Use of sulfonation method to determine DNA contamination of cell cultured monoclonal antibodies produced for human therapeutics.

In the past few years, culture of transformed mammalian cells has been widely used to produce natural or recombinant molecules, as monoclonal antibodies (MAb) and cytokines. For therapeutic use, the cellular DNA level must be determined. A number of techniques have been developed to measure the DNA content, based on sequential extraction blotting and hybridization with a labeled DNA probe. The sulphonate marker has been recently introduced by PBS-Orgenics; it allows the determination of picograms (pg) quantities of purified DNA. However, it is not simple to measure in complex biological samples especially when a large amount of protein is present. In considering the following points: 1. Precautions in handling the samples at different steps of preparation; 2. Modifications of the original technique 3. Concentration of samples expected at very low level, we are able to dose up to 2 pg of contaminant DNA per mg of MAb with a satisfactory reproducibility and reliability. This level is required not only to qualify final MAb but also to evaluate the efficiency of the purification process. Efforts are being made to achieve a better sensitivity.

[Requirements for standards for monoclonal anti-A and anti-B antibodies]. / K otázce pozadavku na oborovou normu pro monoklonální anti-A a anti-B protilátky.

On the model of the Soviet monoclonal anti-A and anti-B antibody--erythrocytes of the groups A1, A2, A1B and A2B the conditions for the elaboration of the branch standard for anti-A and anti-B monoclonal antibodies were worked out. In comparison with the standard for human sera determining the ABO groups, the requirement for avidity can be increased, furthermore it seems necessary to determine the percentage of non-A blood cells in the A2B group, which should not exceed 30%, and the percentage of non-B blood cells in A1B erythrocytes, which should not exceed 10%.

A preliminary analysis of the 11th International Histocompatibility Workshop monoclonal antibodies.

In the 11th International Histocompatibility Workshop prescreening study, HLA Class I and Class II monoclonal antibodies (mAb) were tested on panels of peripheral T and B cells and homozygous typing cell (HTC) lymphoblastoid lines. The 102 Class I mAb, of which 64 were new, were screened on 63 HTC lines and 20 peripheral T cells. The 144 Class II mAb were tested on 63 HTC lines, 20 peripheral B and 10 peripheral T cells. For Class I, 45 of the 102 mAbs tested proved to have clear and identifiable reaction patterns on both PBL and HTC lines and included several interesting new HLA-A and -C locus antibodies. Of the 144 Class II mAb, of which 99 were new, 18 DR, 22 DQ and 7 DP antibodies were judged excellent. The DR antibodies included some which were antigen-specific but the majority were complex antibodies, while the DQ antibodies, as previously, identified the majority of DQ antigens both singly and in combinations. The encouraging number of DP antibodies was interesting in the limited range of sites they appear to detect.

How to deal with potential contaminations of monoclonal antibody preparations.

The message of this contribution is that simple and concrete regulatory rules can not yet be established. We may have added vagueness. We would welcome a dialogue between researchers, manufacturers and regulatory authorities. Prerequisite for a fruitful discussion among all parties will be the availability of information on several aspects of this problem.

Toxicological testing of monoclonal antibodies.

Toxicity studies with monoclonal antibodies for in vivo diagnostic or therapeutic administration must be designed case by case to take into account the intended use in humans and the type of structure (nude or coupled to immunotoxins or radionuclides). The design of experiments is influenced by the origin of the clone (murine or human with different problems of antigenicity) and the type of culture (ascites or fermentation with various kinds of possible contaminations). Therefore, routine animal tests must be supplemented by analytical procedures and assays of pharmacological quality control (pyrogenicity, abnormal toxicity). The antigenicity of these antibodies for experimental animals imposes restrictions concerning duration of experiments and extrapolation of the results to humans. Selection of appropriate species is difficult, they should have identical target cells if possible. Sometimes the choice is limited to non-human primates. Toxicological experiments should be based on pharmacokinetic studies. Formal tests on mutagenicity, teratogenicity and cancerogenicity are, in most cases, irrelevant. Antibody formation may make it impossible to carry out longer-term studies.

Monoclonal antibodies for use in man: current regulatory situation in the Federal Republic of Germany.

The article addresses the requirement to be met for approval of monoclonal antibodies with special emphasis on products coupled with radionuclides and on principles for the conduct of clinical trials. According to the German Drug Law monoclonal antibodies are considered as being sera. Therefore, the Paul-Ehrlich-Institut, Federal Office for Sera and Vaccines, is responsible for marketing authorizations. Sera and vaccines need a special manufacturing licence which is given by the competent authority of the Federal State. Batches of monoclonal antibodies can only be marketed if they have been released by the Paul-Ehrlich-Institut; in connexion with batch control the importance of reference preparations is stressed. The standard requirements for the data to be submitted with the applications for marketing authorizations are in accordance with the EEC Council Directives and Notes for Guidance. For the testing of radioactive monoclonal antibodies in clinical trials, compliance with both the Drug Law and The German Radiation Protection Ordinance must be ensured. In addition to the authorizations required for non-labelled monoclonal antibody products, the use of radioactive substances in diagnosis and therapy requires an authorization by the competent Federal State authority. The main purpose of the planning and performance of clinical trials with new monoclonal antibody in diagnosis and therapy must be the comparison with established diagnostic tools and/or established medicinal products of known effect.

Quality control of anti human CD3 and CD4 monoclonal antibodies.

