Engineering Commons^™

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 …

Determining The Most Efficient Type Of Growth Light For An Aquaponics System Using Yellow Lantern Chilies (Capsicum Chinense), Travis Mceachern

Undergraduate Theses

Abstract

Aquaponics, a type of urban agriculture, shows potential to produce large amounts of food with little water and land requirements. Thus, aquaponics could help address the issue of feeding the growing worldwide population. However, multiple challenges, both technical and economical, are associated with aquaponics, making large-scale implementation of these systems difficult – these systems can require tremendous amounts of energy. This study sought to determine the most efficient types grow lights in aquaponics systems by comparing the growth rates of yellow lantern chilies (Capsicum chinense) when grown under four different types of growth lights: light-emitting diode (LED), …

The Utilization Of Mobile Technology For Crime Scene Investigation In The San Francisco Bay Area, Marc Logrande

Themis: Research Journal of Justice Studies and Forensic Science

The research presented aims to explore factors affecting the decision to adopt a mobile crime scene investigation application in police departments throughout the San Francisco Bay Area. To accomplish this goal, the mobile technology acceptance model was used in designing a survey for data collection. This model utilizes four categories to interpret the factors that influence a police officer’s decision to accept or reject mobile technologies: performance, security and reliability, management style, and cognitive acceptance. Nine police departments were sampled through a series of in-person and over-the-phone interviews to obtain data regarding factors affecting the adoption of a mobile crime …

Effect Of The Inherent Eccentricity In Single-Lap Direct-Shear Tests Of Pbo Frcm-Concrete Joints, Tommaso D'Antino, Lesley Sneed, Christian Carloni, Carlo Pellegrino

Civil, Architectural and Environmental Engineering Faculty Research & Creative Works

Investigation of the bond between fiber reinforced composites and the substrates onto which they are applied is of critical importance to understand their failure mechanisms. The bond behavior can be studied using different experimental test set-ups; the most commonly used are the single-lap and double-lap direct-shear tests. Although single-lap shear tests are simpler to carry out than double-lap shear tests, the presence of an eccentricity between the pulling and restraining forces leads to a mixed mode fracture process at the interface, which may influence the results. This study investigates the eccentricity effect on the bond behavior of fiber reinforced cementitious …

Building Quality Novel Formats And Development Processes, Steven Lang

Cell Culture Engineering XV

There has been an explosion of interest in bispecific or multi-specific bio therapeutic formats. Monospecific antibodies remain the foundation for most bispecific formats. However, monospecific strategies cannot be directly translated into the discovery and development of bispecific antibodies. Bispecifics present unique challenges especially for efficient and consistent assembly/production processes and analytical methods to ensure high quality drug products. Improvements in selection of bispecific combinations/architecture, yield and efficiency and common analytical issues and resolutions will be discussed. Applying improved methods learned from Fab-arm exchange bispecifics and alternative scaffold-antibody fusions, we have established a foundation of knowledge that enables accelerated development

Generation Of A Stable Pluripotent Cell Line From Chinese Hamster Embryonic Fibroblasts, Haiyun Pei, Dong Seong Cho

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells are aneuploidy and exhibit a high degree of genomic instability. Many studies having a cell line with a stable karyotype and genome structure is highly desirable. We have derived a stable cell line from Chinese hamster embryonic fibroblasts by transduction of mouse transcription factors M3O (a fusion gene which is chimeric of Oct4 and the effective transactivation domain of MyoD), Sox2, Klf4, and n-Myc using a lentivirus vector. The cells showed morphology of typical murine pluripotent stem cells, stained positively with alkaline phosphatase, expressed endogenous Sox2, Nanog and surface antigen SSEA1, resembling mouse embryonic stem …

Assessment Of Genomic Instability In Chinese Hamster Ovary (Cho) Cells, Sabine Vcelar, Martina Baumann, Michael Melcher, Nicole Borth

Cell Culture Engineering XV

CHO cells are the number one production system for therapeutic proteins due to their ease of handling, their fast growth in suspension culture and their capability to perform complex protein folding and human-like post-translational modifications. This flexibility is in part due to, but at the same time set off by the frequent occurrence of chromosomal rearrangements and other genomic variants, which influences individual cell line performance and the stability of industrial producer cell lines, resulting in prolonged screening phases in order to isolate cells with sufficiently stable properties. Furthermore producer cell properties are also frequently lost again over time and …

A Novel Bispecific Antibody For Her2+ Breast Cancer: The Beat Gbr 1302, Pierre Moretti, Julie Macoin, Amelie Croset, Darko Skregro, Romain Ollier, Stanislas Blein, Martin Bertschinger, Samuel Hou, Jonathan Back

Cell Culture Engineering XV

While the idea of bispecific drugs was brought up over 30 years ago, the development of formats mature enough for the clinic remained for a long time a challenge. The whole field has been hampered by major problems of manufacturability (e.g. product purity and yields) and immunogenicity. With the recent arrival of new bispecific formats, either as antibody–like molecules (containing an Fc) or scFv fragments, at least 18 bispecific molecules have entered clinical trials showing very promising results. The BEAT® format has been developed as bispecific antibodies maintaining the pharmacokinetics and the low immunogenicity of human IgG with excellent manufacturability …