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The chromatography process of large-scale plasmid purification with high efficiency and low cost has always been a major challenge. We established a two-step plasmid chromatography purification process combining multimodal and thiophilic chromatography with an overall chromatography yield of nearly 70%. Capto Core 700, a multimodal core-shell particle, was firstly used to remove the impurities from the crude lysate. The effects of different experimental conditions on chromatography recovery and impurity removal were screened. Compared to conventional size exclusion chromatography, the sample load and flow rate of this step were enhanced by 40-fold and 5-fold, respectively, while maintaining a 90% yield. For the thiophilic chromatography (Capto PlasmidSelect), the method of Design of Experiments (DoEs) was used to study the influence of parameters on the results. The effects of ammonium sulfate concentration, sodium chloride concentration and flowrate in the elution phase were studied and optimized with a central composite design model consisting of 17 experiments. The versatility of this process was demonstrated by successfully purifying three different lentiviral packaging plasmids (pLP1, pLP2 and pLP/VSVG) and the target plasmid containing green fluorescent protein (GFP). Purified plasmids consistently achieved a supercoiled purity of at least 90% with endotoxin levels below 5 EU/mg. Lentiviral vectors packaged using these plasmids exhibited high infectious titers of 1 × 10 7  TU/mL, thereby verifying the process applicability for diverse plasmid purification requirements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Approval of induced pluripotent stem cells (iPSCs) for the manufacture of cell therapies to support clinical trials is now becoming realized after 20 years of research and development. In 2022 the International Society for Cell and Gene Therapy (ISCT) established a Working Group on Emerging Regenerative Medicine Technologies, an area in which iPSCs-derived technologies are expected to play a key role. In this article, the Working Group surveys the steps that an end user should consider when generating iPSCs that are stable, well-characterised, pluripotent, and suitable for making differentiated cell types for allogeneic or autologous cell therapies. The objective is to provide the reader with a holistic view of how to achieve high-quality iPSCs from selection of the starting material through to cell banking. Key considerations include: (i) intellectual property licenses; (ii) selection of the raw materials and cell sources for creating iPSC intermediates and master cell banks; (iii) regulatory considerations for reprogramming methods; (iv) options for expansion in 2D vs. 3D cultures; and (v) available technologies and equipment for harvesting, washing, concentration, filling, cryopreservation, and storage. Some key process limitations are highlighted to help drive further improvement and innovation, and includes recommendations to close and automate current open and manual processes., (Copyright © 2024. Published by Elsevier Inc.)

Electrospun cellulose adsorbents are an emergent class of materials applied to a variety of bioprocess separations as an analogue to conventional packed bed chromatography. Electrospun adsorbents have proven to be effective as rapid cycling media, enabling high throughput separation of proteins and viral vectors without compromising selectivity and recovery. However, there is a current lack of knowledge in relation to the manipulation and control of electrospun adsorbent structure with function and performance to cater to the separation needs of emerging, diverse biological products. In this study, a series of electrospun cellulose adsorbents were fabricated by adjusting their manufacturing conditions. A range of fiber diameters (400 to 600 nm) was created by changing the electrospinning polymer solution. Additionally, a range of porosities (0.4 to 0.7 v/v) was achieved by varying the laminating pressures on the electrospun sheets. The adsorbents were functionalized with different degrees of quaternary amine ligand density to create 18 prototype anion exchangers. Their morphology was characterized by BET nitrogen adsorption surface area, X-ray computed tomography, capillary flow porometry and scanning electron microscopy measurements. The physical characteristics of the adsorbents were used in an adapted semi-empirical model and compared to measured permeability data. Permeabilities of prototypes ranged from 10 -2 to 10 -4 mDarcy. The measured data showed good adherence to modelled data with possible improvements in acquiring wet adsorbent characteristics instead of dried material. Finally, the electrospun adsorbents were characterized for their binding