By testing two monoclonal antibodies (MoAb), we tried out two methods, which might be suitable for quality control of anti human T cell MoAb. RIV6 and RIV9 were investigated by flow cytometry and Enzyme-linked Immunosorbent Assay (ELISA). The characteristics, specificity and biological activity of RIV6 and RIV 9 were determined and compared respectively with other CD4 specific MoAb, OKT4, OKT4a, LEU3a and the CD3 specific MoAb OKT3 and LEU4 by flow cytometry. An ELISA was set up to quantify mouse IgG3 antibody. Specificity, plate variability and day to day effects were determined. The results illustrate that ELISA and flow cytometry are valuable tools in the quality control of these antibodies.

An enzyme-linked immunosorbent assay for erythropoietin using monoclonal antibodies, tetrameric immune complexes, and substrate amplification.

We recently reported the development of several monoclonal antibodies (MoAbs) to native human erythropoietin (Ep). In the present study we have used the two antibodies with highest affinity to develop a two-sided or sandwich enzyme-linked immunosorbent assay (ELISA) to measure Ep in human serum. In this assay Ep is incubated in microtiter wells precoated with the first (IgE) anti-Ep antibody. Assay wells are then incubated with the second (IgG1) anti-Ep antibody, which is labeled noncovalently with the enzyme alkaline phosphatase (AP) by means of bispecific tetrameric antibody complexes consisting of IgG1 anti-Ep cross-linked to IgG1 anti-AP using rat MoAbs specific for mouse IgG1. Application of this noncovalent labeling procedure, in combination with substrate amplification, results in a detection sensitivity of 0.5 to 1.0 mU/sample (5 to 10 mU/mL), which makes this assay suitable for measuring normal serum Ep levels. The validity of this ELISA for quantitating Ep in biological fluids was demonstrated by the parallelism obtained between pure recombinant Ep dose-response curves and those obtained with plasma and serum from healthy donors and patients with various hematologic disorders. Normal plasma Ep levels detected with this ELISA ranged from 9 to 101 mU/mL with a mean of 32 +/- 23 (SD) mU/mL. Ep levels in sera from patients with polycythemia vera were in the low to normal range, whereas Ep levels in sera from patients with secondary polycythemia and patients with aplastic anemia were moderately to strongly elevated. These results demonstrate that the Ep-ELISA is a sensitive, reliable, and nonradioactive immunologic method for quantitating Ep levels and should prove useful in a variety of clinical and laboratory settings.

Direct detection of Neisseria gonorrhoeae with monoclonal antibodies characterized by serotyping reagents.

A panel of monoclonal antibodies (MAbs) directed to outer membrane protein I were generated with the ultimate aim of detecting Neisseria gonorrhoeae in patient samples by a direct immunofluorescence (IF) test. In an initial evaluation of the sensitivity of these reagents, a cocktail of six IF MAbs recognized 491 (91%) of 540 gonococci isolates from several centers in Sydney, Australia. IF MAbs designated 185 and 228 recognized serovars of WI serogroup and IF MAbs 208, 210, and 312 recognized serovars of WII/III serogroup. IF MAb 198 recognized serovars within both serogroups. Three additional IF MAbs, designated 322, 323, and 330, were then generated by using strains which failed to react with the original MAb cocktail and which belonged to particular serovars. The new cocktail of nine IF MAbs recognized 96% of the gonococcal isolates, which incidentally contained representatives of serovars shown to have a worldwide distribution in previous studies. Although subtle differences were apparent in the reaction patterns found with coagglutination (serotyping) and IF, there nonetheless seems to be merit in the approach of continually evaluating the sensitivity of diagnostic reagents such as MAbs. This is especially true with an organism such as N. gonorrhoeae, which has the capacity to regularly alter the antigenic structure of its outer membrane proteins.

Practical considerations in the production, purification, and formulation of monoclonal antibodies for immunoscintigraphy and immunotherapy.

Issues associated with the large-scale production of monoclonal antibodies for pharmaceutical applications are examined. The development of a commercial monoclonal antibody production process involves much more than just scaling-up the laboratory process and making it cost-effective. It involves establishing the hybridoma cell bank with cells that are free of adventitious agents such as viruses and mycoplasma, that have stability in continuous culture for antibody-production rate and cell viability, and that do not have unusual or expensive media requirements. The style and mode of operation of the bioreactor used to produce the antibody must be explored. The antibody-based product must be processed to high levels of purity, and specific contaminants such as DNA and endotoxin must be reduced to extremely low levels. Appropriate labeling or drug conjugation chemistries must also be developed. The product must be formulated so that it has performance characteristics that are stable over a reasonable period of time. Adequate test procedures must be developed to assure product purity, activity, stability, and safety on a lot-to-lot-basis. Compliance with federal regulations, guidelines, and procedures must be guaranteed. In the coming decade, it is likely that the two arms of biotechnology, hybridoma technology and recombinant DNA technology, will be used together to generate unique protein molecules. These new reagents will face the same practical considerations summarized in this review.

[Aspergillus fumigatus: evaluations of the standardization of diagnostic extracts]. / Aspergillus fumigatus: Ansätze zur Standardisierung von Extrakten für die Diagnostik.

Extracts of Aspergillus fumigatus used for serodiagnosis of allergic diseases are of unknown and nonstandardized composition. Applying immunoprint technique subsequent to isoelectric focusing commercial and self-prepared extracts are compared. By use of an ABPA-pool serum and monoclonal antibodies recognizing antigens of this fungus, different immunological reactivities of the extracts can be demonstrated. It is suggested to use monoclonal antibodies as secondary standards as well as to improve standardization.