Jean-François P. Hamel

Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG2a Monoclonal Antibody in a Wave Bioreactor‐Perfusion Culture System

A novel wave bioreactor‐perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell‐retention filter, and a weight‐based perfusion controller. A polyethylene membrane filter with a pore size of 7 μm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight‐based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 × 105 and 200.5 × 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 × 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L·day) in perfusion culture were much higher than those (i.e., 22.3 × 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L·day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter‐size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.

Determination of monoclonal antibody production in cell culture using novel microfluidic and traditional assays

This study compares microfluidic technology (Protein 200 LabChip® Assay kit, Agilent 2100 Bioanalyzer, referred to here as Protein 200) to the traditional approach for protein analysis, one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), for the sizing and quantification of immunoglobulin G (IgG) in hybridoma cell cultures. Internal references differ between each method: purified IgG was used alone in SDS‐PAGE while myosin (the upper marker) was added to each sample in Protein 200. The IgG used here were produced in cultures propagated in either a serum‐free or a serum‐containing medium. With serum‐containing samples, there was a significant difference in the IgG concentrations (p < 0.05) between SDS‐PAGE and Protein 200. The concentration determined by SDS‐PAGE was significantly higher (> 30%) than by Protein 200 or by high‐pressure liquid chromatography (HPLC) because the large amounts of serum albumin in the samples affect the accuracy of SDS‐PAGE. Protein 200 can determine size similarly to SDS‐PAGE in serum‐free samples (standard error of the mean, SEM, < 1%, 95% confidence < ±1%), unlike in serum‐containing samples. The Protein 200 assay was more effective than the traditional one‐dimensional SDS‐PAGE in determining concentration and size of IgG in cell culture samples and it provided a miniaturized and convenient platform for rapid analysis.

A perfusion culture system using a stirred ceramic membrane reactor for hyperproduction of IgG2a monoclonal antibody by hybridoma cells.

A novel perfusion culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred‐tank reactor during the perfusion culture. Furthermore, the significance of the control strategy of perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (ΔD, vvd per day, vvdd) of perfusion rate, the maximal viable cell density of 3.5 × 107 cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower ΔDapos;s, the cell growth became limited. Under nutrient‐limited condition, the specific cell growth rate (μ) was regulated by ΔD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 ± 0.02 pg/cell·h in all runs. During the cell growth‐limited phase, qmAb was regulated by ΔD within the range of 0.25–0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell·h was obtained during the growth‐limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L·day and 340 mg/L·medium, respectively. This study demonstrated that this novel perfusion culture system for suspension mammalian cells can support high cell density and efficient mAb production and that ΔD is an important control parameter to regulate and achieve high mAb production.

PURIFICATION AND CHARACTERIZATION OF ACETATE KINASE FROM CLOSTRIDIUM THERMOCELLUM

Abstract Acetate kinase (EC 2.7.2.1), an enzyme involved in the wasteful production of acetate during conversion of cellulose to ethanol by Clostridium thermocellum , was purified 144-fold. The enzyme has an M r of 84 kD by non-denaturing gradient gel electrophoresis, and an M r of 46 kD when estimated with a denaturing gel; thus it appears to be a homodimer. Optimum enzyme activity occurs at 50°C and between pH 7.2 and 8.0. Acetate kinase is stable to temperatures up to 60°C, but is completely inactivated at 80°C after two h. The enzyme is stable between pH 7.0 and 9.0 when incubated at 50°C for two h. Optimum acetate kinase activity occurs at a MgCl 2 :ATP ratio of 2:1, which indicates an interaction between Mg 2+ and ATP and that between Mg 2+ and acetate kinase. Enzyme activity is partially inhibited by KCl, an inorganic salt frequently used in chromatography and fermentation media, losing 60% activity in the presence of 0.2 M KCl. Sigmoidal enzyme kinetics were observed from the velocity plot of acetate kinase when either the acetate (S 0.5 = 285 mM) or ATP (S 0.5 = 11 mM) concentration was varied, suggesting cooperative binding of the two substrates.

On-Line Purification of Monoclonal Antibodies Using an Integrated Stirred-Tank Reactor/Expanded-Bed Adsorption System

