Strategy for selecting disposable bags for cell culture media applications based on a root‐cause investigation

The use of disposable bags for cell culture media storage has grown significantly in the past decade. Some of the key advantages of using disposable bags relative to non‐disposable containers include increased product throughput, decreased cleaning validation costs, reduced risk of cross contamination and lower facility costs. As the scope of use of disposable bags for cell culture applications increases, problematic bags and scenarios should be identified and addressed to continue improving disposables technologies and meet the biotech industry's needs. In this article, we examine a cell culture application wherein media stored in disposable bags is warmed at 37°C before use for cell culture operations. A problematic bag film was identified through a prospective and retrospective cell culture investigation. The investigation provided information on the scope and variation of the issue with respect to different Chinese hamster ovary (CHO) cell lines, cell culture media, and application‐specific parameters. It also led to the development of application‐specific test methods and enabled a strategy for disposable bag film testing. The strategy was implemented for qualifying an alternative bag film for use in our processes. In this test strategy, multiple lots of 13 bag film types, encompassing eight vendors were evaluated using a three round, cell culture‐based test strategy. The test strategy resulted in the determination of four viable bag film options based on the technical data. The results of this evaluation were used to conclude that a volatile or air‐quenched compound, likely generated by gamma irradiation of the problematic bag film, negatively impacted cell culture performance. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1535–1549, 2013     

Effect of ambient light on monoclonal antibody product quality during small‐scale mammalian cell culture process in clear glass bioreactors

During a small‐scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small‐scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:562–570, 2014     

A cytotoxic leachable compound from single‐use bioprocess equipment that causes poor cell growth performance

A current trend in the production of biopharmaceuticals is the replacement of fixed stainless steel fluid‐handling units with disposable plastic bags. Such single‐use systems (SUS) offer numerous advantages, but also introduce a new set of materials into the production process and consequently expose biomanufacturers to a new set of risks related to those materials, not to mention reliance on an entirely new supply chain. In the course of developing and conducting a cell‐growth‐based test for suitability of disposable plastic components destined for use in cell culture operations, we discovered that the cytotoxic compound bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP) leaches out of certain bags and into cell culture media in concentrations that are deleterious to cell growth. Specifically, media held in certain bags for several days at 37°C was found to contain bDtBPP, and use of those held‐media samples in cell growth experiments provides data that overlap neatly with cell growth experiments using media spiked directly with bDtBPP, proving that bDtBPP leaching is responsible for the reduced growth attributable to those SUS bags. Overall, this issue represents a risk to the production of biopharmaceuticals in SUS, a risk that must be managed by diligent collaboration among companies along the entire supply chain for SUS components. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:332–337, 2014     

X-ray sterilization of biopharmaceutical manufacturing equipment—Extractables profile of a film material and copolyester Tritan™ compared to gamma irradiation

The biopharmaceutical industry gains enormous flexibility in production processes by using sterilized preassembled single-use devices. Gamma irradiation is an established sterilization technology that may be restricted in the future by the availability of ⁶⁰Co as irradiation source and irradiation capacities. X-ray technology is considered an alternative type of radiation for sterilizing SU equipment. In the context of extractables and leachables—one concern connected with the use of single-use process equipment—the effect of X-ray irradiation on the extractables profile of the materials needs to be compared to established gamma irradiation to qualify this alternative technology. An approach is presented to obtain robust and comprehensive extractables data for materials used in SU devices after sterilization either using X-ray or gamma irradiation. A careful selection of the test items and the test design allows a one-to-one comparison of data obtained from a combination of orthogonal analytical techniques. The extractables of a modern SU film material and the copolyester Tritan™ are evaluated. The data presented allow a risk evaluation on the safety of this new sterilization modality for biopharmaceutical applications. It is demonstrated that the extractables profile of a polymer is not affected by the type of irradiation used for sterilization.     

