Biomedical Engineering and Bioengineering Commons^™

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 …

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 …