material.

A heuristic approach to handling missing data in biologics manufacturing databases.

The biologics sector has amassed a wealth of data in the past three decades, in line with the bioprocess development and manufacturing guidelines, and analysis of these data with precision is expected to reveal behavioural patterns in cell populations that can be used for making predictions on how future culture processes might behave. The historical bioprocessing data likely comprise experiments conducted using different cell lines, to produce different products and may be years apart; the situation causing inter-batch variability and missing data points to human- and instrument-associated technical oversights. These unavoidable complications necessitate the introduction of a pre-processing step prior to data mining. This study investigated the efficiency of mean imputation and multivariate regression for filling in the missing information in historical bio-manufacturing datasets, and evaluated their performance by symbolic regression models and Bayesian non-parametric models in subsequent data processing. Mean substitution was shown to be a simple and efficient imputation method for relatively smooth, non-dynamical datasets, and regression imputation was effective whilst maintaining the existing standard deviation and shape of the distribution in dynamical datasets with less than 30% missing data. The nature of the missing information, whether Missing Completely At Random, Missing At Random or Missing Not At Random, emerged as the key feature for selecting the imputation method.

Adaptive modeling optimized by the data fusion strategy: Real-time dying cell percentage prediction using capacitance spectroscopy.

The previous research showcased a partial least squares (PLS) regression model accurately predicting cell death percentages using in-line capacitance spectra. The current study advances the model accuracy through adaptive modeling employing a data fusion approach. This strategy enhances prediction performance by incorporating variables from the Cole-Cole model, conductivity and its derivatives over time, and Mahalanobis distance into the predictor matrix (X-matrix). Firstly, the Cole-Cole model, a mechanistic model with parameters linked to early cell death onset, was integrated to enhance prediction performance. Secondly, the inclusion of conductivity and its derivatives over time in the X-matrix mitigated prediction fluctuations resulting from abrupt conductivity changes during process operations. Thirdly, Mahalanobis distance, depicting spectral changes relative to a reference spectrum from a previous time point, improved model adaptability to independent test sets, thereby enhancing performance. The final data fusion model substantially decreased root-mean squared error of prediction (RMSEP) by around 50%, which is a significant boost in prediction accuracy compared to the prior PLS model. Robustness against reference spectrum selection was confirmed by consistent performance across various time points. In conclusion, this study illustrates that the data fusion strategy substantially enhances the model accuracy compared to the previous model relying solely on capacitance spectra.

Digital application for drug product potency target evaluation in biopharmaceutical manufacturing.

Biopharmaceutical manufacturing entails a series of highly regulated steps. The manufacturing of safe and efficacious drug product (DP) requires testing of critical quality attributes (CQAs) against specification limits. DP potency concentration, which measures the dosage strength of a particular DP, is a CQA of great interest. In order to minimize the DP potency out-of-specification (OOS) risk, sterile fill finish (SFF) process adjustments may be needed. Varying the potency targets can be one such process adjustment. To facilitate such evaluation, data acquisition and statistical calculations are required. Regularly conducting the OOS risk assessment manually using commercial statistical software can be tedious, error-prone, and impractical, especially when several alternate potency targets are under consideration. In this work, the development of a novel framework for OOS risk assessment and deployment of cloud-based statistical software application to facilitate the risk assessment are presented. This application is intended to streamline the assessment of alternate potency targets for DP in biologics manufacturing. The major aspects of this potency targeting application development are presented in detail. Specifically, data sources, pipeline, application architecture, back-end and front-end development as well as application verification are discussed. Finally, several use cases are presented to highlight the application's utility in biologics manufacturing.

Experimental and computational characterization of mass transfer in high turndown bioreactors.