Modulation of mAb quality attributes using microliter scale fed‐batch cultures

A high‐throughput DoE approach performed in a 96‐deepwell plate system was used to explore the impact of media and feed components on main quality attributes of a monoclonal antibody. Six CHO‐S derived clonal cell lines expressing the same monoclonal antibody were tested in two different cell culture media with six components added at three different levels. The resulting 384 culture conditions including controls were simultaneously tested in fed‐batch conditions, and process performance such as viable cell density, viability, and product titer were monitored. At the end of the culture, supernatants from each condition were purified and the product was analyzed for N‐glycan profiles, charge variant distribution, aggregates, and low molecular weight forms. The screening described here provided highly valuable insights into the factors and combination of factors that can be used to modulate the quality attributes of a molecule. The approach also revealed specific intrinsic differences of the selected clonal cell lines ‐ some cell lines were very responsive in terms of changes in performance or quality attributes, whereas others were less affected by the factors tested in this study. Moreover, it indicated to what extent the attributes can be impacted within the selected experimental design space. The outcome correlated well with confirmations performed in larger cell culture volumes such as small‐scale bioreactors. Being fast and resource effective, this integrated high‐throughput approach can provide information which is particularly useful during early stage cell culture development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:571–583, 2014     

Allogeneic cell therapy bioprocess economics and optimization: Single‐use cell expansion technologies

For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot‐sizes capable of meeting commercial demands of up to 10⁹ cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost‐effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier‐based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost‐effective and where microcarrier‐based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier‐based systems. These data are presented using a technology S‐curve as well as windows of operation to identify the combination of cell productivities and scale of single‐use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision‐making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. Biotechnol. Bioeng. 2014;111: 69–83. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

Application of multivariate analysis and mass transfer principles for refinement of a 3‐L bioreactor scale‐down model—when shake flasks mimic 15,000‐L bioreactors better

Characterization of manufacturing processes is key to understanding the effects of process parameters on process performance and product quality. These studies are generally conducted using small‐scale model systems. Because of the importance of the results derived from these studies, the small‐scale model should be predictive of large scale. Typically, small‐scale bioreactors, which are considered superior to shake flasks in simulating large‐scale bioreactors, are used as the scale‐down models for characterizing mammalian cell culture processes. In this article, we describe a case study where a cell culture unit operation in bioreactors using one‐sided pH control and their satellites (small‐scale runs conducted using the same post‐inoculation cultures and nutrient feeds) in 3‐L bioreactors and shake flasks indicated that shake flasks mimicked the large‐scale performance better than 3‐L bioreactors. We detail here how multivariate analysis was used to make the pertinent assessment and to generate the hypothesis for refining the existing 3‐L scale‐down model. Relevant statistical techniques such as principal component analysis, partial least square, orthogonal partial least square, and discriminant analysis were used to identify the outliers and to determine the discriminatory variables responsible for performance differences at different scales. The resulting analysis, in combination with mass transfer principles, led to the hypothesis that observed similarities between 15,000‐L and shake flask runs, and differences between 15,000‐L and 3‐L runs, were due to pCO₂ and pH values. This hypothesis was confirmed by changing the aeration strategy at 3‐L scale. By reducing the initial sparge rate in 3‐L bioreactor, process performance and product quality data moved closer to that of large scale. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1370–1380, 2015     

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology‐derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer‐based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro‐scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro‐scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX‐Cell Advanced, and OptiCHO media, and 204, C, EX‐Cell, SE‐15, and Y‐30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX‐Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25‐35 × 10⁶ cells‐d/mL, while maintaining specific antibody production (Q_p > 2 pg/cell‐d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX‐Cell, and SE‐15 were capable of providing adequate control of foaming while antifoam 204 and Y‐30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262–270, 2018     

Verification of energy dissipation rate scalability in pilot and production scale bioreactors using computational fluid dynamics

Suspension mammalian cell cultures in aerated stirred tank bioreactors are widely used in the production of monoclonal antibodies. Given that production scale cell culture operations are typically performed in very large bioreactors (≥ 10,000 L), bioreactor scale‐down and scale‐up become crucial in the development of robust cell‐culture processes. For successful scale‐up and scale‐down of cell culture operations, it is important to understand the scale‐dependence of the distribution of the energy dissipation rates in a bioreactor. Computational fluid dynamics (CFD) simulations can provide an additional layer of depth to bioreactor scalability analysis. In this communication, we use CFD analyses of five bioreactor configurations to evaluate energy dissipation rates and Kolmogorov length scale distributions at various scales. The results show that hydrodynamic scalability is achievable as long as major design features (# of baffles, impellers) remain consistent across the scales. Finally, in all configurations, the mean Kolmogorov length scale is substantially higher than the average cell size, indicating that catastrophic cell damage due to mechanical agitation is highly unlikely at all scales. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:760–764, 2014     