capacity of model proteins of different sizes (diameters of 3.5 nm and 8.9 nm) and plasmid DNA. Static binding capacities ranged from 5 mg/ml to 25 mg/ml for the proteins and plasmid DNA and showed <20 % deviation from monolayer coverage based on BET surface area. Therefore, it was concluded that the electrospun adsorbents most likely adsorb monolayers of proteins and plasmid DNA on the surface with minimal steric hindrance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gyorgy Ovari reports financial support and equipment, drugs, or supplies were provided by Cytiva Stevenage. Thomas F. Johnson reports financial support was provided by Medical Research Council (MRC) Innovation Scholarship Grant. Thomas F. Johnson reports equipment, drugs, or supplies was provided by National Research Facility for Lab X-ray CT (NXCT). Gunnar Malmquist reports a relationship with Cytiva Sweden AB that includes: consulting or advisory and employment. Matthew Townsend reports a relationship with Cytiva that includes: employment. Farzad Foroutan reports a relationship with Cytiva that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background Aims: The production of commercial autologous cell therapies such as chimeric antigen receptor T cells requires complex manual manufacturing processes. Skilled labor costs and challenges in manufacturing scale-out have contributed to high prices for these products., Methods: We present a robotic system that uses industry-standard cell therapy manufacturing equipment to automate the steps involved in cell therapy manufacturing. The robotic cluster consists of a robotic arm and customized modules, allowing the robot to manipulate a variety of standard cell therapy instruments and materials such as incubators, bioreactors, and reagent bags. This system enables existing manual manufacturing processes to be rapidly adapted to robotic manufacturing, without having to adopt a completely new technology platform. Proof-of-concept for the robotic cluster's expansion module was demonstrated by expanding human CD8+ T cells., Results: The robotic cultures showed comparable cell yields, viability, and identity to those manually performed. In addition, the robotic system was able to maintain culture sterility., Conclusions: Such modular robotic solutions may support scale-up and scale-out of cell therapies that are developed using classical manual methods in academic laboratories and biotechnology companies. This approach offers a pathway for overcoming manufacturing challenges associated with manual processes, ultimately contributing to the broader accessibility and affordability for personalized immunotherapies., Competing Interests: Declaration of competing interest BS, ALJ, YA, AR, DC, ED, KP, KH, DS, US, VB, AS, NK, AH, MG and AT, wish to disclose that they are current employees of Multiply Labs, Inc. or were employed with the company at the time of this study's execution. They hold equity in the company; AM and FP wish to disclose that they are co-founders of Multiply Labs, Inc. and hold the position of Chief Scientific Officer and Chief Executive Officer, respectively; SC wishes to disclose that she is employed by Cytiva, which is a company active in the cell therapy manufacturing space; MH wishes to disclose that he is employed by Charles River Laboratories, which is a company active in the cell therapy manufacturing space; HA and XJ wishes to disclose that they are employed by Thermo Fisher Scientific, which is a company active in the cell therapy manufacturing space; BDR is a paid advisor to Multiply Labs, Inc. and holds equity in the company; JHE is a paid advisor to and receives sponsored research funding from Multiply Labs, Inc. He serves on its scientific advisory board and holds equity in the company. He receives sponsored research funding from Lonza, Inc. for the development of cellular therapy manufacturing devices., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Thuy Tran reports financial support was provided by Marie Sklodowska-Curie grant agreement No. 841,373. Daniel Aili reports financial support was provided by The Swedish Innovation Agency (VINNOVA), grant numbers 2016–04120 and 2019–00130. Daniel Aili reports a relationship with ArgusEye AB that includes: board membership and equity or stocks. Erik Martinsson reports a relationship with ArgusEye AB that includes: equity or stocks. Ingemar Lundstrom reports a relationship with ArgusEye AB that includes: equity or stocks. Carl-Fredrik Mandenius reports a relationship with ArgusEye AB that includes: equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing "empty" capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a C C -intein tag is engineered into a protein of interest for binding to a N C -intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the N C -intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the C C -intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Johan Ohman has patent #WO2021099607A1 licensed to CYTIVA BIOPROCESS R&D AB. