High throughput chromatography and analytics can inform viral clearance capabilities during downstream process development for biologics.

Demonstration of continuous gradient elution functionality with automated liquid handling systems for high-throughput purification process development

Surface Structuring with Ultra-short Laser Pulses: Basics, Limitations and Needs for High Throughput

A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high-throughput process development (HTPD) strategy implementing several high-throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high-throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high-throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high-throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance.

Elisa Manzotti speaks to Ying Cai, Nathalie Clement, Chantelle Gaskin, Matt Roach & Ashish Saksule. Ying Cai is the Sr. Director of Process Development at Ultragenyx Pharmaceutical. She heads AAV downstream process development and formulation development functions, also a CMC lead of AAV clinical programs. Prior to joining Ultragenyx, Ying worked at Sanofi, Biogen, Merck, and a few CDMOs. Ying has over 20 years’ experience in the development, validation, manufacturing and commercialization of different modalities including AAV, plasmid DNA, oligonucleotides, antibodies, antibody conjugates, and fusion proteins. Ying holds a Ph.D. in Chemical Engineering from the University of Arkansas at Fayetteville and a B.S. in Biochemical Engineering from Zhejiang University in China. Nathalie Clement has more than 25 years of experience in the field of Gene Therapy, with a strong expertise in viral vectors, specifically adeno-associated vectors, in the academic and industry settings. Her research focus has strongly been focused on optimizing processes to support large-scale production of high quality rAAV stocks and their implementation into the GMP settings. During her thesis work at the University Libre of Brussels, Belgium, she developed new recombinant viruses derived from the parvovirus Minute Virus of Mice (MVM) for cancer-selective gene therapy treatments. She then joined Dr. Michael Linden’s laboratory at Mount Sinai School of Medicine, New York, where she developed novel recombinant AAV vectors and directed the AAV Vector Core. She next joined the Powell Gene Therapy Center in 2008 as the Associate Director to supervise AAV production and testing at research, preclinical and clinical grades. She led the Process and Development Group and the Quality Control group responsible for the production and release of all AAV pre-clinical and clinical lots. During her time at UF she oversaw manufacturing, release and stability campaigns of more than 7 AAV INDs from start to finish, including CMC preparations and interactions with FDA. More recently, she spent several months at Resilience, Alachua, Florida, as the Director of Process and Development of the Viral Vaccines and Gene Therapy franchises. IN that role she oversaw viral vaccine and AAV production scales up to 200L in suspension format and in the icellis 500 platform for adherent platforms of a variety of viruses and AAV vectors. Currently Nathalie is taking a break before starting a new adventure in 2022. Chantelle Gaskin is a Field Applications Scientist, specializing in protein and viral vector purification and downstream process development. She held leadership positions at Applied Genetic Technology Corporation and Brammer Bio, prior to joining the Thermo Fisher Scientific Bioproduction Division in 2020. With over 10 years of experience in gene therapy, Chantelle has accumulated comprehensive knowledge of standard industry practices and regulatory standards, applying this knowledge to advance development of therapies for a variety of indications including ocular, CNS and systemic disease. Chantelle holds a Master’s degree in Chemistry from University of Florida and a Bachelor’s in Chemistry from Smith College. Matt Roach leads the AAV Process Development group at Precision BioSciences, which is focused on designing and implementing new strategies for the production and purification of adeno-associated virus. Matt completed his Bachelor’s degree in Biological Sciences at North Carolina State University and his Master’s degree in Microbiology and Cell Science at the University of Florida. Prior to Precision, Matt spent time at Pfizer working on the purification of AAV and the Biomanufacturing Training and Education Center training industry professionals on downstream bioprocessing operations. Ashish Saksule is the Cell and Gene Therapy process development lead and technical expert on bioprocessing platforms for viral vectors (Lentivirus and Adeno-associated virus vector) and non-viral vectors with more than 7 years of experience. Ashish has graduate degree in Chemical Engineering from Michigan Tech University, and Biotechnology graduate degree from Harvard University. His experience spans research & drug development, clinical stage and CRO/CMO settings. Ashish is currently working at Takeda within Global Gene Therapy and have previously worked at MilliporeSigma and Miltenyi Biotec.