Spectroscopic methods and their applicability for high‐throughput characterization of mammalian cell cultures in automated cell culture systems

The number and use of automated cell culture systems for mammalian cell culture are steadily increasing. Automated cell culture systems require miniaturized analytics with a high throughput to obtain as much information as possible from single experiments. Standard analytics commonly used for conventional bioreactor samples cannot handle the high throughput and the low sample volumes. Spectroscopic methods provide a means of meeting this analytical requirement and afford fast and direct access to process information. In the first part of this review, UV/VIS, fluorescence, Raman, near‐infrared, and mid‐infrared spectroscopy are presented. In the second part of the review, these spectroscopic methods are evaluated in terms of their applicability in the new field of mammalian cell culture processes in automated cell culture systems. Unlike standard bioreactors, these automated systems have special requirements that apply to the use of spectroscopic methods. Therefore, they are compared with regard to cell culture automation, throughput, and required sample volume.     

Serum‐free spheroid suspension culture maintains mesenchymal stem cell proliferation and differentiation potential

There have been many clinical trials recently using ex vivo‐expanded human mesenchymal stem cells (MSCs) to treat several disease states such as graft‐versus‐host disease, acute myocardial infarction, Crohn's disease, and multiple sclerosis. The use of MSCs for therapy is expected to become more prevalent as clinical progress is demonstrated. However, the conventional 2‐dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the MSC‐based indications further exacerbates this manufacturing challenge. In contrast, expanding MSCs as spheroids does not require a cell attachment surface and is amenable to large‐scale suspension cell culture techniques, such as stirred‐tank bioreactors. In the present study, we developed and optimized serum‐free media for culturing MSC spheroids. We used Design of Experiment (DoE)‐based strategies to systematically evaluate media mixtures and a panel of different components for effects on cell proliferation. The optimization yielded two prototype serum‐free media that enabled MSCs to form aggregates and proliferate in both static and dynamic cultures. MSCs from spheroid cultures exhibited the expected immunophenotype (CD73, CD90, and CD105) and demonstrated similar or enhanced differentiation potential toward all three lineages (osteogenic, chondrogenic, adipogenic) as compared with serum‐containing adherent MSC cultures. Our results suggest that serum‐free media for MSC spheroids may pave the way for scale‐up production of MSCs in clinically relevant manufacturing platforms such as stirred tank bioreactors. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:974–983, 2014     

Streamlined production of genetically modified T cells with activation,transduction and expansion in closed-system G-Rex bioreactors

BackgroundGas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes.ResultsThe simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically.DiscussionThis study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.     

Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

N‐linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N‐linked glycosylation patterns in a CHO‐S cell fed‐batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High‐throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N‐linked glycosylation patterns during a fed‐batch process as a function of time as well as the manipulated variables. A constant and varying mAb N‐linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model‐based evaluation of feeding regimes using high‐throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1135–1148, 2016     

Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use,Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel? technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1600–1612, 2015     

Demonstrating the Manufacture of Human CAR‐T Cells in an Automated Stirred‐Tank Bioreactor

Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 10⁶ cells ml‐1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T‐flasks and gas‐permeable culture bags). Importantly, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.     

A simple eccentric stirred tank mini‐bioreactor: Mixing characterization and mammalian cell culture experiments

In industrial practice, stirred tank bioreactors are the most common mammalian cell culture platform. However, research and screening protocols at the laboratory scale (i.e., 5–100 mL) rely primarily on Petri dishes, culture bottles, or Erlenmeyer flasks. There is a clear need for simple—easy to assemble, easy to use, easy to clean—cell culture mini‐bioreactors for lab‐scale and/or screening applications. Here, we study the mixing performance and culture adequacy of a 30 mL eccentric stirred tank mini‐bioreactor. A detailed mixing characterization of the proposed bioreactor is presented. Laser induced fluorescence (LIF) experiments and computational fluid dynamics (CFD) computations are used to identify the operational conditions required for adequate mixing. Mammalian cell culture experiments were conducted with two different cell models. The specific growth rate and the maximum cell density of Chinese hamster ovary (CHO) cell cultures grown in the mini‐bioreactor were comparable to those observed for 6‐well culture plates, Erlenmeyer flasks, and 1 L fully instrumented bioreactors. Human hematopoietic stem cells were successfully expanded tenfold in suspension conditions using the eccentric mini‐bioreactor system. Our results demonstrate good mixing performance and suggest the practicality and adequacy of the proposed mini‐bioreactor. Biotechnol. Bioeng. 2013; 110: 1106–1118. © 2012 Wiley Periodicals, Inc.     

