Engineering Commons^™

Integrated Continuous Bioprocessing - A Gold Mine For Cell Culture Process Understanding?, Mats Akesson, Martin Heitmann, Ditte Skibstrup, Peter Tiainen

Cell Culture Engineering XV

We have developed an integrated continuous processing framework for end-to-end production of complex fragile proteins based on perfusion cultivation and automated multi-step purification [1,2]. Upstream, the integrated system consists of a stirred tank bioreactor with an ATF cell retention system. The clarified harvest directly enters an off-the-shelf ÄKTA chromatography system converted into a continuous purification unit. Two alternating capture columns precede a multi-step purification train with full flexibility and control of individual columns.

The integrated set-up enables compact automated bench-top factories converting cell culture media to purified protein in an efficient manner without intermediate storage. It provides monitoring of the …

Identification Of Cell Culture Levers To Lower Trisulfide Modifications In Monoclonal Antibodies Produced In Cho Cell Culture, Masaru Shiratori, Jessica Wuu, Martin Gawlitzek

Cell Culture Engineering XV

Trisulfide bond formations are a commonly observed modification of monoclonal antibodies produced in cell culture processes. Protein trisulfide bonds are post-translational modifications in which an extra sulfur atom is bound between two cysteines involved in disulfide bonding. This modification has been detected in both recombinant and endogenous plasma-derived IgGs. Inconsistent levels of trisulfides were identified as a source of variability observed during the conjugation process for antibody-drug conjugates (ADCs). To understand the root cause for variable trisulfide levels observed during process development for different molecule projects, cell culture studies aimed at identifying process levers to minimize trisulfide formation were conducted. …

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 …

Evaluation Of Public Genome References For Rna-Seq Data Analysis In Chinese Hamster Ovary Cells, Huong Le, Chun Chen, Chetan Goudar

Cell Culture Engineering XV

Recent advances in next-generation sequencing technologies have led to the emergence of RNA-Seq as the preferred transcriptomic tool in the biopharmaceutical industry. However, an important challenge with deploying RNA-Seq to characterize CHO cells is the absence of a common genomic reference for this species. In most published CHO cell transcriptomic studies, RNA-Seq reads are assembled into de novo genomic references which were subsequently used for mapping of the constituent reads. Such an approach makes it difficult to compare results across studies due to the incomplete and non-universal nature of those assemblies. To address this challenge, we evaluated two publicly available …

Application Of Online Co2 Monitoring To Enable A Better Understanding Of Cell Culture Performance Variation Between Gmp-Scale And Scaled-Down Bioreactors, Ting Kuo Huang, Jacob Mauger, James Dvornicky

Cell Culture Engineering XV

Dissolved carbon dioxide (dCO₂) or partial pressure of carbon dioxide (pCO₂) is an important process parameter that may impact process performance (product titer and viability) and product quality attributes (such as glycosylation) during mammalian cell culture process in a bioreactor. The impact of altered level of pCO₂ on the cell culture process may manifest itself upon process scale-up if the CO₂ removal rate is not consistent between the development scale and the manufacturing scale. The pCO₂ level during cell culture process is normally determined through offline measurement; however, the offset between online and …

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 …

Monitoring Live Stem Cells In Suspension And Attached To Carriers In Conventional And Single Use Bioreactors, John Carvell, Aditya Bhat

Cell Culture Engineering XV

Embryonic stem cells are promising cell sources for regenerative medicine and organ replacement after disease or injury. Traditional single use bioreactors such as the Sartorius Biostat Rocking Motion bag are being used for growing up these cells at the larger scale and a number of companies are developing fully automated, closed bioreactor systems specifically for growth of stem cells for clinical applications eg Xpand (Holland), Pall(USA).

The stem cells in the bioreactors need to be monitored and controlled for both product quality and to satisfyGMPrequirements and one of the most important parameters is the concentration of live cells. On-line monitoring …

Influence Of Cultivation Parameters Or Supplement On Product Qualities And Culture Performances During Perfusion, Kyu Kim, Sam Seol, Soo Ryu, Sang Park, Sun Kim, Seung Lee

Cell Culture Engineering XV

Perfusion processes have been developed with technological advances in single-use bioreactor and cell retention device. Perfusion has advantages such as high cell density culture in compact facilities and media change or culture parameter shifts for protein production with desired qualities. Single use bioreactor (200L or 1000L) with ATF System was used for the clinical production. For the process development, the scale-down model was established with lab-scale (2L) bioreactor with ATF2.