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Increased demand for mRNA-based therapeutics and improved in vitro transcription (IVT) yields have challenged the mRNA purification platform. Hybridization-affinity chromatography with an immobilized oligo-deoxythymidilic acid (oligodT) ligand is often used to capture mRNA through base pairing with the polyadenylated tail. Commercially available oligodT matrices include perfusive cross-linked poly(styrene-divinylbenzene) 50 µm POROS™ chromatography resin beads and convective polymethacrylate CIMmultus® monolithic columns consisting of 2 µm interconnected channels. POROS™ columns may be limited by poor mass transfer for larger mRNAs and slow flowrates, while monoliths can operate at higher flowrates but are limited by modest binding capacity. To enable both high flowrates and binding capacity for mRNA of all lengths, prototype chromatography media was developed by Cytiva using oligodT immobilized electrospun cellulose nanofibers (Fibro™) with a 0.3-0.4 µm pore size. In this work, four polyadenylated mRNAs ranging from ∼1900-4300 nucleotides were used to compare the dynamic binding capacity (DBC) of Fibro™, POROS® and CIMmultus® columns as a function of residence time and binding buffer compositions. Fibro™ improved the DBC ∼2-4-fold higher than CIMmultus® and ∼2-13-fold higher than POROS™ across all residence times, mRNA length, and binding matrix compositions tested. CIMmultus® DBC was least dependent on residence time and mRNA size, while both Fibro™ and POROS™ DBC increased at slower flowrates and with shorter mRNA. Surprisingly, inverse size exclusion (ISE) experiments showed that POROS™ was not limited by diffusion and POROS™ along with CIMmultus® demonstrate higher mRNA permeation however the Fibro™ prototype is not in the final configuration. Lastly, IVT reaction products were subjected to purification and oligodT elution product yield, quality, and purity were consistent across the three matrices investigated. These results highlight the benefits of high DBC and equivalent product profiles offered by the oligodT Fibro™ prototype compared to commercially available oligodT media., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: P.G., S.L., P.L., S.B.S are employees of Cytiva. The Fibro™ technology used in this manuscript is protected by a patent (US Patent 9,802,979) in the United States and in foreign jurisdictions for Cytiva., (Copyright © 2024. Published by Elsevier B.V.)

The quantification of monoclonal antibody (mAb) aggregates and fragments using high pressure liquid chromatography-size exclusion chromatography (HPLC-SEC) typically requires off-line measurements that are time-consuming and therefore not compatible with real-time monitoring. However, it has been crucial to manufacturing and process development, and remains the industrial standard in the assessment of product-related impurities. Here we demonstrate that our previously established intrinsic time-resolved fluorescence (TRF) approach can be used to quantify the bioprocess critical quality attribute (CQA) of antibody product purity at various stages of a typical downstream process, with the potential to be developed for in-line bioprocess monitoring. This was directly benchmarked against industry-standard HPLC-SEC. Strong linear correlations were observed between outputs from TRF spectroscopy and HPLC-SEC, for the monomer and aggregate-fragment content, with R 2 coefficients of 0.99 and 0.69, respectively. At total protein concentrations above 1.41 mg/mL, HPLC-SEC UV-Vis chromatograms displayed signs of detector saturation which reduced the accuracy of protein quantification, thus requiring additional sample dilution steps. By contrast, TRF spectroscopy increased in accuracy at these concentrations due to higher signal-to-noise ratios. Our approach opens the potential for reducing the time and labour required for validating aggregate content in mAb bioprocess stages from the several hours required for HPLC-SEC to a few minutes per sample., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J.E.H. and P.A.D. are co-authors on a patent application which could increase in value if the methods and ideas described in this paper find widespread application., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Multimodal chromatography resins are becoming a key tool in the purification of biomolecules. The main objective of this research was the establishment of an iterative framework for the rapid development of new multimodal resins to provide novel selectivity for the future purification challenges. A large chemically diverse virtual library of 