Collaborative study report: Evaluation of the ATCC experimental mycoplasma reference strains panel prepared for comparison of NAT-based and conventional mycoplasma detection methods

Abstract

The main goal of this collaborative study was to evaluate the experimental panel of cryopreserved mycoplasma reference strains recently prepared by the American Type Culture Collection (ATCC^®) in order to assess the viability and dispersion of cells in the mycoplasma stocks by measuring the ratio between the number of genomic copies (GC) and the number of colony forming units (CFU) in the reference preparations. The employment of microbial reference cultures with low GC/CFU ratios is critical for unbiased and reliable comparison of mycoplasma testing methods based on different methodological approaches, i.e., Nucleic Acid Testing (NAT) and compendial culture-based techniques. The experimental panel included ten different mycoplasma species known to represent potential human and animal pathogens as well as common contaminants of mammalian and avian cell substrates used in research, development, and manufacture of biological products. Fifteen laboratories with expertise in field of mycoplasma titration and quantification of mycoplasmal genomic DNA participated in the study conducted from February to October of 2012. The results of this study demonstrated the feasibility of preparing highly viable and dispersed (possessing low GC/CFU ratios) frozen stocks of mycoplasma reference materials, required for reliable comparison of NAT-based and conventional mycoplasma detection methods.

Introduction

Mycoplasma contamination remains a serious, costly, and challenging problem during the development and manufacture of cell-derived biological and pharmaceutical products [1], [2], [3], [4], [5], [6]. To minimize the risk of product contamination and to ensure the safety of cell-derived biologics, various regulatory agencies require that freedom from mycoplasma contamination to be demonstrated for starting cell substrates, viral seeds (in case of vaccine production), and crude cell harvests prior to further processing [6], [7], [8], [9]. The currently recommended mycoplasma testing protocol, developed several decades ago, includes the use of an agar and broth culture method and an indicator cell culture method to detect cultivable and non-cultivable (in the conventional media) mycoplasma species, respectively [6], [7], [8], [9]. Despite the historically successful use of these two mycoplasma detection methods, the limit of detection (LOD) of these methods continues to not be well-defined. However, from the available historical and personal experience, the LODs of the optimized broth culture and the indicator cell culture methods are in the range 3–10 CFU/10 mL and 10–100 CFU/mL, respectively. Although the combined use of these two methods ensures the required level of product safety, the entire testing procedure is laborious, cumbersome, costly, and time-consuming, requiring a minimum of 28 days to complete [7], [8], [9], [11], [12], [13]. Therefore, the development and implementation of alternative mycoplasma detection methods, which could expedite mycoplasma testing during manufacturing stages when a rapid “release or hold” decision is needed prior to further material processing, would be of obvious benefit. As such, many commercial diagnostics manufacturers and reference laboratories have developed innovative rapid assays for detecting mycoplasma. However, prior to implementation of an alternative method for routine testing of biologics, the method must show equivalency or superiority to the currently approved mycoplasma testing method with regard to limit of detection to ensure the required product safety and purity. A comparison of the LOD of methods with different biological readouts (e.g., genomic copies (GC) vs. colony forming units (CFU)) poses significant methodological and technical challenges. Thus, previous attempts to compare NAT-based methods, which detect the presence of mycoplasma-specific nucleic acids (either genomic DNA or cellular RNA) regardless of cell viability, and culture-based methods, which detect only viable cells, led us to understand that unbiased comparisons require special reference materials with a high percentage of viable cells and a low degree of aggregation [4], [10]. The GC/CFU ratio is an important and valuable parameter to assess both the viability of bacterial cells and their level of aggregation in cultures. In practice, the GC/CFU ratio varies over a wide range due to many factors, e.g., innate features of specific mycoplasma strains, culture/incubation conditions, growth phase at which samples are collected, conditions of freezing/thawing and storage, etc [10]. For that reason, comparability studies can yield compromised results leading to an inadvertent overestimation of the LOD of NAT-based methods when mycoplasma reference materials used were prepared inappropriately. To avoid this problem, the mycoplasma stocks used for comparability studies should have the lowest possible GC/CFU ratios, reflecting high cells viability and low level of their aggregation [4]. Furthermore, the preparation of frozen reference materials may also affect the GC/CFU ratio in culture and thus certainly requires optimizing mycoplasma cell culture freezing conditions including cryoprotectant concentration (%) and cooling rate [14], [15], [16], [17], [18].

The primary goal of this collaborative study was to perform a multi-laboratory evaluation of the quality of the experimental mycoplasma reference culture panel prepared by the American Type Culture Collection [19]. The evaluation included the use of different protocols and methods appropriate to reliably assess (i) the titer and (ii) the number of genomic copies and (iii) ratio between values of these parameters to assess the viability and dispersion of mycoplasma cells in the cryopreserved stocks.