Single-use bioreactors (SUBs, or disposable bioreactors) are extensively used for the clinical and commercial production of biologics. Despite widespread application, minimal results have been reported utilizing the turndown ratio; an operation mode where the working range of the bioreactor can be expanded to include low fluid volumes. In this work, a systematic investigation into free surface mass transfer and cell growth in high turndown single-use bioreactors is presented. This approach, which combines experimental mass transfer measurements with numerical simulation, deconvolutes the combined effects of headspace mixing and the free surface convective mass transfer on cell growth. Under optimized conditions, mass transfer across the interface alone may be sufficient to satisfy oxygen demands of the cell culture. Within the context of high turndown bioreactors, this finding provides a counterpoint to traditional sparge-based bioreactor operational philosophy. Multiple monoclonal antibody-producing cell lines grown using this high turndown approach showed similar viable cell densities to those cells expanded using a traditional cell bag rocker. Furthermore, cells taken directly from the turndown expansion and placed into production showed identical growth characteristics to traditionally expanded cultures. Taken together, these results suggest that the Xcellerex SUB can be run at a 5:1 working volume as a seed to itself, with no need for system modifications, potentially simplifying preculture operations.

Application of platform process development approaches to the manufacturing of Mabcalin™ bispecifics.

Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3 L bioreactors and production was scaled-up to up to 1000 L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic.

In-situ investigation of solid phase evolution during lyophilization of mannitol-based antibody formulations using an XRPD climate chamber.

Crystalline mannitol is commonly used as bulking agent in antibody formulations to provide structure to the lyophilized cake and prevent collapse. Depending on the lyophilization process conditions mannitol can either crystallize as α-, ß-, δ-mannitol, mannitol-hemihydrate, or transition to its amorphous state. While crystalline mannitol helps to create a firmer cake structure this is not true for amorphous mannitol. The hemihydrate is also an undesired physical form as it may reduce the drug product stability by releasing bound water molecules into the cake. Our aim was to simulate lyophilization processes in an X-ray powder diffraction (XRPD) climate chamber. In the climate chamber, the process can be carried out fast with low sample quantities to determine optimal process conditions. Insights on the emergence of desired anhydrous mannitol forms helps to adjust the process parameters in larger scale freeze-dryers. In our study we have identified the critical process steps for our formulations and then varied relevant process parameters, which were the annealing temperature, annealing time and temperature ramp rate of the freeze-drying process. Furthermore, the effect of the presence of antibodies on excipient crystallization was investigated by performing the studies on placebo solutions versus two respective antibody formulations. A comparison of the products obtained in a freeze-dryer and the simulated process in the climate chamber showed good accordance demonstrating the method as suitable tool to identify ideal process conditions on a laboratory scale.

[Practice of Human Resource Development Related to Biopharmaceutical Manufacturing and Quality Control and the International Contribution as Centers of Excellence of Asia-Pacific Economic Cooperation-Life Sciences Innovation Forum-Regulatory Harmonization Steering Committee].

In recent years, the development of biologics such as therapeutic antibodies has increased. Around 1980, Japan was the world's leading country in terms of research and development on biologics. However, after that, the development of biologics in Japan has become stagnant. Especially, research and development of therapeutic antibodies, which are critical medical products, is compared to that being conducted in Europe and the United States. The products distributed in Japan are developed and manufactured overseas. Furthermore, related emerging technologies are developed overseas. Therefore, to improve this situation, Biologics Center for Research and Training (BCRET) was established in August 2017 in Kobe city to develop human resources for biologics with the cooperation of Japan Pharmaceutical Manufacturers Association, Japan Agency for Medical Research and Development, Kobe University, and related ministries and agencies. BCRET will develop human resources by providing useful information and experience related to the development and manufacture of biologics in the form of classroom lectures and practical training for process and analysis developers and pharmaceutical affairs related to manufacturing, quality control, and approval applications in the biologics field of pharmaceutical companies, along with reviewers and inspectors of Pharmaceuticals and Medical Devices Agency and inspectors from countries belonging to Asia-Pacific Economic Cooperation (APEC). This is the first attempt in the world to develop human resources in a field of chemistry, manufacturing and control and Good Manufacturing Practice of biologics for inspectors in countries belonging to APEC, and it is expected to contribute internationally for human resources for biologics.