While expanded‐bed adsorption (EBA) units have been used to recover proteins from whole cell cultures, the development of a more efficient, on‐line process could streamline the traditional multistep process. This study implements a bench‐scale on‐line purification system in which whole cell cultures are loaded directly into a chromatography column to capture a monoclonal antibody (mAb) in a single step. The on‐line purification system used here integrates a stirred‐tank reactor (STR) and an EBA unit into a new hybrid (STR‐EBA) system. To conduct this work, first, column and buffer conditions were optimized to capture immunoglobulin G from a hybridoma cell culture. A high cell removal (>95%) was achieved in part by removing the top flow distributor and mesh. Then, the 95% extent of removal was sustained for four successive cycles, each using PBS. With 20 mM phosphate buffer, however, the removal decreased from 95% to 75% stepwise. Next, the operational constraints of the EBA system were determined for the hybridoma cell culture, focusing on the effects of cell viability and density on cell removal. This study shows that the cell removal was not significantly different in the range of 80% to 0% viability. Cell density was also varied between 1 × 106 and 1 × 108 cells/mL. From 0.1 to 6 × 107 cells/mL, cell retention in the column was less than 5% and product recovery remained high, approximately 95%. After characterizing the working conditions of the EBA system, on‐line purification was performed. With 1.1 L of culture containing 3 × 106 cells/mL and 100 mg/L of IgG, repeated‐batch cultures were implemented. Half of the culture volume (550 mL) was directly sent to the EBA system every day, for 11 days, and the same amount of fresh medium was fed into the STR. During on‐line purification, productivity was 58 mg of IgG/cycle (day) and purity was greater than 95%. Simple batch culture alone produced 17 mg of IgG/day. This result suggests that the on‐line STR‐EBA system can achieve higher and faster production compared with STR batch and off‐line EBA purification. Overall, the STR‐EBA system with repeated‐batch mode was an effective and flexible system for bench‐scale mAb production.

Perfusion Cell Culture in Disposable Bioreactors

A novel disposable bioreactor design is described that incorporates a floating filter for the removal of cell-free perfusate. This design eliminates the need for traditional pumparound flow loops and external cell separators. Experiments with a hybridoma cell line producing a monoclonal antibody show that such a device can support very high cell densities of over 3×107 cells/ml and produce up to 73 mg/day of product. This is 6 fold higher productivity than batch culture. The bioreactor was operated continuously for 25 days without any evidence of clogging. Applicafions include bioprocessing and human cell therapy.

Properties of acetate kinase activity inClostridium thermocellum cell extracts

Acetate kinase (EC 2.7.2.1) is involved in the wasteful production of acetate during conversion of cellulose to ethanol byClostridium thermocellum. The properties of this enzyme activity inC. thermocellum cell extracts were determined. Optimum enzyme activity was at 60°C and between pH 7.5 and 9.0. In the presence of air, acetate kinase was stable to temperatures up to 60°C, retaining 90% activity after 2 h, and was inactivated rapidly at higher temperatures. The enzyme exhibited a wide range of stability to pH (5.0–9.0) when incubated at 50°C for 2 h. As with other acetate kinases, a divalent cation, such as Mg2+, was required for enzyme activity. Optimum activity was observed at 20mM MgCl2 when ATP was held constant at 10 mM. Acetate kinase activity was adversely affected by KCl, a salt commonly used in ion-exchange or affinity chromatography, with 0.3M KCl inhibiting by 50%. These results will be important in optimizing the direct microbial conversion process of cellulose to ethanol usingC. thermocellum in coculture withClostridium thermosaccharolyticum.

Suspension Culture Process of MethA Tumor Cell for the Production of Heat-Shock Protein Glycoprotein 96: Process Optimization in Spinner Flasks

Heat‐shock proteins (HSPs) act like “chaperones”, making sure that the cellapos;s proteins are in the right shape and in the right place at the right time. Heat‐shock protein glycoprotein 96 (gp96) is a member of the HSP90 protein family, which chaperones a number of molecules in protein folding and transportation. Heat‐shock protein gp96 serves as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. Currently, heat‐shock protein gp96 was only isolated from murine and human tissues and cell lines. An animal cell suspension culture process for the production of heat‐shock protein gp96 by MethA tumor cell was developed for the first time in spinner flasks. Effects of culture medium and condition were studied to enhance the MethA tumor cell density and the production and productivity of heat‐shock protein gp96. Initial glucose concentration had a significant effect on the heat‐shock protein gp96 accumulation, and an initial glucose level of 7.0 g/L was desirable for MethA tumor cell growth and heat‐shock protein gp96 production and productivity. Cultures at an initial glutamine concentration of 3 and 6 mM were nutritionally limited by glutamine. At an initial glutamine concentration of 6 mM, the maximal viable cell density of 19.90 × 105 cells/mL and the maximal heat‐shock protein gp96 production of 4.95 mg/L was obtained. The initial concentration of RPMI 1640 and serum greatly affected the MethA tumor cell culture process. Specifically cultures with lower initial concentration of RPMI 1640 resulted in lower viable cell density and lower heat‐shock protein gp96 production. At an initial serum concentration of 8%, the maximal viable cell density of 19.18 × 105 cells/mL and the maximal heat‐shock protein gp96 production of 5.67 mg/L was obtained. The spin rate significantly affected the cell culture process in spinner flasks, and a spin rate of 150 rpm was desirable for MethA tumor cell growth and heat‐shock protein gp96 production and productivity. Not only the cell density but also the production and productivity of heat‐shock protein gp96 attained in this work are the highest reported in the culture of MethA tumor cell. This work offers an effective approach for producing heat‐shock protein glycoprotein 96 from the cell culture process. The fundamental information obtained in this study may be useful for the efficient production of heat‐shock protein by animal cell suspension culture on a large scale.