mAb Production Research Articles

Safety Evaluation of the Novel Cocktail of Monoclonal Antibodies for Postexposure Prophylaxis in Category III Animal Exposures

Abstract Introduction: Rabies monoclonal antibody (mAb) is a life-saving immune-biological for postexposure prophylaxis (PEP) in all Category III animal exposures. A novel cocktail of mAbs derived using recombinant DNA technology is presently available for usage. The WHO recommends monitoring the clinical use and outcomes of mAb products. Methods: An open-label, postmarketing study was conducted at the anti-rabies clinic in the southern part of India. PEP was provided to all the study participants per the national guidelines. All the subjects were assessed for any adverse events (AEs) following PEP up to 35 days; if any, were treated free of cost at the study center. Results: The present study included 309 subjects across all age groups, 59.2% were adults, 33.3% of children, and 7.5% elderly. Majority of the patients were exposed to dogs (89.6%) and most of them had lacerations (86.3%) in different parts of their body. A total of 19 (6.2%) AEs were reported. All the AEs were local reactions, namely pain (2.6%), erythema (1.4%), tenderness (1%), induration (0.6%), and swelling (0.6%). All reported AEs were mild (Grade 1 severity) and resolved completely with symptomatic treatment. Conclusion: The novel cocktail of mAbs was safe for PEP in Category III animal exposures across all the age groups and supports its continued and improved usage for Universal Health Coverage to prevent rabies.

Development, physico-chemical characterization, and in vivo stability of a novel aglycosylated monoclonal antibody targeting FAM19A5

AbstractIntroducing aglycosylation into therapeutic monoclonal antibodies (mAbs) can prevent side effects associated with fragment crystallizable (Fc)-mediated effector functions. This modification induces structural changes in the heavy chain constant domain 2-constant domain 3 within Fc regions, which decreases antibody stability at acidic pH and high temperature. In this study, NS101, a novel aglycosylated mAb targeting family with sequence similarity 19, A5 (FAM19A5) for neurological diseases was evaluated with respect to its developability and in vivo stability as therapeutics. When recombinant CHO cells producing NS101 were cultivated using a fed-batch mode in a 500 L bioreactor, cell growth and mAb production profiles were consistent across three consecutive runs. NS101, thus produced, features an additional intra-disulfide bond in the heavy chain complementarity-determining region 3, contributing strong and sophisticated binding to the cryptic epitope. The melting temperature (Tm) of NS101 was lower than that of commercial glycosylated therapeutic mAbs, but NS101 showed better stability at 4 °C for 36 months. The binding affinity of NS101 to FAM19A5 and neonatal Fc receptor were comparable to those of glycosylated NS101. In addition, in three human cohort groups receiving 6, 12, and 24 mg/kg of NS101, the mean half-life was 22 days, and NS101 exhibited in vivo stability, considering that the half-lives of commercialized therapeutic mAbs and endogenous IgGs are 2–4 weeks and 21 days, respectively. Taken together, the results obtained here demonstrate that NS101, a novel aglycosylated mAb, has potential as a therapeutic agent for neurological diseases.

Hybrid modeling for in silico optimization of a dynamic perfusion cell culture process.

The bio-pharmaceutical industry heavily relies on mammalian cells for the production of bio-therapeutic proteins. The complexity of implementing and high cost-of-goods of these processes are currently limiting more widespread patient access. This is driving efforts to enhance cell culture productivity and cost reduction. Upstream process intensification (PI), using perfusion approaches in the seed train and/or the main bioreactor, has shown substantial promise to enhance productivity. However, developing optimal process conditions for perfusion-based processes remain challenging due to resource and time constraints. Model-based optimization offers a solution by systematically screening process parameters like temperature, pH, and culture media to find the optimum conditions in silico. To our knowledge, this is the first experimentally validated model to explain the perfusion dynamics under different operating conditions and scales for process optimization. The hybrid model accurately describes Chinese hamster ovary (CHO) cell culture growth dynamics and a neural network model explains the production of mAb, allowing for optimization of media exchange rates. Results from six perfusion runs in Ambr® 250 demonstrated high accuracy, confirming the model's utility. Further, the implementation of dynamic media exchange rate schedule determined through model-based optimization resulted in 50% increase in volumetric productivity. Additionally, two 5 L-scale experiments validated the model's reliable extrapolation capabilities to large bioreactors. This approach could reduce the number of wet lab experiments needed for culture process optimization, offering a promising avenue for improving productivity, cost-of-goods in bio-pharmaceutical manufacturing, in turn improving patient access to pivotal medicine.

