Biomedical Engineering and Bioengineering Commons^™

Bioreactor Scale-Up Harmonization - From Process Development To Manufacturing, Claudia Berdugo-Davis

Cell Culture Engineering XV

Successful bioprocess scale up requires an understanding of the microenvironment influenced by bioreactor parametric conditions and the effect on cellular metabolism and expression of a recombinant protein. While, significant understanding of best practices exists in scaling up bioprocesses, Contract Development and Manufacturing Organizations (CDMO) have a unique challenge in requiring a working knowledge of our bioreactor capabilities driven by a diverse client network and thus a need for a ready-to-implement scale up platform for a wide range of molecule types. An effective CDMO must be able to scale-up client platform processes (which differ for each client) as well as utilize …

Metabolomic Analysis For Scale-Down Model Improvement, Eric Garr

Cell Culture Engineering XV

The use of scale-down models is essential for the development, characterization and continuous improvement of commercial cell culture processes. A model demonstrated to be predictive of large-scale performance is advantageous as small-scale data can be used to support process investigations and changes, reduce costs and consequently accelerate new therapies to market. Aligning geometric aspect ratios and dimensionless engineering parameters improved the overall scale-down model representation for key process outputs such as pCO₂ accumulation and cell viability. Despite these changes, the revised scale-down model did not fully match the large-scale process lactate accumulation. Interestingly, it was demonstrated that pCO_{2 …}

Application Of Multivariate Data Analysis In The Monitoring And Control Of Mammalian Cell Processes, Stephen Goldrick, Richard Turner, Marcel Kuiper, Kenneth Lee, Rahul Pradhan, Suzanne Farid

Cell Culture Engineering XV

High throughput (HT) methodologies are increasingly being adopted for bioprocess development activities. However, often the large quantities of data generated from such studies as well as from historical batch records are not fully harnessed for their potential insights. Multivariate data analysis (MVDA) is a well-known technique that can reduce the dimensionality of large data sets and help generate useful correlations of typical process behaviour and determine root causes of process deviations.

This work focuses on the application of various MVDA techniques and data mining tools to investigate the impact of process variations on the critical quality attributes (CQAs) of mammalian …

Cardiac Remodeling And Dysfunction In Childhood Obesity: A Cardiovascular Magnetic Resonance Study, Linyuan Jing, Cassi M. Binkley, Jonathan D. Suever, Nivedita Umasankar, Christopher M. Haggerty, Jennifer Rich, Christopher D. Nevius, Gregory J. Wehner, Sean M. Hamlet, David K. Powell, Aurelia Radulescu, H. Lester Kirchner, Frederick H. Epstein, Brandon K. Fornwalt

Pediatrics Faculty Publications

Background: Obesity affects nearly one in five children and is associated with increased risk of premature death. Obesity-related heart disease contributes to premature death. We aimed to use cardiovascular magnetic resonance (CMR) to comprehensively characterize the changes in cardiac geometry and function in obese children.

Methods and results: Forty-one obese/overweight (age 12 ± 3 years, 56 % female) and 29 healthy weight children (age 14 ± 3 years, 41 % female) underwent CMR, including both standard cine imaging and displacement encoded imaging, for a complete assessment of left ventricular (LV) structure and function. After adjusting for age, LV mass index …

Elucidating Glycosylation Pattern Of Protein Produced In Mammalian Cells, Tung Sy Le

Cell Culture Engineering XV

The glycosylation pathway is a highly branched network. Although only a relatively small number of enzymes are involved in the pathway, a multitude of intermediate and terminal glycans can be formed. Each intermediate glycan in the network can be the substrate of subsequent glycosylation steps. In each step, there can be multiple enzymes acting on a single glycan due to overlapping substrate specificity. These enzymes tend to compete with each other to divert the network flux toward a particular branch. Alternation of their relative ratios can result in different glycan profiles. In mammals, such enzymes are distributed in a tissue-specific …

Reduction Of N-Glycan Profile Variation By Using Capacitance Probes For Optimized Process Control, Christopher Bro, Tom Kristensen, Michael Wahlers, An Zhang, Anne Tollstrup

Cell Culture Engineering XV

The glycan profile of therapeutic monoclonal antibodies frequently plays an important role in their biological function and pharmacokinetics. Therefore, improved control of the glycosylation profile of biopharmaceutical monoclonal antibodies has become an increased priority during late stage and commercial manufacturing of New Biological Entities as well as biosimilars. Two ways to obtain better control are through process parameter optimization and/or through addition of media supplements to the production reactor. Cell culture supplementation with mycophenolic acid is one method to efficiently manipulate N-glycan profiles of monoclonal antibodies, notably the level of fucosylation. We have observed at least for some CHO-based cell …

Impact Of Culture Conditions And Cell Age On Sequence Variant Levels In Monoclonal Antibody Biotherapeutics, Karin Anderson

Cell Culture Engineering XV

Sequence variants (SV) are protein products that contain unintended substitutions in the amino acid sequence. SV are one source of heterogeneity inherent to biological systems. They can arise from multiple sources within CHO cells including mutations in genomic DNA, transcription and/or translation errors, splice variants, and post translational modifications. This poster focuses on SV resulting from single base changes in a copy of the integrated exogenous gene in genomic DNA of single cell cloned CHO cell lines.

