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Biomedical subjects

W Berthold

Publications and source records attributed to W Berthold.

At least 19 recordsLinked to original sources
PubMed1996

Process scale considerations in evaluation studies and scale-up.

This paper focuses on the scale of validation studies that are performed in order to demonstrate viral clearance in downstream processing of biopharmaceutical products. A serious concern for rDNA-derived proteins from recombinant mammalian cell cultures and monoclonal antibodies (MAbs) derived from hybridoma cultures is the potential risk from contaminating retroviral particles or adventitious viruses. Accordingly the downstream process has to be designed to reduce a potential virus load significantly: by virus removal and by virus inactivation methods. Such viral clearance is essential for drug safety- and has to be validated. This means that the design of downstream processing has to take into consideration both the capability for viral clearance and the capability for validation respectively.

Animals↗
PubMed1995

Clarification of animal cell cultures on a large scale by continuous centrifugation.

Animal cells from 80-L and 2000-L fed batch fermentations were removed by a prototype disc stack centrifuge in order to achieve a fast and reliable separation of solids from large quantities of cell culture fluids. The clarification capacity was excellent for animal cells but particles remained in the liquid phase and affected further downstream processing of the cell-free harvest fluid. No significant loss of product was observed. A number of parameters were monitored to optimize process conditions for use with animal cells.

Animals↗
PubMed1994

Protein purification: aspects of processes for pharmaceutical products.

The concept of purity is addressed for proteins as drugs. In addition to a short description of general techniques, emphasis is given to a discussion of special protein impurities and contaminants from the host cell of process additives. This includes the host homologue to the product and biologically active molecules and the quantitation of host cell proteins. Of special concern are closely related non functional proteins from the gene for the protein product itself. They pose the question of how a 'pure' protein can be defined, and to what extent potential heterogeneities caused by the purification method represent a risk. Most relevant are 'invisible' contaminants such as potential virus including the BSE agent. Critical aspects will be discussed of the current rationale for a strategy to arrive at safe biological products. The limitations of current virus enumeration methods are discussed in relation to technical limitations to prove the efficiency of virus removal steps. Another limitation is the nature of the protein product and its individual resistance to denaturing conditions in virus inactivation steps. This is amply demonstrated by the lack of methods for inactivation of the BSE agent maintaining the native state of proteins. As a general recommendation it is proposed to base risk assessment on scientific data and come to scientifically defined criteria of acceptance in both the global and national communities. This will facilitate bringing new biopharmaceuticals to the benefit of patients in the shortest period of time.

Animals↗
PubMed1994

Optimization of vaccine production for animal health.

Vaccines on the basis of mammalian cell cultures are of major importance for human and animal health. Therefore efforts are undertaken for the improved production of more effective vaccines. Of course, the main purpose of all these approaches is to save lives and improve the quality of life for human beings. However, there is also some remarkable effort in the food industry and the associated animal production, especially in the case of some Flaviviridal viruses (BVD), where > 80% of all cattle herds are found to be infected. These viruses can cause tremendous economic losses of calfs and embryos (Ames, 1990). Because of these facts, there is a continuous endeavour for improving the manufacturing of therapeutics or preventing agents such as vaccines for the treatment of cattle. The competitive economic situation and the specific market demands still require effective and high yield production methods, especially in the case of one of the most widespread viral diseases in cattle like BVD (Ames, 1990). We have succeeded in establishing an improved method for the production of BVD on the basis of a continuous fermentation mode, that consist of modifications of the corresponding process and media improvements.

Animals↗
PubMed1994

Interaction of cell culture with downstream purification: a case study.