A Data-Driven Approach for Leveraging Inline and Offline Data to Determine the Causes of Monoclonal Antibody Productivity Reduction in the Commercial-Scale Cell Culture Process.

The monoclonal antibody (mAb) manufacturing process comes with high profits and high costs, and thus mAb productivity is of vital importance. However, many factors can impact the cell culture process, and lead to mAb productivity reduction. Nowadays, the biopharma industry is actively employing manufacturing information systems, which enable the integration of both online data and offline data. Although the volume of data is large, related data mining studies for mAb productivity improvement are rare. Therefore, a data-driven approach is proposed in this study to leverage both the inline and offline data of the cell culture process to discover the causes of mAb productivity reduction. The approach consists of four steps, namely data preprocessing, phase division, feature extraction and fusion, and cluster comparing. First, data quality issues are solved during the data preprocessing step. Next, the inline data are divided into several phases based on the moving window k-nearest neighbor method. Then, the inline data features are extracted via functional data analysis and combined with the offline data features. Finally, the causes of mAb productivity reduction are identified using the contrasting clusters via the principal component analysis method. A commercial-scale cell culture process case study is provided in this research to verify the effectiveness of the approach. Data from 35 batches were collected, and each batch contained nine inline variables and seven offline variables. The causes of mAb productivity reduction were identified to be the lack of nutrients, and recommended actions were taken according to the result, which was subsequently proven by six validation batches.

Feasibility of Laboratory Equipment-Based Simulation Methods to Assess the Impact of Vehicle Transportation on Product Quality of mAb Dosing Solutions.

Therapeutic monoclonal antibody (mAb) products for intravenous (IV) administration generally require aseptic compounding with a commercially available diluent. When the administration site is located away from the preparation site, the prepared dosing solution may need to be transported in a vehicle. The impact of vehicle transportation on the product quality of mAbs needs to be evaluated to define safe handling and transportation conditions for dosing solutions. The design and execution of actual vehicle transportation studies require considerable resources and time. In this study, we systematically developed three different laboratory equipment-based methods that simulate vehicle transportation stresses: orbital shaker (OS), reciprocating shaker (RS), and vibration test system (VTS)-based simulation methods. We assessed their feasibility by comparing the impact on product quality caused by each simulated method with that caused by actual vehicle transportation. Without residual polysorbate 80 (PS80) in the mAb dosing solution, transportation via a cargo van led to a considerable increase in the subvisible particle counts and did not meet the compendial specifications for the light obscuration method. However, the presence of as low as 0.0004%w/v (4 ppm) PS80 in the dosing solution stabilized the mAb against vehicle transportation stresses and met the compendial specifications. Vehicle transportation of an IV bag with headspace resulted in negligible micro air bubbles and foaming in both PS80-free and PS80-containing mAb dosing solutions. These phenomena were found to be comparable to the VTS-based simulated method. However, the OS- and RS-based simulated methods formed significantly more micro air bubbles and foaming in an IV bag with headspace than either actual vehicle transportation or the VTS-based simulated method. Despite the higher interfacial stress (micro air bubbles and foaming) in the dosing solution created by the OS- and RS-based simulated methods, 0.0004%w/v (4 ppm) PS80 in the dosing solution was found to be sufficient to stabilize the mAb. The study shows that under appropriate simulated conditions, the OS-, RS-, and VTS-based simulated methods can be used as practical and meaningful models to assess the impact and risk of vehicle transportation on the quality of mAb dosing solutions.

