Alan Doyle

DNA fingerprinting--a valuable new technique for the characterisation of cell lines.

DNA fingerprinting is an important new development for the authentication of cell lines. Multilocus methods such as those developed by Alec Jeffreys provide information on a wide range of genetic loci throughout the human genome and thus give a useful genetic “snap-shot” of a cell culture. Our work has shown that Jeffreys multilocus fingerprinting method can be applied to cell lines from a wide range of animals including reptiles, birds, fish and diverse mammals. It can also differentiate very closely related cell lines including those from the same mouse strain. Routine fingerprint analysis has enabled an unprecedented level of confidence in the consistency of cell stocks. Our results demonstrate that this straightforward method represents a powerful and readily interpreted system for cell authentication and exclusion of cross-contamination.

Multilocus DNA fingerprint analysis of cellbanks: Stability studies and culture identification in human B-lymphoblastoid and mammalian cell lines

The technique of multilocus DNA fingerprinting has great potential for the authentication of animal cell cultures and in identification of cross-contamination. The Alec Jeffreys probes 33.6 and 33.15 were used as multilocus probes to demonstrate the consistent DNA fingerprint profiles in human peripheral blood and its derivative Epstein-Barr virus (EBV) transformed B-lymphoblastoid cultures maintained by repeated subculture for six months. However, fingerprint analysis of EBV transformed cultures generated from small numbers of cells showed that the majority (seven of eight cultures) had anomalous profiles. Some of these altered profiles shared common features not seen in the peripheral blood pattern. Analysis of seven murine hybridoma clones from a single fusion experiment revealed only two clones which could not be distinguished using probe 33.15. Further studies of master and distribution cell banks for eleven cell lines demonstrated consistent fingerprint profiles in all cases except one (U937). However, this cell line showed only minor differences in the master and distribution bank profiles. These data indicate that, while changes in fingerprint profile may be identified in exceptional instances, the multilocus fingerprinting method using probes 33.6 and 33.15 is a powerful and reliable tool in the quality control of animal cell cultures.

Establishment and characterization of a human CD4 positive cell bank for HIV related studies

The human CD4 positive cell lines JM, CCRF, CEM, U937, HL60 and THP-1 have been cleared of mycoplasma contamination and defined by DNA fingerprinting and cell surface phenotype marker analysis. These cells have been banked and are now available as a source of standardized cell lines for HIV related research.

GENETIC AND BIOCHEMICAL ANALYSIS OF A MURINE HYBRIDOMA IN LONG-TERM CONTINUOUS CULTURE

Abstract Multilocus DNA-fingerprinting was used to monitor the genomic stability of a murine hybridoma grown in a CSTR for 83 days. Minor changes in the fingerprint were observed by day 20 with additional changes seen by day 83. The values of QsmAb at the beginning and end of the culture were not significantly different, neither was there a change in product heterogeneity as shown by IEF affinity immunoblot analysis. There was no change in either the isotype or specificity of the mAb during the culture. Therefore the genetic drift was not associated with marked changes in the product. The minor genetic drift observed by DNA-fingerprinting in long-term continuous cultures is therefore probably not significant.

Quality control and validation

There is increasing acceptance and utilization of human and animal cell lines for the manufacture of biologicals. Continuous cell lines offer more ethically acceptable and reproducible means of production in comparison with the use of animals and primary cells. Furthermore, mammalian cell lines have the potential to provide complex recombinant biologicals with post-translational modifications (i.e. glycosylation) which are typical of native glycoproteins. The proven capabilites of 10000–1 systems has demonstrated that cell culture can be used in industrial-scale operations [1]. In regulatory matters there is a developing trend for emphasis to be placed on defining and validating the end product, rather than all constituents of the production process. This may enable the use of a wider variety of cell lines, thus increasing the opportunities to enhance productivity.

Multilocus DNA Fingerprinting in the Analysis of Cell Stocks: Stability Studies and Application to a Wide Range of Species

DNA fingerprinting is a new technique which provides a highly useful supplement to the standard authentication techniques of isoenzyme analysis and cytogenetics. While fingerprinting does not identify the species of origin it does give specific identification of a cell line. As an alternative, single locus probes can be useful in distinguishing very closely related cell lines. However, they lack the capacity of multilocus DNA fingerprinting whereby a single test can both identify the cell line with good specificty and exclude cross-contamination by cell lines from an extremely wide range of species. We demonstrate the stability of fingerprints of cell lines during repeated sub culture, mycoplasma infection (with two species) and high level antibiotic treatment. This data contributes to the validation of DNA fingerprinting in cell technology and promotes its use for routine quality control of cell stocks of diverse species of origin.

PRODUCT PROCESS VALIDATION WITH HUMAN IMMUNODEFICIENCY VIRUS-I

HIV-I was used to spike a monoclonal antibody preparation which was subsequently purified by affinity chromatography. Presence of HIV-I was assessed at different stages of the process, by a syncitial assay, a p24 antigen ELISA, and by presence of reverse transcriptase. Keywords:- HIV-I, validation, syncitial assay, p24 core antigen, reverse transcriptase.

VALIDATION OF DNA FINGERPRINTING IN ANIMAL CELL TECHNOLOGY: THE DIFFERENTIATION AND IDENTIFICATION OF MURINE HYBRIDOMA CLONES FOR A SINGLE FUSION AND THE STABILITY OF TRANSFORMED CELL LINES

DNA fingerprinting is under assessment for use in the quality assurance of cell stocks: providing information on the consistency and stability of cell stocks as well as screening for cross-contamination. At the European Collection of Animal Cell Cultures (ECACC) Alec Jeffreys probes 33.6 and 33.15 have been used for analysis of cell lines from a wide range of species. We have previously reported our initial assessment of DNA fingerprinting for use in the quality control of cell banks [1, 2]. This report describes further validation of fingerprinting in animal cell technology. DNA fingerprints have demonstrated consistency between human and peripheral blood mononuclear cells and Epstein-Barr virus transformed B lymphoblastoid lines from the same individual. Analysis of long-term cultures of these cell lines (ie up to 6 months) also revealed the stability of their fingerprints. Analysis of murine hybridomas has shown certain limitations of the 33.6 probe in that clones from the same fusion expressing different Ig subclasses, could not be differentiated. Significantly probe 33.15 enabled differentiated of clones producing antibodies of identical heavy chain subclass although it was not possible to differentiate all clones. The work discussed in this report represents the ongoing process of validation of DNA fingerprinting: supporting its use in the authentication of EBV transformed cell lines and the analysis of murine hybridomas.