The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells.

Protein hydrolysates as substitutes for serum havebeen employed by many in cell culture mediumformulation, especially with the shift to low proteinor protein-free media. More recently, vegetablehydrolysates have also been added as nutritionalsupplements to fortify the amino acid content in smallpeptide form for batch and fed-batch fermentations. Several of these new hydrolysates (peptones of soy,rice, wheat gluten etc.) were tested as protein-freemedium supplements for the production of a recombinanttherapeutic protein. Multiple peptone-supplemented,continuous perfusion bioreactor experiments wereconducted, varying dilution rates and basal mediumcomposition over the various runs. Cell specificrates and product quality studies were obtained forthe various peptones and compared with peptone-freemedium. The potential for peptones to decreaseintrinsic and proteolytic degradation of the productwas also investigated.It was found that peptones confer a nutritionalbenefit, especially at low dilution rates, for therecombinant BHK cell line used in this investigation.The specific productivity increased 20-30% comparedto the peptone-free controls. However, this benefitwas also fully delivered by using fortified medium inplace of the peptone-enriched media. Therefore, whilepeptones may be considered as useful medium additiveswhen development time is limited, their addition maybe avoided by systematic medium development ifpermitted by the time line of the project.

Fermentor temperature as a tool for control of high-density perfusion cultures of mammalian cells.

Temperature is a key environmental variable whose potential in animal cell fermentor optimization is not yet fully utilized. The scarce literature data suggests that reduced fermentor temperature results in an improved viability and shear resistance, higher cell density and titer in batch cultures, and reduction in glucose/lactate metabolism. Due to the arrest of the cells in the G1 phase, the specific growth rate was found to decrease at temperatures below 37.0 degrees C. The response of the specific production rate was cell line dependent: in some cases it increased 2-to-3-fold, but decreased in other cases. The controlable slowdown of cell metabolism at lower temperature can be used in optimization of perfusion mammalian cell cultures with several potential advantages, including higher cell density in oxygen limited reactors, lower perfusion rate, improved product quality, simplified pH control, and others. To evaluate this strategy, a series of long-term experiments in 15 L perfusion bioreactors culturing recombinant hamster cells at 20.0 x 10(6) cells/mL were conducted. The temperature was changed over a range of set points, and maintained at each of these for a long period of time. Steady state process data was collected and analyzed. The effect of temperature on the following characteristics of the perfusion process was studied: cell growth, glucose/lactate metabolism, glutamine/ammonia metabolism, cell respiration, cell density at constant oxygen transfer rate, proteolytic activity, and product quality (glycosylation and molecule fragmentation). The results suggest that temperature is a variable with a significant potential in optimization of perfusion cultures. Properly selected temperature set point will contribute to the overall improvement of process performance. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 328-338, 1997.

Real-time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor.

On-line monitoring and control of cell culture fermentation is important for optimal and consistent production of biologicals. In this work, glucose and lactate concentrations are monitored on-line using a commercially available analyzer (Model 2700, Yellow Springs Instruments, Yellow Springs, OH) during batch and perfusion hybridoma cell fermentation. Cell free samples from the reactor are obtained using a 0.45 mum hollow fiber filtering system placed in a circulation loop. The samples were analyzed at specified times and the data are collected on a computer. A process control strategy was developed to control the concentrations of glucose and lactate in a perfusion reactor where the feed rate is adjusted to maintain their concentrations at desired set points. Hybridoma cells (A10G10) were cultivated in a high density perfusion culture where cell density increased from 2 to 14 million cells/mL. During this period the control algorithm successfully adjusted the perfusion rate while maintaining constant glucose and lactate concentrations. Glucose consumption and lactate accumulation rates as well as net lactate yield on glucose were monitored continuously during perfusion culture. These metabolic rates were observed to be independent of cell concentration and were used for the estimation of viable cell density in the reactor. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 372-378, 1997.

Characteristics of the adsorption of immunoglobulin M onto Q Sepharose Fast Flow ion-exchangers.

Measurement of adsorption breakthrough curves in packed beds has shown that the amounts and rates of uptake of immunoglobulin M (IgM) onto the commonly used anionic ion-exchanger Q Sepharose Fast Flow (based on 6% agarose) are severely limited as a result of the large molecular size of this adsorbate (RMM 950,000). A similar ion-exchanger based on a more porous 4% agarose, Q Sepharose 4 Fast Flow was evaluated as an alternative adsorbent for the purification of IgM. Equilibrium adsorption isotherms and the effective diffusivities of IgM within these two adsorbents were measured. Q-Sepharose 4 Fast Flow was found to have a maximum capacity for IgM 2.5 times greater than that of Q Sepharose 6 Fast Flow and the effective diffusivity of IgM was found to be between 6 and 7 times greater than with the latter material. Comparison of the breakthrough curves obtained for these adsorbents at a variety of flow velocities confirm that Q Sepharose 4 Fast Flow is a superior adsorbent for the capture and purification of large proteins.

Real-time monitoring of protein secretion in mammalian cell fermentation: measurement of monoclonal antibodies using a computer-controlled HPLC system (BioCad/RPM).

On-line, "real-time" monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG(1), without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer-controlled 15-L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell-free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real-time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement.

Evaluation and applications of optical cell density probes in mammalian cell bioreactors.

On-line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 x 10(6) cells/mL to 20 x 10(6) cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of "step perfusion adjustments" based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions.

Simplified Procedure for Estimating the Effect of a Change in Heating Rate on Sterilization Value 1.

A general relationship between a relative change in the temperature response parameter, f, and the sterilization value delivered in a thermal process has been developed. The relationship is based on numerical differentiation of Ball's formula method and employs a dimensionless elasticity term to express the relative change in sterilization value due to a relative change in heating rate. The funtion presented can be used for g's of up to 30°F and for changes of 3 to 20% in the value of the temperature response parameter.