A recombinant CHO cell line producing a fusion protein was cultivated using in-house serum-free media. Influence of insulin (0~3mg/L) on qualities was investigated in the established perfusion process using in-house …

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 …

Cho-Specific Recombinant Protein Glycosylation Reaction Network, Benjamin Kremkow, Kelvin Lee

Cell Culture Engineering XV

Protein glycosylation is one of the most important product quality attributes and impacts efficacy, half-life, and immunogenicity. Previous glycosylation models effectively simulated the key parts of the N-glycosylation pathway. Building upon these prior efforts as well as recent CHO-K1 and Chinese hamster (CH) genome sequencing efforts, we share a new model for CHO- and CH- glycosylation. The model contains all N-glycosylation-related genes and all of the metabolic genes associated with central carbon metabolism, nucleotide sugar precursor synthesis, and nucleotide sugar transport annotated from the CHO-K1 and CH genomes. The model predicts both intracellular and secreted glycans for both mAb and …

Poly-Pathway Model Approach: Simulation Of Multiple Metabolic States, Erika Hagrot

Cell Culture Engineering XV

Animal cell lines have a complex and flexible metabolism and can display varied metabolic behavior depending on the culture conditions. A model that simulate and predict these variations would be a precious tool in the development of media, feeds or processes. However, in order to function as a predictive tool such a model has to describe the multiple metabolic states that can occur for a variety of conditions. This leads to the challenge of identifying a flexible model structure of relevant metabolic pathways and kinetics. To address this challenge, we have introduced the poly-pathway model approach aiming at capturing …

Improving The Productivity And Product Quality Of Antibodies Expressed From A Cho Transient System, Athena Wong

Cell Culture Engineering XV

Transient transfection of CHO cells is a widely used tool to express recombinant proteins for non-GLP preclinical studies due to the system’s ability to rapidly produce proteins of similar product quality to CHO stable cell lines. Our high throughput CHO transient transfection process allows for efficient generation of milligram to multi-gram amounts of protein from small scale automated 96-deep well plates and tubespins to large scale 100L bioreactors. In this work, we present a case study describing the influence of media components on PEI-mediated CHO transfections. Given the dependence of PEI transfection on electrostatic interactions for formation and cell internalization …

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 …

Lensless Imaging For Continuous Cho Viable Cell Density Monitoring In Bioreactors, Geoffrey Esteban, Martin Pisaneschi, Jeremie Cubeta, David Sergeant

Cell Culture Engineering XV

During suspension cell cultures in bioreactors, traditional measure for cell count and cell viability still rely on sampling and staining protocol where the Trypan Blue exclusion method is performed once a day. While automatic cell counters have reduced the statistical error of the original manual method, sampling the bioreactor is a risk for contamination and is prohibiting the use of such method for process control as the sampled volume becomes significant. Lensless Imaging Technology is a new breakthrough method for accurately and precisely determine cell concentration and viability without staining. This technique has the unique capability of acquiring microscopy images …

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, …

Enhancing Enveloped Viral Particles Production By Targeted Supplementation Design: Releasing Bottlenecks In Ic-Bevs, Francisca Monteiro

Cell Culture Engineering XV

The increasing demand of Insect Cell-Baculovirus Expression Vector System (IC-BEVS) based biopharmaceuticals raises the interest in developing high-titer production processes. Previously, we addressed the impact of the baculovirus infection on the physiology of High Five and Sf9 host cell lines, stressing out key cellular features that support higher productivities. This information was applied to design tailored supplementation schemes to boost IC-BEVS production yields of three targets with increasing complexity: recombinant influenza neuraminidase (rNeur); enveloped influenza VLPs (Inf-VLP) and the baculovirus itself (BV). Higher rNeur productivities were achieved when supplementing High Five cultures with cholesterol. For Sf9 cells, GSH, antioxidants combined …