Biomedical Engineering and Bioengineering Commons^™

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 …

Development Of Bioreactor Auto-Sampling System For Real Time Product Quality Monitoring In Mammalian Cell Culture, Meena George

Cell Culture Engineering XV

Product quality attributes such as glycosylation, size and charge variants are critical characteristics for the development of biotherapeutics in mammalian cell culture processes in order to target a desired product profile. In an attempt to characterize the entire time-course of a fed-batch culture under different media conditions, a bioreactor autosampling device integrated with cell removal and sample retention systems and at-line instruments such as Patrol UPLC was developed to rapidly analyze the cell culture IPC as well as product quality changes in real time. This information can be rapidly translated for better process understanding and process control to enable optimization …

Accelerate Cell Culture Development Using The Modular Automated Sampling Technology (Masttm) Platform In An Integrated Bioprocess Lab Environment, Clinton Pepper

Cell Culture Engineering XV

Biopharmaceutical companies are continually striving to fundamentally understand and optimize cellular performance within their bioreactors. While on-line process analytical technology (PAT) tools like dielectric and Raman spectroscopy are helping to provide insight into cell processes that impact titer and antibody quality, the integration of these data with off-line measurements such as cell density, viability, titer and glycosylation remains an elusive goal.

Bend Research Inc., in collaboration with Lilly, Pfizer and other major biopharmaceutical companies, is advancing the Modular Automated Sampling Technology (MAST^TM) platform and integrating this system with automated analytical and data retrieval systems. This approach allows application …

Kinetic Physico-Chemical Model For Cell Culture Processes – Applications And Opportunities, Natraj Ram, Kartik Subramanian, John Ponzo

Cell Culture Engineering XV

Cell culture processes for production of recombinant proteins can be modeled to provide critical insights into the interrelationship between several parameters that impact the process performance and control. For the first time, we describe a model that incorporates pH control in combination with gas transfer to provide a more complete description of the physicochemical processes that occur during the entire course of the cell culture process. The model includes multi-component chemical equilibria involving carbonate, lactate and sodium hydroxide coupled with electroneutrality for calculation of pH. Further, the pH feedback control has been incorporated depending on levels of carbonate and lactate …

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 …

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 …

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 …

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 …

Evaluation Of Product Antibody (Mab) Heterogeneity In Non-Clonal Cell Pools For Early Pre-Clinical Development, Gabi Tremml

Cell Culture Engineering XV

During early stage of pre-clinical biologics development, grams of product antibody is needed for process development, formulation development, and analytical assay development. To accelerate the pre-clinical timeline, it's a common practice to use master wells (non-clonal stable cell pools) to generate development material. The advantage of using master wells is that the product antibody is generated from the same host cell line and expression vector in an earlier and shorter time. Thus, the purified product antibody can be representative to the final therapeutic product. However, the non-clonal nature of the cell pools can give rise to potential risk of heterogeneity …

Adjusting Product Quality Attributes Of A Biosimilar Using Process Levers, Brett Belognia

Cell Culture Engineering XV

The development of a biosimilar introduces many unique challenges compared to that of a novel drug. The primary consideration in developing a biosimiliar is that the product quality of the protein of interest be “highly similar” to that of the branded product. Product quality attributes are defined by the manufacturing cell line and the manufacturing process. The ability to manipulate the manufacturing process is key to obtaining a highly similar product. This poster will focus on the development of biosimilars and provide examples of how the product quality can be manipulated with process levers to achieve similarity with that of …

Investigating The Reverse Warburg Effect: How High Extracellular Lactate Alters Breast Cancer Metabolism, Daniel Odenwelder, Sarah Harcum, Arthur Brodksy

Cell Culture Engineering XV

Metabolism plays a critical role in the progression of cancer malignancy, with nutrient source flexibility allowing cancer cells to outcompete normal cells and survive harsh environmental conditions. Lactate accumulation in poorly vascularized tumors has predominately been considered a waste product resulting from the Warburg effect; however, recent studies have shown that lactate can additionally serve as a metabolic substrate for some cancer cells, a process referred to as the Reverse Warburg effect. Lactate accumulation is also associated with cancer stem cell-like gene expression, drug-resistance, metastasis, and poor prognosis in breast cancer patients. Unfortunately, little is known about the role of …