100 multimodal Capto™ MMC ligand analogues was created, and a broad array of chemical descriptors were calculated for each ligand in silico. Principal component analysis (PCA) was used to map the chemical diversity and guide selection of ligands for synthesis and coupling to the Capto ImpRes agarose base matrix. Twelve new ligands were prepared in two groups: 'group one' consist of L00-L07 and 'group two' consist of L08-L12. These ligands are diverse in the influence of varied secondary interactions such as hydrophobic interactions, H-bonding, etc. Additional resin prototypes were also prepared to look at the chromatographic impact of ligand density variation. High-throughput plate-based studies were performed for parallel resin screening for batch-binding of six model proteins at different chromatographic binding pH and sodium chloride concentration conditions. Principal component analysis of the binding data provided a chromatographic diversity map leading to the identification of ligands with improved binding. Further, the new ligands have improved separation resolution between a monoclonal antibody (mAb1) and product related impurities, a Fab fragment and high molecular weight (HMW) aggregates, using linear salt gradient elutions. To quantify the importance of secondary interactions, analysis of the retention factor of mAb1 on the ligands at various isocratic conditions lead to estimations of (a) the total number of water molecules and counter salt ions released during adsorption, and (b) hydrophobic contact area (HCA). The iterative mapping approach of chemical and chromatography diversity maps described in the paper proves to be a promising method for identifying new chromatography ligands for biopharmaceutical purification challenges., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In the biopharmaceutical industry, chromatography resins have a finite number of uses before they start to age and degrade, typically due to losses of ligand integrity and/or density. The "health" of a column is predicted and validated by running multiple cycles on representative scale-down models and can be followed by real-time on-going validation during commercial production. Principal Component Analysis (PCA), Partial Least Square (PLS), Similarity Scores and Single One Point-MultiParameter Technique (SOP-MPT) along with machine learning principles were applied to explore the hypothesis that there is predictive capability of latent variables in chromatography absorbance profiles for process performance (step yield) and product quality (aggregates, fragments, host cell proteins (HCP) and DNA, and Protein A ligand). The first stage of this study is described in this paper: a MabSelect SuRe™ chromatography column was cycled with a method to establish the "normal" baseline for process performance and product quality, followed by runs using a harsher NaOH Cleaning in Place (CIP) procedure (with a higher NaOH concentration than that recommended by the vendor) to accelerate resin degradation. The different mathematical analytical tools correlated with resin degradation of the column (reflected in decreasing step yield and binding capacity with increasing running cycle), specifically when using the Wash, Elution and Strip phases of the chromatography method. Monomer, HCP and DNA content were not significantly impacted and therefore a correlation with product quality was inconsequential. Importantly, this work shows proof-of-concept that while more traditional methods of measuring resin integrity such as the height equivalent to a theoretical place (HETP) and Asymmetry (As) measurements could not detect changes in the integrity of the resin, PCA, PLS, Similarity Scores and SOP-MPT (to a lesser extent) applied to the absorbance data were capable of anticipating issues in the chromatography bed by identifying atypical outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Foam fractionation has recently attracted attention as a low-cost and environmentally benign treatment technology for water contaminated with per- and polyfluoroalkyl substances (PFAS). However, data on the mass balance over the foam fractionation process are scarce and when available, gaps in the mass balance are often identified. This study verified the high treatment efficiency of a pilot-scale foam fractionation system for removal of PFAS from industrial water contaminated with aqueous film-forming foam. ΣPFAS removal reached up to 84 % and the removal of perfluorooctane sulfonic acid (PFOS) up to 97 %, but the short-chain perfluorobutanoic acid (PFBA) was only removed with a mean efficiency of 1.5 %. In general, mobile short-chain PFAS were removed less efficiently when the perfluorocarbon chain length was below six for carboxylic acids and below five for sulfonic acids. Fluctuations in treatment