Biomedical Engineering and Bioengineering Commons^™

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 …

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 …