Separation of product from secreting mammalian cells in the culture both means the transition from product generation to product isolation. This interface within a biotech production process has to perform a proper solid/liquid phase separation of the cell suspension to make the product containing fluid amenable for further purification. These subsequent steps require fluid with low occurrence of contaminants in order to function properly. The goal of this study was to evaluate some economic and fast cell separation methods for the preparation of a product fluid ready for use in further ultrafiltration and chromatographic processes. We have performed experiments to test the usefulness of disc stack centrifuges and tangential flow microfiltration units at large scale. Both systems revealed outstanding prospects with regard to throughput and scale up properties. However, the centrificgation did not lead to a fluid sufficiently free of particles for direct ultrafiltration or chromatography. Thus, an additional filtration step was necessary. On the other hand microfiltration led to an acceptable quality of process fluid directly. By optimisation of process parameters an effective, reproducible and robust cell separation can be obtained. However, our experience has been that such optimal conditions are somewhat specific for a narrow range. Thus, even the equipment functioning well with one type of cell would possibly not perform as well with another cell or even with the same cell under conditions slightly different to the usual situation.

Animals↗
PubMed1994

Pentapeptide identified as a monoclonal antibody binding site in the serine-protease domain of t-PA.

The first defined sequential epitope of the tissue plasminogen activator (t-PA) was determined by a monoclonal antibody against a synthetic peptide segment corresponding to peptide sequence 341-354 of t-PA. This segment was selected by computer assisted epitope prediction. Balb/c mice were immunized with catalase-peptide and tripalmitoyl-S-glyceryl-cys-teinyl-seryl-peptide conjugates. A monoclonal antibody derived from this immunization was reactive with native recombinant t-PA (rt-PA) and reduced carboxymethylated recombinant t-PA (RCM rt-PA). The sequential epitope was detected by Pepscan method using overlapping octa- and nonapeptides. By fine epitope mapping with tetra-, penta-, hexa- and heptapeptides the epitope was minimized to the pentapeptide EEEQK (347-351). Replacement set analysis confirmed the importance of this amino acid sequence, especially of the amino acid E348, for antibody binding. Functional assays of rt-PA were not affected by this antibody indicating that the epitope has no influence on the enzymatic center and the binding site of the inhibitor. The analysis demonstrates that the predicted recognition site of the monoclonal antibody 17-134/11 is exposed on the surface of the native rt-PA molecule.

Amino Acid Sequence↗
PubMed1994

Gene stability in mammalian cells and protein consistency.

The safety of a patient who is the recipient of protein drugs has to be assured. A "wrong" protein is thought to represent a great risk. The philosophy of testing strategies related to gene stability with product safety will be discussed in the light of experimental data available today. Although all mammalian cell lines used in the production of biologicals including recombinant DNA-derived lines have been produced from individual clones (functional monoclonality) they have been found to be heterogenous with regard to the genomic content (number of chromosomes, characteristics of identifiable chromosomes and position and number of integrated recombinant sequences). The verification of the presence of correct gene in a production cell line constitutes a well accepted and useful test, especially if derived by "population sequencing". A batch not related repeated confirmation of this fact cannot lead to any additional assurance for the correctness of all proteins constituting a given product beyond the level provided by cheminal testing. In contrast to this obvious and unavoidable heterogeneity in cellular genomes, the coding regions of genes have not been shown to change. Evidence is available to demonstrate the consistency of protein products originating from recombinant (and hybridoma) cell lines, e.g. more than 500,000 patients have received and tolerated rtPA well.

Aneuploidy↗
PubMed1992

Experimental approaches to guarantee minimal risk of potential virus in purified monoclonal antibodies.

Regarding biological products, increasing awareness of potential side effects have placed great importance not only at protein purity regarding other proteins but on the removal of biologicals such as DNA and especially virus the importance of which may not be known. Monoclonal antibodies (Mab) have come to be an important class of molecules obtained from hybridoma cells, i.e., nonrecombinant cells in culture. It has been noted during the last years, that with rare exceptions hybridoma cell lines contain retrovirus like particles. The infectious nature of the EM-visible particles has been tested for, however, in most cases not been substantiated. In order to bring these valuable biological reagents, Mab's, to good use in man for imaging or therapy, the remaining concern about a potential retroviral infection has to be reduced to an acceptable minimum. We describe experimental approaches for the validation of chromatographic and ultrafiltration steps used in the production of monoclonal antibodies to remove and inactivate murine retrovirus. Present day biotechnological manufacturing processes have been devised incorporating a number of strategic preventive measures that have found wide spread acceptance. They permit to answer the question: how can a potentially harmful infection by an unknown virus be excluded. Knowledge of the efficacy of purification steps to clear infectious model virus is fundamental to devise biotechnological manufacturing processes yielding a purified antibody for use in man.