efficiency due to natural variations in the chemistry of the influent water were minor, confirming the robustness of the technology, but significant positive correlations between PFAS removal and influent metal concentration and conductivity were observed. Over all experiments, the mass balance closure did not differ significantly from 100 %. Nonetheless, PFAS sorption to the walls of the reactor was measured, as well as high PFAS emissions by the air exiting the reactor. PFAS emissions in aerosols correlated positively with mass balance closure. The elevated aerial PFAS concentrations measured in the experimental facility have implications for worker safety and prevention of PFAS-emissions to the atmosphere, and demonstrate the importance of installing appropriate filters on the air outlet of foam fractionation systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

With growing demands for therapeutic monoclonal antibodies, in silico downstream process development based on mechanistic modeling of chromatography separation process is being increasingly used for process optimization and process characterization. Application of mechanistic modeling in biopharmaceutical industry has been sparse due to the significant investment of time and resources that are required for performing model calibration. Mechanistic modeling of the chromatography process involves a large number of mass transport and binding parameters and their initial input values are required for simulations. These input values of column parameters can be easily obtained either from experiments or from empirical correlations available in literature. On the other hand, obtaining the model input valves for binding kinetic parameters is usually a cumbersome process as it involves performing batch experiments which are not only tedious but also require significant quantities of purely isolated main product and its related impurities, which is challenging as the product related impurities are typically present in smaller quantities and hence are difficult to obtain as pure species. In the present work, a mechanistic model that is based on the general rate model coupled with extended Langmuir binding model has been used for prediction of linear gradient elution peaks of monoclonal antibody on cation exchanger chromatography. The present work describes an accelerated approach for obtaining the input values for binding kinetic parameters in the extended Langmuir binding model from the two Yamamoto coefficient A and B values obtained by Yamamoto method directly from the model calibration linear gradient elution runs of different gradient slopes and at low to moderate protein loadings. The equations that can relate the two coefficients to the extended Langmuir model equation binding kinetic parameters were derived. Therefore, once A and B are determined, the binding kinetic parameter values were determined straightforward, thereby avoiding the problem of multiple solutions for the model parameters. The estimated binding parameters were successfully validated from isocratic elution experiments performed at low loading. What we demonstrate is that the proposed approach allows us to estimate binding kinetic parameters in a significantly more efficient and accelerated manner than presently used approaches, thereby accelerating development and implementation of mechanistic modeling for process chromatography., Competing Interests: Declaration of Competing Interest Anurag S. Rathore reports financial support was provided by DBT Centre of Excellence for Biopharmaceutical Technology (BTCOE34SP150972015). Shuichi Yamamoto reports financial support was provided by AMED (Japan Agency for Medical Research and Development (Grant Number JP21ae0121016)., (Copyright © 2022. Published by Elsevier B.V.)

Activities involved in the production of certain advanced therapy medicinal products (ATMPs) require standardized approaches to mononuclear cell procurement to ensure the highest product quality, safety and process efficiency. These aims must be achieved while meeting regulatory and accreditation requirements for the procurement of mononuclear cells as starting materials. Mononuclear cells constitute the starting materials for many ATMPs, and this article sets out recommendations for procurement by clinical apheresis, addressing the variation among existing working practices and different manufacturers' requirements that currently poses a challenge when managing multiple different protocols., Competing Interests: Declaration of Competing Interest JS is an employee of and has stock ownership in bluebird bio, Inc. DH was employed by Autolus Therapeutics plc during the drafting of the manuscript. WS is employed by Autolus Therapeutics plc., (Copyright © 2022 International Society for Cell & Gene Therapy. All rights reserved.)

