Manfred Reiter

Comparison of protein A, protein G and copolymerized hydroxyapatite for the purification of human monoclonal antibodies

Protein A Superose, protein G Sepharose fast flow and copolymerized hydroxyapatite were used for the purification of human monoclonal antibodies against HIV 1. Both desalted culture supernatant and a prepurified protein solution were used as starting materials. The different runs were compared with respect to yield and recovery of biological activity. The biological activity (specific reactivity) was checked by antigen enzyme-linked immunosorbent assay with recombinant antigen. The human monoclonal antibodies could not be selectively eluted from the hydroxyapatite but elution could be effected from the protein A Superose at pH 4.0 and from protein G at pH 3.0. The eluted immunoglobulin G was distributed over a broad pH range when protein G Superose was used. Biologically active material could be obtained from protein A Superose and protein G Sepharose fast flow.

The use of macroporous gelatin carriers for the cultivation of mammalian cells in fluidised bed reactors

A fluidised bed system for the cultivation of mammalian cells on a new type of macroporous gelatin microcarrier is described. The volumetric cell densities achieved under controlled conditions for two ‘standard cell lines’ (VERO, CHO) were one order of magnitude higher compared to conventional techniques using spherical microcarriers. The system can be potentially used for both anchorage dependent and independent cells.

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.

High-performance liquid chromatographic determination of metabolic products for fermentation control of mammalian cell culture: analysis of carbohydrates, organic acids and orthophosphate using refractive index and ultraviolet detectors

A method for the determination of carbohydrate substrates and excreted metabolic end-products of cell culture supernatants using a strong cation-exchange column in the H+ form has been developed. Organic acids and carbohydrates can be determined in addition to orthophosphoric acid. Pyrrolidone carboxylic acid, resulting from chemical conversion of the amino acid glutamine during the incubation of fresh medium and during the fermentation process, can be determined. The chromatographic method allows the correction of glutamine uptake values for physiological studies. Measured values of pyrrolidone carboxylic acid in supernatants of human hybridoma cell line show that it cannot be consumed by the cells. This technique allows the separation of major metabolites used in process optimization. Peak homogeneity is proved by on-line monitoring of the effluent with an ultraviolet (214 nm) and a refractive index detector connected in series.

Isolation of human monoclonal antibody isoproteins by preparative isoelectric focusing in immobilized pH gradients

A method for preparative isolation of human monoclonal antibody isoproteins is described in the present paper. A human monoclonal antibody directed against the transmembrane protein gp 41 from the human immunodeficiency virus (HIV-1) was used in this study. The antibody belongs to the IgG1 subtype and exhibits antibody dependent cellular cytotoxicity. The resolving power of conventional preparative protein separation techniques such as ion-exchange chromatography, chromatofocusing and lectin affinity chromatography is too poor for a complete separation of isoproteins. The more sophisticated technique of chromatofocusing on FPLC-based material (Mono P, Pharmacia) did not satisfy our expectation. With semipreparative IEF in immobilized pH gradients we were able to prepare the different isoproteins of a human monoclonal antibody in milligram amounts. No significant difference between the single isoproteins with respect to specificity and avidity to the recombinant antigen (rec gp 160) was detected. Therefore, we assume that the separation conditions did not influence the immunochemical nature of the antibody and significant denaturation and/or precipitation of the IgG did not occur. Furthermore the method affords preparative separation with resolution equivalent to analytical runs. Experiments for scale up and further characterization of isoproteins (carbohydrate composition, amino acid analysis, half life times etc.) are in progress.

Large-scale mammalian cell culture.

Over the past year, progress has been made toward a better understanding of the physiological and biochemical responses of cell cultures to various environmental changes. Much of the theoretical and experimental work that has been reported will help improve expression in mammalian cell culture, which is one of the key steps in biopharmaceutical manufacturing.

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells. Two-dimensional electrophoresis in animal cell culture technology

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-termin vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extendedin vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years.Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.

Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads

Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 108 cells/ml a stable growth rate of 0.004 h−1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.

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.

High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures

A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 107 cells per ml were obtained. The described system is scaleable.