Biomedical Engineering and Bioengineering Commons^™

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, …

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 …

Improving The Metabolic Efficiency Of Mammalian Cells And Its Impact On Glycoproteins Quality, Eric Karengera, Gregory De Crescenzo, Yves Durocher, Olivier Henry

Cell Culture Engineering XV

Glycosylation is a critical quality attribute for recombinant therapeutic proteins, which can be impacted by a number of process conditions, including waste metabolite accumulation. While fed-batch strategies that consist in substituting or controlling the main substrates at low concentrations have proven generally effective at improving protein titer, they can also adversely affect product glycosylation. Metabolic engineering strategies aiming at reducing by-product formation may thus be beneficial for ensuring product quality consistency. In this work, we have specifically investigated the impact of PYC2 overexpression on the quality of a recombinant glycoprotein of therapeutic interest, the interferon α2b (IFNα2b) that has one …

Monitoring Live Biomass In Disposable Bioreactors In Range Of Vessel Formats, Dan Kopec

Cell Culture Engineering XV

The detection of biomass is one of the most requested parameters in industrial cell cultivation. The knowledge of the biomass progress during cultivation gives deeper process knowledge and control capabilities in order to define harvest or infection points. Offline methods like visual cell counting or semi-automated systems still dominate the biomass detection in industrial cell cultivation. But these offline methods based on taking a representative sample cannot practically monitor the process continuously.

The radio frequency (RF) impedance method for online in-situ detection of viable biomass has already become well established in biopharmaceutical applications using traditional reusable stainless steel equipment. On …

Use Of An Automated, Integrated Laboratory Environment To Enable Predictive Modeling Approaches For Identifying Critical Process Parameters And Controlling Key Quality Attributes, Brandon Downey, John Schmitt, Jeffrey Breit, Brian Russell, Justin Beller, Liz Herman, Anthony Quach, David Lyon

Cell Culture Engineering XV

An essential part of ensuring a high quality medicine is being able to reliably control Critical Quality Attributes (CQA’s). In the cell culture process, bioreactor conditions, feeds, cell state are some of the many variables that affect CQA’s. Out of this very large set of possible variables, the small subset of these (i.e., critical process parameters, or CPP’s) that have a large effect on the CQA’s must be identified and understood such that those CPP’s can be controlled to ensure quality product. Here, we demonstrate the use of predictive modeling techniques to supplement experimental bioreactor studies when defining critical process …

Advancement Of Cell Culture Process Understanding And Control Through Real-Time Multivariate Process Monitoring, Use Of Statistical Process Modes And Deployment Of Process Analytical Technologies, Patrick Gammell, Thomas Mistretta, Cenk Undey, Eric Kwei, Tonny Wang, Greg Naugle

Cell Culture Engineering XV

Commercial bioprocess manufacturing facilities generate considerable amounts of process data which are routinely used for process monitoring. Through the analysis of batch over batch trends it is possible to ensure that the process remains in a state of continuous statistical control. Real time process data can also be used to drive rapid root cause analysis and to enable timely interventions to prevent performance parameter excursions. Finally, these process data also provide significant opportunities to further understand the interaction between operating parameters, process equipment and raw materials to enable improved process control and optimization.

To use the available process data effectively, …

Agent-Based Model Predictive Framework To Control Cell Culture Bioreactors, Elif Bayrak, Tony Wang, Myra Coufai, Ali Cinar, Cenk Undey

Cell Culture Engineering XV

Bioprocesses require unique operational conditions and highly specialized process knowledge to obtain consistent product quality and productivity. Optimization and control of these processes are challenging due to the nonlinearities and uncertainties involved, and cell-bioreactor interactions are poorly understood. Automated control of bioreactors using model predictive control (MPC) technologies is less common as translating complex process specific interactions to linear models is challenging. Accurate models of the process are needed for MPC to succeed. Due to the complexity and heterogeneity involved in the culture environment, conventional mechanistic modeling efforts are often incomplete for describing the interactions of cell physiology and environmental …

Cho Cell Culture Process Impacts Monoclonal Antibody Trisulfide Modification And Sulfhydryl-Drug Conjugation, Michael Hippach

Cell Culture Engineering XV

During the production of monoclonal antibodies using a CHO cell culture process, important parameters are generally controlled by a feedback mechanism (PID) in order to ensure consistency in both productivity and product quality. These parameters typically include pH, dissolved oxygen, and temperature. While most of these parameters are controlled very well within their specific dead band range, dissolved oxygen at times can be a little more difficult to control. Oscillations in dissolved oxygen concentration are not uncommon and these fluctuations can sometime be exacerbated when using an aeration strategy with a high mass transfer rate.

Recently, we observed a cell …

A Stochastic Model To Study Genetic And Metabolic Effects On N-Linked Protein Glycosylation, Phillip Spahn, Anders Hansen, Henning Hansen, Johnny Arnsdorf, Helene Kildegaard, Nathan Lewis

Cell Culture Engineering XV

Glycosylation is a vital processing step for a large number of cellular proteins as it critically affects protein stability and solubility as well as protein-protein interactions. As a consequence, glycosylation is a major quality attribute of recombinant proteins in biopharmaceutical applications. However, since glycosylation does not follow a template, but instead involves a complex interplay of various influencing factors in the Golgi, tailoring glycosylation towards certain desired attributes is challenging and usually requires trial-and-error experimentation. Computational modeling offers an intriguing option to understand and rationally engineer the complex reaction network underlying glycosylation. Here we present a computational model that describes …