The increased sensitivity and selectivity of analytical instruments and methods has revealed across industry that a number of cloned CHO cell lines, including …

Continuous Suspension Cell Culture Monitoring In Bioreactors Using Quantitative Imaging, Ann D'Ambruoso, Jeremie Barbau, Denis Haumont

Cell Culture Engineering XV

Monitoring of suspension cell cultures often relies on sampling followed by a staining procedure. Estimations of cell count and cell viability are traditionally performed once a day using Trypan-Blue cell exclusion as a method of choice. Stained samples are destroyed afterwards creating toxic waste. Sampling a bioreactor and counting cells involve manual operations and weekend work is regularly needed.

Differential Digital Holographic Microscopy (DDHM) is a new quantitative imaging technique that allows cell counting as well as cell viability monitoring in a continuous, label-free set-up. No need for sampling (thus eliminating the risk of contamination), staining and waiting for results …

Impact Of Harvest Conditions On The Glycosylation Profile Of A Therapeutic Antibody, Raghavan Venkat

Cell Culture Engineering XV

It is essential to maintain the glycosylation profile of a protein therapeutic as it can affect its efficacy (ADCC or CDC), pharmacokinetics/pharmacodynamics, safety, and/or immunogenicity. Pilot scale experiments for an antibody manufacturing process, which used low pH during harvest step to improve the robustness of a subsequent downstream filtration step, revealed the presence of certain peaks that were not present in the harvested material without such low pH treatment. Analytical characterization of these peaks identified them to be associated with M6, M7, M8, and M9 high mannose glycoforms. Further experiments, which looked into the individual effects of the feed pump …

Real Time Prediction And Control Of Glycoform Profile Of Mammalian Cell Cultures Using In Silicoglycosylation Model Coupled With Extracellular Metabolites, Sha Sha, Seongkyu Yoon

Cell Culture Engineering XV

In silico mechanistic models for glycosylation are investigated for biotherapeutics development and production. The models systematically describe the transport-kinetics of glycosylation process and serve as a prediction framework for glycan profile. However, inputs for those models are generally limited to intracellular parameters, such as enzymatic activity and nucleotide sugar concentration. These intracellular metabolites are unfortunately inaccessible in process. To modulate those models for glycoform prediction in culture process, linkage between measurable culture variations and the intracellular metabolites change will be thoroughly investigated. The measurable metabolites and intracellular variables associated with in silico glycosylation model are integrated in the modeling. Despite …

A Biphasic Cultivation Strategy To Optimize Protein Expression And Minimize Aggregation Of The Final Product, Andreas Gili

Cell Culture Engineering XV

The effects of specific culture parameters are cell line-dependent and need to be investigated to allow optimization of productivity and product quality. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. In biphasic cultivation, culture conditions are changed after accumulating biomass during an initial growth phase to allow maximum recombinant protein expression in the production phase. Cultures were run in batch mode, and after an initial growth phase, cultivation temperature and pH were shifted to allow optimized protein production and quality. Using a design of experiments …

Seamless Scalability, Consistency And Quality Of Transient Protein Production In Cho Cells By Using Maxcyte Flow Electroporation Technology, Weili Wang, Rama Shivakumar, Pachai Natarajan, Angelia Viley, Madhusudan Peshwa, James Brady

Cell Culture Engineering XV

Recombinant protein production often suffers from process inconsistencies as cell cultures are scaled up. High levels of process consistency and scalability are important not only for GMP stage manufacturing, but they are also critical for early stage R&D studies since good predictivity of any scale production can shorten timelines and minimize costs. There are many parameters that influence consistency of production during scale up. These include agitation rates, dissolved oxygen levels and pH. Consistency of production following transient gene expression (TGE) is further impacted by process variabilities that are inherent to many transient transfection methodologies. The aim of this study …

Scale-Up And Scale-Down Challenges For A High Density Long-Term Perfusion Suspension Cell Culture In Large-Scale Single Use Bioreactors, Weichang Zhou, Hang Zhou

Cell Culture Engineering XV

As part of efforts to develop a continuous processing platform for biologics manufacturing using single use bioreactors, we have been focusing on development of several initial unit operations: high density perfusion suspension cell cultures and early product capture steps, in order to realize its potentials of being flexible, improving product quality and lowering costs. These initial unit operations require large volumes and represent the most important part of this processing platform. By integrating these steps into a continuous operation, we can deliver the largest benefits of this processing platform. In this presentation, we will discuss our efforts towards continuous biologics …

