K. Strutzenberger

Selective Retention of Viable Cells in Ultrasonic Resonance Field Devices

A double‐chamber ultrasonic resonance field device was used for the separation and retention of animal cells. By controlling operational parameters such as flow and power input, the device can retain viable cells more efficiently, allowing for selective removal of nonviable cells and cell debris. A simple model describing the forces acting on spherical particles in a sound field (primary radiation force, Bernoulli force, secondary radiation force) is presented. Field stability increases with decreasing average flow rates and increasing power input. At very high field stability, as achieved with low flow rates and high power input, the selectivity for viable cells is reduced, due to the efficient retention of all types of particles. At high flow rates and resulting low field stability, selectivity is also reduced, due to poor separation efficiency, resulting in equally low retention of viable cells, nonviable cells, and cell debris.

Changes during subclone development and ageing of human antibody-producing recombinant CHO cells

Some of the problems encountered with human or human-mouse heterohybridomas, such as low growth rates and high serum requirements, have led to the increased use of recombinant cell lines for production of human antibodies. To evaluate the suitability of such alternative cell lines for the production of human antibodies we have analysed several subclones with differing specific production rates of a recombinant CHO cell line. Gene copy number and site of chromosomal integration for the light and heavy chain and the dhfr gene were determined by in-situ hybridisation. Specific mRNA content was analysed by Northern blot. In addition the intracellular content in light and heavy chain was measured by flow cytometry and the specific secretion rates were determined. The stability of gene expression was followed in the highest producing subclone for over a year. As previously seen in heterohybridoma cells a high expression rate of light chain is beneficial in speeding up secretion rates of whole antibody. When grown in the presence of G418 and methotrexate the amplified gene copies in the genome of recombinant CHO cells were stable over more than 100 passages. However, the expression of light chain, and with it the secretion rate, decreased with time. The low intracellular concentration of light chain resulted in accumulation of heavy chain in the endoplasmic reticulum due to retention by chaperones. The specific secretion rate decreased by 50% after 100 passages. When no G418 or methotrexate were present 75% of the gene copies were lost after 100 passages.

Analysis of changes during subclone development and ageing of human antibody-producing heterohybridoma cells by Northern blot and flow cytometry

The economic importance of obtaining high-producing subclones for large scale production of pharmaceutical proteins is self-evident. However, few papers have studied the changes that occur during subclone development. This information would be important for further improvement of screening and subcloning protocols. We have therefore compared subclones of a human-mouse heterohybridoma cell line producing a human antibody againt HIV-1. Three subclones with low, medium and high specific production rates were selected for this study and their light and heavy chain mRNA content, the intracellular content of light and heavy chain and the specific secretion rates compared. In addition the long time stability of antibody expression in the highest producing subclone was analysed for one year. For the three subclones a correlation between the intracellular content in light chain and the secretion rate was found, while the intracellular content in heavy chain was the same for all three subclones. These results indicate that the assembly in the endoplasmic reticulum (ER) is one of the major rate limiting factors in antibody production. During long time cultivation of the heterohybridoma cell line a continuous decrease in light and heavy chain production was seen without the appearance of a non producing sub-population.

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.

Comparison of the production of a human monoclonal antibody against HIV-1 by heterohybridoma cells and recombinant CHO cells: A flow cytometric study