Monoclonal antibodies (mAb) are used as therapeutics and for diagnostics of a variety of diseases, and novel antibodies are continuously being developed to find treatments for new diseases. Therefore, the manufacturing process must accommodate a range of mAb characteristics. Acid-sensitive mAbs can severely compromise product purity and yield in the purification process due to the potential formation of aggregates. To address this problem, we have developed an integrated downstream process for the purification of pH-sensitive mAbs at mild conditions. A calcium-dependent Protein A-based ligand, called Z Ca , was used in the capture step in a 3-column periodic counter-current chromatography operation. The binding of Z Ca to antibodies is regulated by calcium, meaning that acidic conditions are not needed to break the interaction and elute the antibodies. Further, the virus inactivation was achieved by a solvent/detergent method, where the pH could remain unchanged. The polishing steps included a cation and an anion exchange chromatography step, and screening of the capture and polishing steps was performed to allow for a seamless integration of the process steps. The process was implemented at laboratory scale for 9 days obtaining a high yield, and a consistently pure drug substance, including high reduction values of the host cell protein and DNA concentrations, as well as aggregate levels below the detection limit, which is attributed to the mild conditions used in the process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SH has filed a patent application regarding the novel Z(Ca) domain., (Copyright © 2022. Published by Elsevier B.V.)

The transition from preclinical biological drug development into clinical trials requires an efficient upscaling process. In this context, bispecific antibody drugs are particularly challenging due to their propensity to form aggregates and generally produce low titers. Here, the upscaling process for a tetravalent bispecific antibody expressed by a piggyBac transposon-mediated stable HEK293 cell pool has been evaluated. The project was performed as a case study at Testa Center, a non-GMP facility for scale-up testing of biologics in Sweden, and encompassed media adaptation strategies, fed-batch optimization and a novel antibody purification technology. The cell pool was adapted to different culture media for evaluation in terms of cell viability and titers compared to its original Expi293 Expression Medium. These parameters were assessed in both sequential stepwise adaption and direct media exchanges. By this, a more affordable medium was identified that did not require stepwise adaptation and with similar titers and viability as in the Expi293 Expression Medium. Fed-batch optimizations resulted in culture densities reaching up to 20 × 10 6 viable cells/mL with over 90 % viability 12 days post-inoculum, and antibody titers three times higher than corresponding batch cultures. By implementing a novel high-speed protein A fiber technology (Fibro PrismA) with a capture residence time of only 7.5 s, 8 L of supernatant could be purified in 4.5 h without compromising the purity, structural integrity and function of the bispecific antibody. Results from this study related to medium adaptation and design of fed-batch protocols will be highly beneficial during the forthcoming scale-up of this therapeutic antibody., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

A fundamental understanding of the protein retention mechanism in preparative ion exchange (IEX) chromatography columns is essential for a model-based process development approach. For the past three decades, the mechanistic description of protein retention has been based predominantly on the steric mass action (SMA) model. In recent years, however, retention profiles of proteins have been reported more frequently for preparative processes that are not consistent with the mechanistic understanding relying on the SMA model. In this work, complex elution behavior of proteins in preparative IEX processes is analyzed using a colloidal particle adsorption (CPA) model. The CPA model is found to be capable of reproducing elution profiles that cannot be described by the traditional SMA model. According to the CPA model, the reported complex behavior can be ascribed to a strong compression and concentration of the elution front in the lower unsaturated part of the chromatography column. As the unsaturated part of the column decreases with increasing protein load density, exceeding a critical load density can lead to the formation of a shoulder in the peak front. The general applicability of the model in describing preparative IEX processes is demonstrated using several industrial case studies including multiple monoclonal antibodies on different IEX adsorber systems. In this context, the work covers both salt controlled and pH-controlled protein elution., Competing Interests: Declaration of Competing Interest The authors affiliated to GoSilico declare that the software ChromX used in this study is a commercial product of GoSilico. The presented research does not depend on the usage of ChromX, the model can be implemented in any kind of modeling software., (Copyright © 2021. Published by Elsevier B.V.)