Changes In Product Quality – What Is Comparable “Enough” And What Is “Similar Enough?”, David Robinson

Cell Culture Engineering XV

Regulatory guidance documents clearly outline the requirements for demonstrating analytical comparability to support process changes and analytical similarity to support the approval of biosimilars. These include demonstration that process changes do not produce differences in product characteristics that might lead to an adverse impact on the safety or efficacy of the product¹. For biosimilar products, a stepwise approach is required that starts with sponsors demonstrating that the product characteristics of the proposed biosimilar are either highly similar to that of the originator reference product or that any observed differences can be demonstrated to not impact the ability to …

Strategies For Optimizing A Cell Culture Platform To Achieve High Recombinant Protein Titer Without Impacting Product Quality, Natarajan Vijayasankaran, Robin Gruver, Steven Meier

Cell Culture Engineering XV

Recent advances in cell line development and in the design of culture media have significantly increased both the volumetric and specific productivity of fed batch processes, which is our standard platform technology for synthesis of therapeutic proteins. Cell culture processes are generally simplified when needed as a robust platform technology to meet the imperatives of speedy timelines for early process development and ease of manufacturing. However, both culture media and feeding strategies need to be customized for specific cell lines when there is a need to achieve higher titer due to higher product demand. This presentation will focus on the …

Demonstrating Process Performance Comparability Of The Keytruda® Upstream Process After Transfer And Scale-Up To Different Manufacturing Sites, Jurgen Van De Lagermaat

Cell Culture Engineering XV

The Keytruda® drug substance manufacturing process was transferred and scaled up from a donor manufacturing site (12,500 L bioreactor WV) to two additional recipient sites (12,000 L and 22,000 L bioreactor WV) to further expand drug substance production capacity of Merck’s novel monoclonal antibody against PD-1. Process facility fit into the recipient manufacturing facilities was required due to differences in equipment and operation as compared to the donor site and therefore upstream process performance comparability needed to be demonstrated (regulatory requirement). A three-level comparability approach was used, whereby upstream intermediate product quality (Level 1), cell culture performance KPAs (Level 2) …

Critical Process Parameter Identification Using The Ambr15(Tm) For Process Characterization, Matthew Zustiak, Michael Bennett, Stephanie Chakravarty, Matt Caple

Cell Culture Engineering XV

Process characterization is a critical phase in the development of a commercial process for biotherapeutic production. Knowing the critical quality attributes of your molecule prior to beginning process development and/or characterization is imperative when using a quality by design (QbD) approach. Here we use a (QbD) approach for the characterization of a fed-batch process using an NSO cell line to express an IgG. For this molecule, the glycosylation profile, and in particular, the total fucosylation was identified as a critical quality attribute. After performing a primary hazard analysis, several process inputs were determined to potentially have an impact on this …

Targeted Sequencing For Comprehensive Genetic Characterization Of A Recombinant Cho Cell Line, Brian Mickus

Cell Culture Engineering XV

Next generation sequencing has revolutionized genomics, catalyzing an era of personally-tailored therapeutics with enhanced efficacy and safety profiles. This technology also holds great promise for bioprocess and cell line development. The combination of Targeted Locus Amplification (TLA) and next generation sequencing is an emerging approach for the characterization of transgene integration and genetic stability for recombinant cell lines. TLA [de Vree et al., Nature Biotechnology 32, 1019-1025 (2014)] is based on the crosslinking of physically proximal sequences and enables the targeted complete amplification and sequencing of transgenes and their integration sites with greatly increased sequence coverage and depth. Information …

Controller Design For Effective Glycosylation Control In Mabs, Devesh Radhakrishnan

Cell Culture Engineering XV

Monoclonal antibodies (mAbs), a class of commercially viable biotherapeutics, undergo post-translational modifications when expressed in mammalian cell lines, resulting in structural and pharmacological changes in the protein. One such post translational modification is N-linked glycosylation, where the non-template driven enzymatic attachment of different sugar moieties (glycans) to the mAb can alter the product quality of the mAb, compromising the efficacy and safety of the drug product. While significant research effort has been devoted to developing techniques for characterizing and monitoring the glycosylation pattern in mAbs, a robust technique for controlling the glycan distribution and ensuring consistent glycosylation does not currently …

Investigating The Impact Of Process Optimization On Productivity, Product Quality, Cell Metabolism, And Intracellular Environment, Shailendra Singh

Cell Culture Engineering XV

One of the key goals in process development for monoclonal antibodies is to improve productivity and product quality as needed. The early stage cell culture process developed for an antibody had titers averaging 4 g/L and variable aggregates levels within cell culture. Through process optimization work, involving changes in media formulations, feeding strategy, and process parameters, the final optimized process achieved industry leading titers (greater than 10 g/L) with consistently lower aggregate levels. To understand the impact of process optimization on the CHO cell metabolism and intracellular environment we evaluated 4 conditions: Early stage cell culture process, 2 intermediate processes, …