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning.

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process.

Occupational Exposure Risks When Working with Protein Therapeutics and the Development of a Biologics Banding System.

Background: As the pharmaceutical industry advances its understanding of biological processes and how they relate to (the causes and treatments of) disease, many new modalities such as protein therapeutics (PTs) are emerging as breakthrough therapies to treat both rare and common diseases. As PTs become more prevalent, occupational health and safety professionals are challenged with identifying potential occupational exposure risks, health hazards, and assessing best practice recommendations for workers who develop, manufacture, and administer PTs. Methods: To characterize airborne exposures to PTs, we conducted a retrospective analysis of industrial hygiene (IH) data for PTs spanning >15 years. This information was used to support the development of an occupational exposure control banding system designed for and applicable to biologically derived PTs (produced in living cells). Overall, 403 IH samples were evaluated that included exposure data for monoclonal antibodies, fusion proteins, PEGylated proteins, and surrogates. Results: Our evaluation of historical IH PT sample data indicated low exposure potential across manufacturing activities with >99% (400/403) being below an airborne concentration of 1 µg/m3. Processes with the highest potential for airborne exposure included high-energy operations (e.g., homogenization) and maintenance activities (e.g., cleaning and repairs). Conclusion: The observed low exposure potential is expected given that many biological manufacturing activities are closed to maintain product sterility. This evaluation indicated that the banding systems historically utilized for small molecules could benefit from being revisited for PTs.

Low-Cost, Large-Scale Production of the Anti-viral Lectin Griffithsin.

Griffithsin, a broad-spectrum antiviral lectin, has potential to prevent and treat numerous viruses including HIV, HCV, HSV, SARS-CoV, and SARS-CoV-2. For these indications, the annual demand for Griffithsin could reach billions of doses and affordability is paramount. We report the lab-scale validation of a bioprocess that supports production volumes of >20 tons per year at a cost of goods sold below $3,500/kg. Recombinant expression in engineered E. coli enables Griffithsin titers ∼2.5 g/L. A single rapid precipitation step provides > 90% yield with 2-, 3-, and 4-log reductions in host cell proteins, endotoxin, and nucleic acids, respectively. Two polishing chromatography steps remove residual contaminants leading to pure, active Griffithsin. Compared to a conventional one this process shows lower costs and improved economies of scale. These results support the potential of biologics in very large-scale, cost-sensitive applications such as antivirals, and highlight the importance of bioprocess innovations in enabling these applications.

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines.

Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS-/- CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10-19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500-L and 1000-L bioreactors replicated laboratory results using 5-L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

Cell-Based Therapy Manufacturing in Stirred Suspension Bioreactor: Thoughts for cGMP Compliance.

Cell-based therapy (CBT) is attracting much attention to treat incurable diseases. In recent years, several clinical trials have been conducted using human pluripotent stem cells (hPSCs), and other potential therapeutic cells. Various private- and government-funded organizations are investing in finding permanent cures for diseases that are difficult or expensive to treat over a lifespan, such as age-related macular degeneration, Parkinson's disease, or diabetes, etc. Clinical-grade cell manufacturing requiring current good manufacturing practices (cGMP) has therefore become an important issue to make safe and effective CBT products. Current cell production practices are adopted from conventional antibody or protein production in the pharmaceutical industry, wherein cells are used as a vector to produce the desired products. With CBT, however, the "cells are the final products" and sensitive to physico- chemical parameters and storage conditions anywhere between isolation and patient administration. In addition, the manufacturing of cellular products involves multi-stage processing, including cell isolation, genetic modification, PSC derivation, expansion, differentiation, purification, characterization, cryopreservation, etc. Posing a high risk of product contamination, these can be time- and cost- prohibitive due to maintenance of cGMP. The growing demand of CBT needs integrated manufacturing systems that can provide a more simple and cost-effective platform. Here, we discuss the current methods and limitations of CBT, based upon experience with biologics production. We review current cell manufacturing integration, automation and provide an overview of some important considerations and best cGMP practices. Finally, we propose how multi-stage cell processing can be integrated into a single bioreactor, in order to develop streamlined cGMP-compliant cell processing systems.