Potential application of aqueous two‐phase systems and three‐phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus

Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidant enzyme that represents the primary cellular defense against superoxide radicals and has interesting applications in the medical and cosmetic industries. In the present work, the partition behavior of SOD in aqueous two‐phase systems (ATPS) (using a standard solution and a complex extract from Kluyveromyces marxianus as sample) was characterized on different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt, and ionic liquid (IL)–salt). The systems composed of PEG 3350‐potassium phosphate, 45% TLL, 0.5 M NaCl (315 U/mg, 87% recovery, and 15.1‐fold purification) and t‐butanol‐20% ammonium sulfate (205.8 U/mg, 80% recovery and 9.8‐fold purification), coupled with a subsequent 100 kDa ultrafiltration stage, allowed the design of a prototype process for the recovery and partial purification of the product of interest. The findings reported herein demonstrate the potential of PEG‐salt ATPS for the potential recovery of SOD. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1326–1334, 2014     

A predictive high‐throughput scale‐down model of monoclonal antibody production in CHO cells

Multi‐factorial experimentation is essential in understanding the link between mammalian cell culture conditions and the glycoprotein product of any biomanufacturing process. This understanding is increasingly demanded as bioprocess development is influenced by the Quality by Design paradigm. We have developed a system that allows hundreds of micro‐bioreactors to be run in parallel under controlled conditions, enabling factorial experiments of much larger scope than is possible with traditional systems. A high‐throughput analytics workflow was also developed using commercially available instruments to obtain product quality information for each cell culture condition. The micro‐bioreactor system was tested by executing a factorial experiment varying four process parameters: pH, dissolved oxygen, feed supplement rate, and reduced glutathione level. A total of 180 micro‐bioreactors were run for 2 weeks during this DOE experiment to assess this scaled down micro‐bioreactor system as a high‐throughput tool for process development. Online measurements of pH, dissolved oxygen, and optical density were complemented by offline measurements of glucose, viability, titer, and product quality. Model accuracy was assessed by regressing the micro‐bioreactor results with those obtained in conventional 3 L bioreactors. Excellent agreement was observed between the micro‐bioreactor and the bench‐top bioreactor. The micro‐bioreactor results were further analyzed to link parameter manipulations to process outcomes via leverage plots, and to examine the interactions between process parameters. The results show that feed supplement rate has a significant effect (P < 0.05) on all performance metrics with higher feed rates resulting in greater cell mass and product titer. Culture pH impacted terminal integrated viable cell concentration, titer and intact immunoglobulin G titer, with better results obtained at the lower pH set point. The results demonstrate that a micro‐scale system can be an excellent model of larger scale systems, while providing data sets broader and deeper than are available by traditional methods. Biotechnol. Bioeng. 2009; 104: 1107–1120. © 2009 Wiley Periodicals, Inc.     

Micro sequential injection system as the interfacing device for process analytical applications

It is an important and desirable capability to be able to control the quality and quantity of biological product by maintaining and adjusting bioreactor performance throughout its production duration. Amino acids are the building blocks of proteins. Scientists will need to ensure sufficient supply of amino acids as the substrates in the bioreactors as well as to control the excess level of undesirable free amino acid byproducts to maintain an optimum growth environment for cell culture. We have developed a compact and robust sample preparation platform capable of interfacing with analytical instruments to achieve bioreactor amino acids monitoring. We demonstrated the feasibility of this concept by incorporating an automatic amino acid sample preparation protocol to a micro sequential injection (μSI) system connected to an ultra‐performance liquid chromatography system for real‐time, at‐line amino acid separation, and quantitation. The μSI system was configured into a “platform‐like” sample preparation system that is able to accommodate future wet chemistry‐type sample preparations. Its real‐time amino acid results can be readily available to bioprocess scientists for quick decision making and design of their next experiment. Potential automatic feedback control mechanisms can be established through trigger events based on predetermined analytical signal thresholds so the system can communicate with facility infrastructure to control bioreactors in near real‐time fashion. The proposed μSI system described in this paper can be widely used as an automatic sample preparation system connected to the front‐end of analytical instruments to enable process analytical technology applications. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:607–613, 2015