F. Unterluggauer

Pilot scale production of a human monoclonal antibody against human immunodeficiency virus HIV-1

Human monoclonal antibodies against the transmembrane protein gp41 of HIV-1 were isolated and purified on a pilot scale. A purification scheme was established for the production of human monoclonal antibodies on the gram scale. 50 1 of culture supernatant can be treated in one purification cycle. The hybridomas were mass cultured in an airlift fermenter. The culture broth was clarified by microfiltration and chromatographed on CM-Sepharose fast flow and protein A Superose. Scale up of the high performance affinity chromatography from 1 ml protein A Superose up to 40 ml is described. All desalting steps were performed by gel filtration on Sephadex G-25 coarse. The yield of the whole purification procedure is in the range of 50-60%. The purity is higher than 99.9%. DNA and reverse transcriptase could not be detected. The whole method is designed as a basis for scale up to industrial scale. Results from quality control assays have proven the validity of this approach.

Combination of zetaprep mass ion-exchange media and high-performance cation-exchange chromatography for the purification of high-purity monoclonal antibodies

A procedure involving diafiltration, mass ion exchange on a QAE Zetaprep disk, gel chromatography and cation-exchange chromatography was used for the purification of mouse monoclonal antibodies from hybridoma culture supernatant. Prior to the separation steps, the starting solution was adjusted to the desired pH and conductivity. Diafiltration was used for this purpose in order to keep the volume constant or even to reduce the volume of sample. A QAE Zetaprep disk was used to remove the main protein contaminants from the culture supernatant. After washing unbound proteins out of the Zetaprep disk, slightly bound protein was eluted with a buffer solution containing 50 mM sodium chloride. The monoclonal antibody was eluted with a solution containing 150 mM sodium chloride. The purity of the eluted antibody was 50%, and was increased to 99% by subsequent high-performance cat-ion-exchange chromatography. The purity was determined by means of sodium-dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. The advantage of the two high-performance techniques, mass ion exchange and high-performance cation-exchange chromatography, are the high-flow-rates and the high resolution that can be obtained. These techniques are suitable for the production of injectable therapeutic preparations.

Long-Term Stability Of Continuously Perfused Animal Cells Immobilized On Novel Macroporous Microcarriers

The ultimate goal of optimizing biological production is the establishment of simple, safe, cheap, consistent, and easily manageable production systems. Various complicated techniques have been used for animal cell culture. In practice, however, only the simple ones are used for production purposes. A high cell density continuous perfusion in vitro system simulates a situation that most closely exists in the in vivo maintenance of differentiated cells arrested in the G1/G0-phase of the cell cycle. In order to make this a technological reality, a macroporous open structured carrier has been designed using high pressure polyethylene as a primary matrix material. During long-standing continuous perfusion of cultured mammalian cells such as Chinese hamster ovary (CHO) cells with a protein-free medium, the cells are maintained at high cell density (> 10 8 per ml) in a fully productive but nonpropagating state. Using the fluidized bed porous bead technology we do not expect any limitation in the scale-up of any technology using continuous mammalian cell lines.

SCALE-UP OF A FLUIDIZED BED REACTOR OPERATED WITH POROUS GLASS CARRIERS

ABSTRACT The IAM modular fluidized bed reactor was scaled up from laboratory size (7 1) to pilot scale (80 1). Hardeware was designed to fit the modular IAM pilot plant structure and software was adapted to the new reactor design. The new reactor was tested using porous glass beads and CHO cells as model cell line. After inoculation and batch phase a continuous perfusion system using protein free-medium was set up and operated over a period of three weeks. At the final dilution rate (approx. 12 times the carrier volume) a cell density up to 5 × 10 7 cells/ml carrier bed could be achieved. Fermentation control was achieved using an industrial direct digital process control system (Honeywell TDC 3000).

PILOT-SCALE MODULAR HARD-AND SOFTWARE CONCEPT FOR ANIMAL CELL FERMENTATION

The design, process control and operation of a modular pilot-plant with various convertible fermenter designs (stirred tank, airlift, spin-filter and fluidized bed) with a working volume up to 300 l are described. Hardware and process control software are carefully designed for maximum operability and adaptability.

OXYGENATION IN FLUIDIZED BED BIOREACTORS USING THE MICROSPARGING TECHNIQUE

ABSTRACT An oxygenation system based on a microsparger with a stainless steel mesh with a pore size of 0.5 micron for oxygen supply in high density fluidized bed bioreactors is described. The design of cell culture processes can be improved with respect to sterility and simplicity. The microsparger applied in the 80 liter pilot scale fluidized bed bioreactor has an oxygen transfer capacity of 5 000 mg O2/h. Under standard conditions one cm2 of the microsparger can support 1012 cells.

Comparison of Fluidized Bed and Fed Batch Reactor Cultures for Production of Anti-HIV-Antibody

High product yield, product quality and stable reactor culture conditions are a key to economically viable pharmaceutical bioprocesses. Two types of culture methods for production of recombinant anti-HIV-antibody were applied within this study. The tested cell line was a DHFR- recombinant CHO cell line expressing anti-HIV antibodies (CHO-4E10; Polymun Scientific, Vienna, Austria).Cells were cultured in a perfused lab-scale fluidized bed Cytopilot Mini™ reactor (Amersham Biosciences, Uppsala, Sweden) attached to macroporous Cytoline 1™ (Amersham Biosciences, Uppsala, Sweden) microcarriers and as suspension cells in a pilot scale stirred tank bioreactor fed-batch process. Both processes were performed using protein-free media. The systems were compared in terms of cell growth, product yield and product quality. Both systems were equally well suited for production of the recombinant protein in terms of product yield and product quality, however demonstrating an economic advantage of the fluidized bed process.