Animals↗
PubMed1988

Purification of proteins produced by biotechnological process.

Proteins maintain functions important to life. Faulty functioning or deficiency gives rise to pathological reactions. These proteins can now be produced, using the methods of recombinant DNA technology and administered to patients for replacement therapy. Many proteins as active ingredients are already available for use as immunomodulators, agents for tumour treatment, plasma proteins and hormones. They are in various stages of development, ranging from cloning of the producing cells to marketing of the finished products. Since the active substances are proteins synthesized by recombinant cells, their purification presents a particular challenge to protein chemists. Purification of recombinant DNA-derived proteins intended for human use is an essential part of the biotechnical process. It starts immediately after the fermentation of the host cell. The characteristics of the protein determine which microorganisms or cell cultures are used and this in turn defines the first purification step. The microorganisms are disrupted, and the insoluble protein, desposited in "inclusion bodies" has to be renatured, or the proteins secreted by cells and have to be concentrated. The subsequent strategy for purification of the protein does not depend on the fermentation process but is entirely determined by the physiochemical properties of the proteins. The goal of the first purification step is to isolate as fast and quantitatively as possible the recombinant protein from the culture filtrate, in order to minimize potential changes brought about by proteases or glycosidases. Immunoaffinity or ligand-affinity chromatography is used preferentially for this purpose. The concentration of protein and buffer changes are carried out by precipitation followed by reconstitution or, preferably, by dialysis and ultrafiltration/diafiltration.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1986

A sensitive radioimmunoprecipitation assay for the detection of antibody to recombinant human gamma-interferon: comparison to a bioassay neutralization test.

This report describes a specific radioimmunoprecipitation (RIP) assay for the detection of antibodies to recombinant DNA (rDNA) derived human gamma-interferon (rHuIFN-gamma). The assay was shown not to detect antibodies to rHuIFN-alpha, rHuIFN-beta, human lymphotoxin, or E. coli proteins and was reproducible with intraassay and interassay coefficients of variation of 1.6 and 3%, respectively, for the log titer of a high positive control. Comparison of this assay with a standard bioassay for detection of neutralizing antibody (abrogation of the inhibitory effect of rHuIFN-gamma on EMC virus replication in A549 cells) demonstrated that the RIP assay was more sensitive for detection of HuIFN-gamma neutralizing monoclonal antibody. Nonneutralizing monoclonal antibody was detectable in the RIP assay but not in the bioassay neutralization test. Examination of polyclonal antisera (rabbit and monkey) that contained neutralizing antibodies also demonstrated the RIP system to be a more sensitive indicator of the presence of antibodies than the bioassay neutralization test. In preliminary studies of human samples (86 patients) from clinical trials using an assay precipitation system capable of detecting antibody of the IgG, IgM, IgA, and IgE classes, no antibody to rHuIFN-gamma was observed. These patients were also found negative for neutralizing antibody to rHuIFN-gamma.

Antibodies↗
PubMed1985

Selective inhibitory effect of Hu-IFN-gamma on the agarose clonability of tumor-derived lymphoid cell lines.

Recombinant human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were compared for their ability to influence the proliferative capacity of tumor-derived cell lines and of normal B lymphocytes infected in vitro by Epstein-Barr virus (EBV). EBV-induced B-cell proliferation was suppressed almost completely when 10(2) U/ml IFN-alpha were added to the culture medium while the same dose of IFN-gamma had significantly lower inhibitory activity. The pure IFNs differed in their ability to influence the growth of three Burkitt lymphoma-derived cell lines, Raji, Daudi, and Namalwa, depending on whether the cells were propagated in suspension or in semisolid cultures. IFN-alpha inhibited cell proliferation under both culture conditions with thresholds of sensitivity characteristics for each cell line. In contrast, IFN-gamma had no effect on the growth in suspension but it abolished the clonogenic potential of tumor cell lines in semisolid agarose. The results suggest that the two IFN types may exert their growth inhibitory activity through different mechanisms of action.

B-Lymphocytes↗