In the sessions of Materials Genome Initiative (MGI) and Materials Informatics (MI) at recent international and domestic conferences, majority of research presentations are on material prediction using a combination of computational chemistry and machine learning. On the other hand, the contents incorporating high-throughput synthesis and evaluation experiments are only occasionally seen. It is also true that the number of samples that can be handled per hour is small compared to the former data science.However, if a series of experimental processes linked to synthesis, evaluation, and analysis is made user-friendly and high-throughput, it is possible to efficiently generate multi-condition and multi-component data in a unified experimental environment such as starting materials. As a result, even when creating a data set by extracting text from a paper, the data shortage can be supplemented with experimental data.Unlike thin films and polymers, powder synthesis is a heterogeneous reaction that requires the use of a wet process to perform high-throughput experiments. We have hitherto developed powder synthesis apparatus equipped with a micropump for solution dispensing, and high-throughput exploration system for powder and thin-film library based on electrostatic spray deposition consisted by combining multiple syringe pumps and a high-voltage power supply. By using these system, we have been exploring multi-component cathode materials for lithium ion battery and thermoelectric materials. In addition, recently, high-throughput experiments in a high-pressure environment of 200 MPa and 500ºC are possible.If the time required for physical property evaluation and data analysis is the same as before, even if only the synthesis based on experiments becomes high throughput, the attractiveness of MI will be weakened. Therefore, it is necessary to continue to develop jigs and software for efficient evaluation and analysis of huge libraries obtained in high-throughput experiments.In this presentation, we will introduce the exploration for A-site and B-site substitutes of perovskite-type CaMnO3, which is expected as an n-type thermoelectric material. In the preliminary search, it was confirmed that Ca1-x Bi x Mn1-y Ni y O3 contributes to the improvement of thermoelectric power by substituting elements at each site. As the next step, the amount of Bi substitution was determined to be within 10% from the combination of high-throughput synthesis and high-throughput Seebeck coefficient measurement. Synchrotron XRD and XAFS measurements were used to investigate the correlation between physical properties and crystal structure. We have succeeded in collecting data without filling the capillaries and making pellets by developing special tools. Synchrotron XRD and XAFS measurements were used to investigate the correlation between physical properties and crystal structure. We have succeeded in collecting data without filling the capillaries and making pellets by developing special tools. We also developed software that automates Rietveld analysis, although we need basic knowledge to set up an initial structural model. As a result, we have made it easier to generate data sets for visualizing crystallographic data and physical property data at once. In the obtained Bi-substituted CaMnO3 powder, when Bi was 8% or less, the conductivity and power factor increased due to the increase in carrier concentration. At Bi8% and higher, PF reaches its limit value, which involves a large change in the MnO6 octahedral plane bond distances Mn-O1 (1) and Mn-O1 (2), that is, a significant increase in octahedral distortion. High-throughput experiments including the collection of crystallographic information through the development of these technologies are expected to be an effective tool for future machine learning.

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process-related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non-infectious retrovirus-like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96-well batch-binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow™ (QSFF) and Capto adhere™, a mixed mode resin. QSFF batch-binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product-specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96-well batch-binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins.

DEVELOPMENT OF MAMMALIAN CELL MANUFACTURING PROCESSES

Systematic downstream process development for purification of baccatin III with key performance indicators