The production of human monoclonal antibodies for therapeutic use is of increasing importance for treatment of viral infections such as AIDS. As human x mouse heterohybridomas rarely reach the growth rates and cell specific production rates of mouse hybridomas the transfection of standard cell lines, such as CHO or BHK, is a promising alternative. This has the additional advantage that the IgG subtype can be changed to suit the desired application. However, the use of a cell line that has not originally developed to produce antibodies, as lymphocytes and myeloma cells have, might have unrecognised drawbacks. This will be especially significant in the case of antibodies as each molecule consists of 4 chains linked by disulphide bonds which require specific intracellular factors to be properly folded and processed (Heavy chain binding protein, Protein Disulfide Isomerase a.o.). In this study we have therefore compared two cell lines: a human x mouse heterohybridoma producing IAM-2F5, a human IgG3 antibody specific for HIV-1 with neutralising properties and a Chinese Hamster Ovary cell transfected with dihydrofolate reductase and with the heavy and light chain genes of IAM-2F5 modified to IgG1. From each cell line three subclones were selected with low, medium and high specific production rates. Batch cultures were performed and the following cellular parameters analysed by flow cytometry; 1) total RNA content (translational activity); 2) total protein content; 3) cell cycle phase distribution; 4) concentration of light and heavy chains; 5) concentration of helper proteins such as BiP and PDI. The production rate of heterohybridoma cells was best reflected in the intracellular concentration of kappa chain, while the gamma chain concentration was comparable for all three subclones. In the CHO cells the gamma chain expression and thus gene copy number appeared to be the limiting factor. The GRP78/BiP concentration in CHO remained unchanged in spite of a 5-fold higher concentration of gamma chain in the high producing subclone. The PDI concentration in CHO cells was much lower compared to the heterohybridoma cells, irrespective of production rates.

Human endothelial cell lines established by mutated forms of the simian virus 40 large T oncogene.

The large T oncoprotein of Simian Virus 40 is widely used to improve the growth characteristics of primary cells in culture. Beside growth stimulation and immortalization, expression of the large T protein in human cells frequently leads to a loss of differentiated characters and changes in the karyotype. We have constructed mutated forms of the large T protein by deletion of various fragments of the DNA binding domain to test, whether this region is responsible for undesired influences on cell differentiation. After transfection into human umbilical vein endothelial cells, the resulting cell lines showed no improvement in expression of the differentiation marker von Willebrand factor compared to cell lines transfected with the wild type oncogene. Changes in the karyotype were still observed. Our results contribute to the mapping of functional domains of the large T protein. The truncated large T proteins retained growth stimulating activity after removal of 111 and 241 amino acids of the DNA binding region.

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.

Monoclonal Antibody Production Using the Porous Glass Bead Immobilization Technique

The increase of cell density in bioreactors and thus immobilization techniques have received a substantial amount of attention.s2 The technique for mass production and maintenance culture of anchorage-dependent mammalian cells at high cell densities is established for the production of a wide range of biological^.^^^ There are a number of reasons claimed beneficial for using immobilization techniques for the propagation of hybridomas, for example, high cell density, decreased biomass separation cost, lower media costs, the use of protein-free media, and higher volumetric production rate^.^-^

Scale-Up of Ultrasonic Resonance Field Cell Separation Devices Used in Animal Cell Technology

Ultrasonic standing waves have been used for the separation of suspended particles, such as animal cells (Kilburn et al., 1989). This technology can be used for cell retention within the fermenter for high density cell culture or as cell removal step in downstream processing. Devices for bench scale applications have been developed and evaluated in experimental set-ups and on laboratory fermenters (Trampler et al., Doblhoff et al., 1994). To scale-up this promising technology for industrial processes, a number of criteria, such as aseptic and hygienic design, steam sterilisability, sufficient flow rates and separation efficiency have to be taken into account. In this paper we will discuss the theoretic limitations of different resonator designs and will describe experimental results with a new type of resonator with an internal cooling loop. The modular, aseptic device is mounted on top of the fermentation equipment and can be steam sterilised in-situ. High amplitudes necessary to achieve separation of animal cells cause thermal and acoustic flow within the separation chamber. The internal cooling loop reduces this temperature increase and thus disrupting forces, which would otherwise limit the achievable flow rate and separation efficiency.

Protein Mass Production in Hybridomas and Recombinant CHO Cells

Hybridomas (immortalised lymphocytes) represent a single gene-copy number expression system for antibodies in the native host cell. Antibodies represent a heterodimeric protein encoded by two genes. We expressed antibodies in hybridomas as well as in recombinant CHO cells. Also other recombinant CHO cells were established under comparable conditions to express other glycoproteins encoded by single genes.