Implementation of a Macroporous Polyhydroxyethylmethacrylate Cryogel-Based Mini-Bioreactor System to Improve Monoclonal Antibody Production.

Monoclonal antibodies (mAbs) are pioneers in the diagnosis and treatment of many diseases, such as cancer, asthma, poisoning, viral infections, etc. As the market value of mAbs increases in the biopharma industry, the demand for high quantities is met by upscaled production using bioreactor systems. Thus, disposable, porous matrices called cryogels have gained the primary focus for adherent support in the proliferation of hybridoma cells. In this study, a gelatin-immobilized polyhydroxyethylmethacrylate-based cryogel material (disc-shaped, 9 mL bed volume) was synthesized, and a mini-bioreactor set up developed for culturing hybridoma cells to produce mAbs continuously. The hybridoma clone, 1B4A2D5, secreting anti-human serum albumin monoclonal antibodies, was immobilized in the cryogel matrix (2 discs, 18 mL bed volume). The hybridoma cells were attached to the matrix within 12 h after inoculation, and the cells were in the lag phase for seven days, where they were secreted mAb into the circulation medium. During the initial exponential phase, the glucose consumption, lactic acid production, and mAb production were 3.36 mM/day, 3.67 mM/day, and 55.61 µg/mL/day, respectively. The medium was refreshed whenever the glucose in the media went below 50% of the initial glucose concentration. The cryogenic reactor was run continuously for 25 days, and the mAb concentration reached a maximum on the 17th day at 310.59 µg/mL. The cumulative amount of mAbs produced in 25 days of running was 246 µg/mL, 7.7 times higher than the mAbs produced from T-flask batch cultivation. These results demonstrate that the developed polyhydroxyethylmethacrylate-based cryogel reactor can be used efficiently for continuous mAb production.

Production of a monoclonal antibody specific to the IgM heavy chain of Asian seabass (Lates calcarifer Bloch, 1790) and its application in assessing health status following vaccination and challenges with Flavobacterium covae and Streptococcus iniae

Immunoglobulin M (IgM) shows potential as a significant immunological marker in Asian seabass (Lates calcarifer), accentuating the importance of developing specific monoclonal antibodies (mAbs) for immunological studies. In this study, we investigated the antigenicity of the IgM heavy chain (IgMH), particularly the Cμ3 region of IgMH (IgMHCμ3), and synthesized a peptide for anti-IgMHCμ3 mAb production. The synthesized peptide was used to generate a specific mAb in rabbits. The produced mAb demonstrated high specificity and affinity for Asian seabass IgMH, as confirmed through indirect ELISA, Western blot analysis, indirect immunofluorescence assay (IIFAT) and flow cytometry analysis. In addition, an indirect ELISA was developed and optimized to measure anti-Flavobacterium covae and anti-Streptococcus iniae antibody titers in Asian seabass serum. The monoclonal antibody (mAb) was subsequently used to assess the health status of Asian seabass post-vaccination and challenge with these pathogens, demonstrating its potential as a valuable tool for immunological studies and health assessments in Asian seabass. Vaccination and postchallenge fish exhibited a pronounced increase in specific antibodies and total serum IgM and IgM+ cell populations. Thus, this study establishes a foundational platform for using the anti-IgMHCμ3 mAb as an essential tool for evaluating and understanding the immune responses and health status of Asian seabass, especially in the context of bacterial infections and vaccinations, and paves the way for more studies in fish immunology.