Improved Scale-Down Model Development Case Study For Raw Materials Screening, Angela Au, Davanna Marks, Hugh Graham

Cell Culture Engineering XV

The lack of an adequate scale-down model for the cell culture stages of legacy processes is an ongoing issue across the industry. This presentation will describe an existing scale-down model, the modifications made to it, and an example of the utility of the new model. The scale-down model for Process X was originally developed based on power per unit volume, with minimal modification to accommodate other input parameters. This scale-down model performed significantly differently from the manufacturing-scale process with respect to cell growth, metabolites, and productivity. For example, the productivity in the scale-down model was 3 to 4 times higher …

Life In The Fast Lane: Developing And Commercializing Keytruda®, A Novel Breakthrough Therapy Designation Oncology Therapy, In Three Years From First Patient Dosed To Us Approval, Gargi Maheshwaru, Athena Nagi, Ronald Smulders, Fergal O´Donovan, Kimberly May, Tamas Blandi, Marc Bastiaansen

Cell Culture Engineering XV

In order to accelerate the availability of novel potentially life-saving therapies, the FDA has developed multiple programs, including recently assigning breakthrough therapy designation to promising candidates in development. At the time of breakthrough therapy designation, Merck had only manufactured supplies at a small clinical scale facility. In order to meet forecasts for projected commercial and clinical demand and to ensure uninterrupted supply for Keytruda®, a novel monoclonal

antibody against PD-1, Merck commercialized two drug substance facilities in parallel. Through multiple interactions with the FDA, both facilities were licensed, one that was the initial clinical supply site, and, a …

A Carbon Dioxide Stripping Model For Mammalian Cell Culture In Manufacturing Scale Bioreactors, Zizhuo Xing, Nicholas Abuabsi, Michael Borys, Zheng Jian

Cell Culture Engineering XV

Achieving adequate CO₂ stripping rates in large scale bioreactors is an important consideration during the scale up of animal cell cultures to large scale bioreactors due to the use of relatively low power input and gas sparging rates. It has previously been reported that cell growth, productivity, and product quality attributes such as glycosylation can be significantly impacted when cells are exposed to high CO₂ environments. CO₂ stripping models that depend on the CO₂ mass transfer coefficient have been applied to simulate CO₂ profiles in cell cultures using varied sparger types, reagents for pH adjustment, …

Softsensors: New Approach For Process Monitoring Cell Growth In Small Scale Fermentation Systems, Wolfgang Paul, Daniel Christofori, Bernhard Keil, Arthur Mohr

Cell Culture Engineering XV

Process development and in particular the use of high throughput systems required sampling for controlling. One of the most important parameter is the cell growth, but sampling, sample dilution and analyzing is time consuming and generates high efforts in the case of high throughput fermentation systems. Sampling allows also only a look in the culture status at a certain time point, the information between two sample points is missing. Therefore we develop a new softsensor, which takes online signals of the bioreactor, which are correlated to cell growth to estimate the cell growth. The new approach based on multiple linear …

Genetic Engineering Of Mmv Virus Resistance Into Cho Cells: Probing The Role Of Various Cho Sialyltransferases In Virus Binding And Internalization, Kevin Kayser, Joaquina Mascarenhas, Trissa Borgschulte, David Onions

Cell Culture Engineering XV

Contamination by the parvovirus Mouse Minute Virus (MMV) remains a continuing challenge in CHO biopharmaceutical production processes. As part of developing a risk mitigation strategy against such events our group has evaluated the genetic engineering of Chinese Hamster Ovary (CHO) cell lines to create a new host cell line that would be resistant to MMV infection e.g by inhibiting viral attachment to a cell surface receptor. While the exact functional receptor for MMV binding to CHO cell surface is unknown, previous work in our group has validated the role of sialic acid on the cell surface as important for cell …

Bioreactor Perfusion Via Single-Use Centrifugation Has Fewer Product Quality Implications Than Tangential Flow Filtration, Rustin Shenkman, Douglas Gunzelmann, Ting Yang, Matthew Traylor, Kenneth Manning

Cell Culture Engineering XV

During development of a perfusion cell culture process for the production of a therapeutic protein, tangential flow filtration (TFF) technology was evaluated for cell retention in addition to Shire’s platform single-use centrifugation technology. Unlike centrifugation TFF is based on a microfiltration membrane and thus has the potential to partially retain many biological compounds, especially when exposed to extracellular matrix proteins and antifoam emulsions (Routledge, 2012; Wu et al., 2011). Retention increases the mean residence time of product at process temperature. In the case of heat-labile molecules longer exposure to bioreactor culture temperature may correlate with changes in quality attributes.

Therapeutic …

Polysorbate 20 And 80 Degradation By Group Xv Lysosomal Phospholipase A2 Isomer X1 In Monoclonal Antibody Formulations, Troii Hall, Stephanie Sandefur, Christopher Frye, Lihua Huang

Cell Culture Engineering XV

Decreases in the intact polysorbate (PS-20 and PS-80) content were observed while evaluating the long-term storage stability of CHO derived, purified monoclonal antibodies. It was determined that polysorbate had been enzymatically degraded; therefore, studies were performed to identify and characterize the protein(s) responsible. Polysorbate degrading activity was enriched from CHO media leading to the identification of Group XV phospholipase A₂ Isomer X1 (PLA2) by LC-MS/MS. Recombinant phospholipase A₂ was expressed, purified and conformational integrity confirmed against a phosphatidylcholine substrate. Incubation of recombinantly produced PLA2 with PS-20 and PS-80 resulted in hydrolysis of both monoester and higher order PS-20 …