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/7e5b1831f3db4118af08131cb11c856b.png" data-image="1"></div><div class="author-det"><h3 class="author-name">René Gantier</h3><h5 class="author-title">Director of Technology for Gene Therapy, Repligen</h5> René Gantier is the Director of Technology for Gene Therapy at Repligen. He has almost 20 years of experience developing production and purification processes for biotherapies. René holds a MSc in Cell Biology from the University of Nantes (France) and a PhD in Biochemistry from the University of Rouen (France). He started his career in the biotechnology industry as a scientist developing long-lasting therapeutic cytokines at Nautilus Biotech (France), before joining Pall Life Sciences as a downstream processing field application specialist, and then expanded his role to lead the Biotech Process R&amp;D team developing end-to-end upstream-downstream continuous bioprocessing solutions for mAbs and cell/gene therapies. In his current role at Repligen, he provides technology guidance to develop high productivity next generation gene therapy manufacturing processes. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/293d59b48641407ab2c636baca03ea7c.png" data-image="1"></div><div class="author-det"><h3 class="author-name">Ramsey Shanbaky</h3><h5 class="author-title focus-visible" data-focus-visible-added="">Bioprocess Applications Manager, C Technologies Inc. - a Repligen Company</h5> Ramsey Shanbaky is the Bioprocess Applications Manger for C Technologies, Inc. He spent the first 8 years of his career working in R&amp;D, Manufacturing and Product Management designing and building fiber optic products for high powered laser delivery in the medical device industry. In the first part of his tenure at C Technologies, he worked alongside his customers to implement the SoloVPE product with biopharmaceutical accounts primarily in the western region. For the last 5 years, he has been partnering with customers to implement the FlowVPE system for inline concentration measurements both in the process development areas and GMP manufacturing areas for bioprocessing. He holds an BSEE from Purdue University and multiple patents and applications. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Jay Headshot_200x200.png" data-image="1"></div><div class="author-det"><h3 class="author-name">Jay West</h3><h5 class="author-title">Principal Scientist at Bristol Myers Squibb</h5> Jay West is Principal Scientist at Bristol Myers Squibb in the Process Development Analytics group in Devens, MA. He obtained his BS in Chemistry from Rensselaer, MS in Chemistry from University at Albany, PhD in Biochemistry from Boston College, and performed postdoctoral research at Northeastern University. He has 8 years pharmaceutical industry experience, having worked at Regeneron Pharmaceuticals precious to BMS. His focus the past three years has been on Process Analytical Technology, particularly in downstream process development. </div></div></div>

Porous silicon structures were observed in laser-assisted etching in a TMAH solution. The porous structures significantly increase the active surface areas and therefore are of great interest to the pharmaceutical and fine chemicals industry, in the areas of high throughput drug discovery, reaction optimization and process development. Laser-assisted etching of silicon using a Nd :YAG laser with a TMAH solution is rarely studied but desirable as the solution is nontoxic. To the best of our knowledge, the porous structures directly produced this way have not been reported previously. In this paper, we discuss our experimental results and the possible mechanism of formation of the porous structures. A dummy micro-reactor was fabricated with selective porous surface structures to demonstrate its potential industrial applications.

Toward unbiased identification and comparative quantification of host cell protein impurities by automated iterative LC–MS/MS (HCP-AIMS) for therapeutic protein development

In the biopharmaceutical industry, process development has to overcome the paradox between increasing demands on product quality and aggressive overall development time lines. Streamlining development and scale‐up has become a requirement in order to cope with these constraints. Hydrophobic interaction chromatography (HIC) has gained popularity as an efficient tool in downstream process development, which represents an orthogonal method compared to other widely used chromatography principles. Thus, host cell protein (HCP) removal combined with the separation of product variants based on subtle differences in hydrophobicity are major strengths of this separation technique. The development and scale‐up of a preparative HIC step for a therapeutic protein for human use is described. Issues like the removal of product‐ and process‐related impurities have been addressed. The direct transferability of the results, from scale‐down optimization experiments via the pilot scale to the final production scale, clearly show the robustness and power of HIC in this process context.

Protein engineering enables Serratia marcescens nuclease A to hydrolyze nucleic acids under high-salt conditions.

In the post-genomic era, the demand for faster and more efficient protein production has increased, both in public laboratories and industry. In addition, with the expansion of protein sequences in databases, the range of possible enzymes of interest for a given application is also increasing. Faced with peer competition, budgetary, and time constraints, companies and laboratories must find ways to develop a robust manufacturing process for recombinant protein production. In this review, we explore high-throughput technologies for recombinant protein expression and present a holistic high-throughput process development strategy that spans from genes to proteins. We discuss the challenges that come with this task, the limitations of previous studies, and future research directions.