Data-driven parameterization and development of mechanistic cell cultivation models in monoclonal antibody production processes: Shifts in cell metabolic behavior

Representative kinetic models to describe monoclonal antibody (mAb) production processes are needed for effective process design. The development of mechanistic models can be impeded by the lack of complete understanding of changes in cell metabolism, e.g., lactate metabolic shifts. State-estimation-based methods were applied to assess the fit of available kinetic models over experimental runs. The results indicated the regions where model parameter updates were required. Different clustering strategies were applied to isolate the variations in the culture environment and correlate them to the lactate shifts. Alternative formulations for the specific lactate consumption/production term were provided for each identified phase. Two case studies are presented for pilot-scale data in different reactor types. The results show the improvement in modeling accuracy and highlight the role of oxygen and nutrient levels on the shifts. The approach showcases the use of data-driven insights to effectively utilize limited experimental data to develop robust mechanistic models.

A Complete Sojourn of Monoclonal Antibodies: AI, Rare Diseases / Disorders And Immunotoxic Effects

Abstract: Monoclonal antibodies (mAbs) are magic bullets proved to be a wonder in the pharmaceutical as well as medical fields. These are produced by various methods like hybridoma technology, phage display technology, YAC technology, and transgenic animals and plants. Based on the percentage of animal origin, mAbs are divided into chimeric, murine, humanized, and fully human. This review covers the history and methods of mAb production, immunotoxicity (Immunosuppression, immunostimulant, autoimmunity, hypersensitivity) associated with mAbs, and targets of mAbs. It also compiles mAb production using AI, new modifications, and novel mAbs, with its various clinical trial information ensuring the use of mAbs in rare diseases and disorders.

High accuracy discriminant analysis of preparation from 22 commercial antibodies based on UV spectroscopy combined with chemometrics

The demand for monoclonal antibodies (mAbs) in hospitals is continually rising due to the advancement of targeted therapy or immunotherapy. Typically, mAbs are produced in centralized chemotherapy production units and require rigorous control to ensure the right drug at the right dose before administration to patients.This study aimed to develop a robust discriminant analysis method using a large dataset comprising 22 mAbs used in cancer therapy. The discriminant analysis was based on UV spectroscopy coupled with chemometrics. A dataset consisting of 404 samples was prepared and analyzed using various preprocessing methods, wavelength selection and discriminant analysis algorithms to maximize overall accuracy. Validation of the newly developed discriminant analysis was conducted using samples collected from real manufactured preparations for patient (over 1,380 samples). The study achieved an overall accuracy of 99.6% and 99.8% on the 22 mAbs using partial least squares-discriminant analysis (PLS-DA) and k-nearest neighbors (kNN), respectively. The utilization of chemometrics for discriminating mAbs based on their UV spectra, following total area normalization, has been successfully demonstrated. This method has undergone validation, affirming its capability to swiftly and dependably distinguish different mAbs. Moreover, it effortlessly integrates within the workflow involved in the preparation of mAbs drugs within a cancer hospital setting. This advancement holds promise for streamlining processes and enhancing efficiency in the production and use of mAbs, thereby potentially benefiting patient care and treatment outcomes.

Enhanced efficacy of glycoengineered rice cell-produced trastuzumab.

For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 μg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

Harnessing the potential of microalgae for the production of monoclonal antibodies and other recombinant proteins.

Monoclonal antibodies (mAbs) have become indispensable tools in various fields, from research to therapeutics, diagnostics, and industries. However, their production, primarily in mammalian cell culture systems, is cost-intensive and resource-demanding. Microalgae, diverse photosynthetic microorganisms, are gaining attention as a favorable option for manufacturing mAbs and various other recombinant proteins. This review explores the potential of microalgae as a robust expression system for biomanufacturing high-value proteins. It also highlights the diversity of microalgae species suitable for recombinant protein. Nuclear and chloroplast genomes of some microalgae have been engineered to express mAbs and other valuable proteins. Codon optimization, vector construction, and other genetic engineering techniques have significantly improved recombinant protein expression in microalgae. These accomplishments demonstrate the potential of microalgae for biopharmaceutical manufacturing. Microalgal biotechnology holds promise for revolutionizing the production of mAbs and other therapeutic proteins, offering a sustainable and cost-effective solution to address critical healthcare needs.