Advanced Process Monitoring And Feedback Control To Enhance Cell Culture Process Production And Robustness, An Zhang, Valerie Tsang, Brandon Moore, Vivian Shen, Yao Ming Huang, Rashmi Kshirsagar

Cell Culture Engineering XV

It is common practice in biotherapeutic manufacturing to define a fixed-volume feed strategy for nutrient feeds based on historical cell demand. However, once the feed volumes are defined, they are inflexible to batch-to-batch variations in cell growth and physiology and can lead to inconsistent productivity and product quality. In an effort to control critical quality attributes and to apply Process Analytical Technology (PAT), we demonstrated three different and novel approaches for implementing online monitoring and feedback control to improve the performance and/or robustness of cell culture processes. First, we describe the first reported fed-batch process utilizing online amino acid measurements …

Implementation Of A Recirculating Tff N-1 Perfusion System At Manufacturing Scale: Conquering Process Hurdles And Scaling Challenges, Alex Doane, Weiwei Hu, William Yang, Kelly Wiltberger, Haofan Peng

Cell Culture Engineering XV

In order to increase the output of a traditional large scale stainless steel fed-batch manufacturing facility without major engineering work, we investigated the implementation of N-1 perfusion technology to feed a more intensified fed-batch production process. Perfusion N-1 enables cells to grow to high densities in the seed train bioreactor. By shifting cell growth from production to seed train, we can increase seed train occupancy per batch and reduce production bioreactor occupancy per batch, which improves facility throughput by bringing the run duration ratio of N-1/N closer to unity. Instead of using the popular ATF technology, an in-house TFF system …

Efforts To Reduce Impact Of Media Variability On Product Quality For A Commercial Perfusion Process, Nirel Rillera, Kevin Yeh, Benjamin Youn, Jerome Acuna, John Henstrand, Sean Forestell

Cell Culture Engineering XV

Strict control of raw materials used in bioprocesses is necessary to ensure consistent product quality. However, use of poorly characterized complex components makes achieving such strict control difficult. A strong correlation between product quality variability among bulk drug substance lots and changes in a complex medium component was observed in a commercial perfusion process. This correlation was subsequently confirmed experimentally at both large and small scales. A multifaceted strategy was applied to investigate and mitigate the impact of the media variability on product quality, which included (1) studies in small scale perfusion bioreactors; (2) development of a simple cell based …

To Clone Or Not To Clone? – Wrong Question! An Investigation On Genome Heterogeneity And Stability And On What Controls Cell Behavior, Nicole Borth

Cell Culture Engineering XV

The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. Potential causes include i) the high number of chromosomal rearrangements, including variation in chromosome numbers observed in CHO or any other rapidly growing cell line; ii) mutations including SNPs or InDels that change the activity or function of enzymes; iii) epigenetic changes that alter the gene expression pattern of a cell without impacting the genome sequence itself. To understand the relative contribution of these towards phenotype …

A Systematic Development Approach To Optimize And Control Biopharmaceutical Product Quality, Min Zhang, Leisha Youngblood, Carrie Weymer, Gian Arias, Tim Hill

Cell Culture Engineering XV

The complexity of recombinant protein manufacturing processes, including expression systems (i.e. host cell line, expression vector, cell line engineering process), cell culture process conditions, and choice of media and feeds, present significant challenges to achieve desired product quality of biopharmaceuticals. Significantly more challenging is to develop processes for biosimilar proteins, which must match product quality attributes of the originator molecule, show comparable in-vitro biological activity, and have no clinically meaningful differences as compared to innovator products. To address these challenges, and provide appropriate, flexible, and quick solutions to govern product quality control during manufacturing, Fujifilm Diosynth Biotechnologies (FDB) has developed …