A direct RT qPCR method for quantification of retrovirus-like particles in biopharmaceutical production with CHO cells.

Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing biologic drugs, like monoclonal antibody, in the biopharmaceutical industry. Retrovirus-like particles (RVLPs) are made during the manufacturing process with CHO cells and it is incumbent upon the manufacturer to perform risk assessment based on levels of RVLP in unprocessed bulk. Quantification of RVLP using electron microscopy (EM) is the standard method. However, reverse transcription based real-time PCR (RT qPCR) is an alternative method available. This method involves RNase digestion of cell culture fluid to remove free RNA, followed by extraction of total nucleic acid and digestion with DNase to remove extracted DNA molecules, and then finally reverse transcription and PCR. Here we report a method where the nucleic acids extraction step is eliminated prior to qPCR. In this method the cell-free culture supernatant sample is digested with thermolabile DNase and RNase at the same time in a 96-well PCR plate; subsequently the enzymes are heat-denatured; then RT qPCR reagents are added to the wells in the PCR plate along with standards and controls in other wells of the same plate; finally the plate is subjected to RT qPCR for analysis of RVLP RNA in the samples. This direct RT qPCR method for RVLP is sensitive to 10 particles of RVLP with good precision and accuracy and has a wide linear range of quantification. The method has been successfully tested with different production batches, shown to be consistent, and correlates well with the extraction-based method. However, the results are about 1-log higher compared to EM method. This method simplifies the RVLP quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Additionally, the method can be an added tool for viral clearance studies, by testing process-intermediate samples like Protein A column and ion-exchange column eluates, for increased confidence in purification of biologics manufactured in CHO cell culture.

Process Analytical Technology for Advanced Process Control in Biologics Manufacturing with the Aid of Macroscopic Kinetic Modeling.

Productivity improvements of mammalian cell culture in the production of recombinant proteins have been made by optimizing cell lines, media, and process operation. This led to enhanced titers and process robustness without increasing the cost of the upstream processing (USP); however, a downstream bottleneck remains. In terms of process control improvement, the process analytical technology (PAT) initiative, initiated by the American Food and Drug Administration (FDA), aims to measure, analyze, monitor, and ultimately control all important attributes of a bioprocess. Especially, spectroscopic methods such as Raman or near-infrared spectroscopy enable one to meet these analytical requirements, preferably in-situ. In combination with chemometric techniques like partial least square (PLS) or principal component analysis (PCA), it is possible to generate soft sensors, which estimate process variables based on process and measurement models for the enhanced control of bioprocesses. Macroscopic kinetic models can be used to simulate cell metabolism. These models are able to enhance the process understanding by predicting the dynamic of cells during cultivation. In this article, in-situ turbidity (transmission, 880 nm) and ex-situ Raman spectroscopy (785 nm) measurements are combined with an offline macroscopic Monod kinetic model in order to predict substrate concentrations. Experimental data of Chinese hamster ovary cultivations in bioreactors show a sufficiently linear correlation (R² ≥ 0.97) between turbidity and total cell concentration. PLS regression of Raman spectra generates a prediction model, which was validated via offline viable cell concentration measurement (RMSE ≤ 13.82, R² ≥ 0.92). Based on these measurements, the macroscopic Monod model can be used to determine different process attributes, e.g., glucose concentration. In consequence, it is possible to approximately calculate (R² ≥ 0.96) glucose concentration based on online cell concentration measurements using turbidity or Raman spectroscopy. Future approaches will use these online substrate concentration measurements with turbidity and Raman measurements, in combination with the kinetic model, in order to control the bioprocess in terms of feeding strategies, by employing an open platform communication (OPC) network-either in fed-batch or perfusion mode, integrated into a continuous operation of upstream and downstream.