In-line fiber optical sensor for detection of IgG aggregates in affinity chromatography

Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.

Ectoine enhances recombinant antibody production in Chinese hamster ovary cells by promoting cell cycle arrest

Chinese hamster ovary (CHO) cells represent the most preferential host cell system for therapeutic monoclonal antibody (mAb) production. Enhancing mAb production in CHO cells can be achieved by adding chemical compounds that regulate the cell cycle and cell survival pathways. This study investigated the impact of ectoine supplementation on mAb production in CHO cells. The results showed that adding ectoine at a concentration of 100 mM on the 3rd day of cultivation improved mAb production by improving cell viability and extending the culture duration. RNA sequencing analysis revealed differentially expressed genes associated with cell cycle regulation, cell proliferation, and cellular homeostasis, in particular promotion of cell cycle arrest, which was then confirmed by flow cytometry analysis. Ectoine-treated CHO cells exhibited an increase in the number of cells in the G0/G1 phase. In addition, the cell diameter was also increased. These findings support the hypothesis that ectoine enhances mAb production in CHO cells through mechanisms involving cell cycle arrest and cellular homeostasis. Overall, this study highlights the potential of ectoine as a promising supplementation strategy to enhance mAb production not only in CHO cells but also in other cell lines.

Modeling of cell cultivation for monoclonal antibody production processes considering lactate metabolic shifts.

Demand for monoclonal antibodies (mAbs) is rapidly increasing. To achieve higher productivity, there have been improvements to cell lines, operating modes, media, and cultivation conditions. Representative mathematical models are needed to narrow down the growing number of process alternatives. Previous studies have proposed mechanistic models to depict cell metabolic shifts (e.g., lactate production to consumption). However, the impacts of variations of some operating conditions have not yet been fully incorporated in such models. This paper offers a new mechanistic model considering variations in dissolved oxygen and glutamine depletion on cell metabolism applied to a novel Chinese hamster ovary (CHO) cell line. Expressions for the specific rates of lactate production, glutamine consumption, and mAb production were formulated for stirred and shaken-tank reactors. A deeper understanding of lactate metabolic shifts was obtained under different combinations of experimental conditions. Lactate consumption was more pronounced in conditions with higher DO and low glutamine concentrations. The model offers mechanistic insights that are useful for designing advanced operation strategies. It can be used in design space generation and process optimization for better productivity and product quality.

A case study application of AQbD to the re-development and validation of an affinity chromatography analytical procedure for mAb titer quantitation

Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R2 values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.

A Comprehensive Review of Monoclonal Antibodies in Modern Medicine: Tracing the Evolution of a Revolutionary Therapeutic Approach.

Monoclonal antibodies (mAbs) have emerged as potent therapeutic agents, revolutionizing the landscape of modern medicine. This comprehensive review traces the evolution of mAbs from their inception to their current prominence, highlighting key milestones in their development and exploring their diverse therapeutic applications. Beginning with an overview of their molecular structure and mechanisms of action, we delve into the production and engineering of mAbs, including hybridoma technology and recombinant DNA techniques. Therapeutic applications across various medical disciplines, including cancer treatment, autoimmune diseases, and infectious diseases, are examined in detail, showcasing the significant clinical successes of mAbs. Furthermore, this review discusses the challenges and opportunities in manufacturing scalability, cost-effectiveness, and access to therapies. Looking ahead, the implications of mAbs in future research and clinical practice are explored, emphasizing the potential for next-generation mAbs, personalized medicine, and integration with emerging modalities such as immunotherapy and gene therapy. In conclusion, the evolution of monoclonal antibodies underscores their transformative impact on healthcare and their continued promise to advance the frontiers of medicine.