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Modular virus-like particles and capsomeres are potential vaccine candidates that can induce strong immune responses. There are many described protocols for the purification of microbially-produced viral protein in the literature, however, they suffer from inherent limitations in efficiency, scalability and overall process costs. In this study, we investigated alternative purification pathways to identify and optimise a suitable purification pathway to overcome some of the current challenges. Among the methods, the optimised purification strategy consists of an anion exchange step in flow through mode followed by a multi modal cation exchange step in bind and elute mode. This approach allows an integrated process without any buffer adjustment between the purification steps. The major contaminants like host cell proteins, DNA and aggregates can be efficiently removed by the optimised strategy, without the need for a size exclusion polishing chromatography step, which otherwise could complicate the process scalability and increase overall cost. High throughput process technology studies were conducted to optimise binding and elution conditions for multi modal cation exchanger, Capto™ MMC and strong anion exchanger Capto™ Q. A dynamic binding capacity of 14 mg ml -1 was achieved for Capto™ MMC resin. Samples derived from each purification process were thoroughly characterized by RP-HPLC, SEC-HPLC, SDS-PAGE and LC-ESI-MS/MS Mass Spectrometry analytical methods. Modular polyomavirus major capsid protein could be purified within hours using the optimised process achieving purities above 87% and above 96% with inclusion of an initial precipitation step. Purified capsid protein could be easily assembled in-vitro into well-defined virus-like particles by lowering pH with addition of calcium chloride to the eluate. High throughout studies allowed the screening of a vast design space within weeks, rather than months, and unveiled complicated binding behaviour for Capto TM MMC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

In this study, the enhanced alkaline stability of Protein A ligands and resins designed by protein engineering approaches is demonstrated. High throughput PreDictor™ plates were used to evaluate and compare the human Immunoglobulin G (IgG) static binding capacities (SBC) of MabSelect SuRe™ and MabSelect™ PrismA affinity chromatography (AC) resins after continuous incubation in 0.1-2.0 M NaOH for 1-72 h. The alkaline effect on the Protein A affinity ligand was studied by high resolution mass spectrometry (MS). The IgG binding capacity of the investigated AC resins show expected declining trends with increasing NaOH concentrations and incubation times. The decrease is larger for MabSelect SuRe than for MabSelect PrismA and occur at lower NaOH concentrations. MabSelect SuRe display high remaining binding capacity even after 72 h continuous incubation in 0.1 M NaOH, while higher concentrations induce an accentuated decline with incubation time. The MabSelect PrismA resin shows almost no effect on the binding capacity even after 72 h incubation in 0.5 M NaOH. Decline in capacity is first observed after 48 h incubation in 1.0 M NaOH, thus displaying the extreme alkaline stability of the PrismA affinity ligand. The MS analysis of the ligands, including a Protein A single B-domain, SuRe-domain and PrismA-domain clearly illustrate the increasing alkaline stability (B-domain < SuRe < PrismA) as the ligand has been refined using a protein engineering approach. Deamidation and ligand degradation could be monitored in relation to NaOH incubation conditions. Enzymatic digestion of MabSelect SuRe and MabSelect PrismA resins after alkaline incubation and LC-MS/MS peptide mapping facilitates identification and quantification of specific deamidation sites on the affinity ligand., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As discovery research organizations push more molecules and new modalities through their company pipelines, there comes a need to widen purification development and production bandwidth by increasing automation and throughput. Continuous processing technologies have the unique property of reducing manufacturing floor space and reducing costs. We can speed development and production by implementing automation and continuous process technologies early in discovery research. Here we describe an automated continuous instrument made up of an ÄKTA™ pcc for initial capture by protein A, an ÄKTA pure 150 retrofitted to automatically condition protein A eluate, and a second ÄKTA pure 150 built for flow-through anion exchange chromatography. The continuous instrument we have designed and built recirculates protein A eluate from the ÄKTA pcc in a closed loop while signals from the pH and conductivity meters direct addition of titrant for accurate and precise adjustments to the pH and salt concentration. The instrument is run without user intervention and can be used continuously for production or for development as a tool for screening running conditions on the anion exchange step., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)