Dynamic optimization of an integrated cultivation-aggregation model for mAb production.

Due to their proteinaceous structure, monoclonal antibodies (mAbs) are susceptible to irreversible aggregation, with harmful consequences on drug efficacy and patient safety. To mitigate this risk in modern biopharmaceutical processes, it is critical to comply with current good manufacturing practices (cGMP) and pursue operating strategies minimizing irreversible aggregation whilst also maximizing mAb throughput. These conflicting objectives are targeted in this study by formulating and analyzing an integrated dynamic model accounting for both cultivation and aggregation of mAbs from a Chinese Hamster Ovary (CHO) cell line. Two manipulated dynamic variables are considered here in simulation studies: firstly temperature manipulation within a batch reactor, and secondly feed flow manipulation within a series of isothermal fed-batch reactors. Following this, dynamic optimization investigations have been conducted, firstly with the single objective of maximizing mAb throughput and secondly with multiple (two) objectives of maximizing mAb throughput while also minimizing irreversible aggregate content, simultaneously. The study provides key insight into tradeoffs of how simultaneous temperature and feed flowrate manipulation affects mAb throughput and aggregation inside bioreactors.

Analytical Quality by Design as applied to the development of a SEC-HPLC platform procedure for the determination of monoclonal antibody purity without mobile phase additives

This work presents the application of AQbD principles to the development of a size exclusion chromatography (SEC) HPLC procedure for the determination of monoclonal antibody (mAb) product purity using state-of-the-art column technology available via the Waters™ XBridge Premier Protein SEC column. Analytical Quality by Design (AQbD) emphasizes a systematic, risk-based lifecycle approach to analytical procedure development based on sound statistical methodologies. It has recently become increasingly recommended by regulatory agencies as a response to the need for greater efficiency, improved reliability, and increased robustness among modern analytical procedures in the pharmaceutical industry. Use of an Analytical Target Profile (ATP) and formal risk assessments informed the application of Design of Experiments (DoE) to optimize this analytical procedure, as well as assess its robustness and ruggedness. Importantly, our ruggedness results demonstrated the transferability of this procedure between two laboratories within the Catalent Biologics Global Network. Application of this analytical procedure as a platform approach for evaluating mAb purity is expected to support expedited, first-in-human timelines of mAb molecules by enabling great quantitative performance with simple mobile phase buffer compositions. Taken together, this case study demonstrates the utility of adopting AQbD principles in analytical procedure development.

Evaluation of various membranes at different fluxes to enable large-volume single-use perfusion bioreactors.

The growing demand for biological therapeutics has increased interest in large-volume perfusion bioreactors, but the operation and scalability of perfusion membranes remain a challenge. This study evaluates perfusion cell culture performance and monoclonal antibody (mAb) productivity at various membrane fluxes (1.5-5 LMH), utilizing polyvinylidene difluoride (PVDF), polyethersulfone (PES), or polysulfone (PS) membranes in tangential flow filtration mode. At low flux, culture with PVDF membrane maintained higher cell culture growth, permeate titer (1.06-1.34 g/L) and sieving coefficients (≥83%) but showed lower permeate volumetric throughput and higher transmembrane pressure (TMP) (>1.50 psi) in the later part of the run compared to cultures with PES and PS membrane. However, as permeate flux increased, the total mass of product decreased by around 30% for cultures with PVDF membrane, while it remained consistent with PES and PS membrane, and at the highest flux studied, PES membrane generated 12% more product than PVDF membrane. This highlights that membrane selection for large-volume perfusion bioreactors depends on the productivity and permeate flux required. Since operating large-volume perfusion bioreactors at low flux would require several cell retention devices and a complex setup, PVDF membranes are suitable for low-volume operations at low fluxes whereas PES membranes can be a desirable alternative for large-volume higher demand products at higher fluxes.