Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques

Abstract

Mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are known to be occasional microbial contaminants of cell cultures that produce biologics. This presents a serious concern regarding the risk of mycoplasma contamination for research laboratories and commercial facilities developing and manufacturing cell-derived biological and biopharmaceutical products for therapeutic use. Potential undetected contamination of these products or process intermediates with mycoplasmas represents a potential safety risk for patients and a business risk for producers of biopharmaceuticals. To minimize these risks, monitoring for adventitious agents, such as viruses and mycoplasmas, is performed during the manufacture of biologics produced in cell culture substrates. The “gold standard” microbiological assay, currently recommended by the USP, EP, JP and the US FDA, for the mycoplasma testing of biologics, involves the culture of viable mycoplasmas in broth, agar plates and indicator cells. Although the procedure enables highly efficient mycoplasma detection in cell substrates and cell-derived products, the overall testing strategy is time consuming (a minimum of 28 days) and requires skilled interpretation of the results. The long time period required for these conventional assays does not permit their use for products with short shelf-lives or for timely ’go/no-go’ decisions during routine in-process testing. PCR methodology has existed for decades, however PCR based and other alternative methods for mycoplasma detection have only recently been considered for application to biologics manufacture. The application of alternative nucleic acid-based, enzyme-based and/or recombinant cell-culture methods, particularly in combination with efficient sample preparation procedures, could provide advantages over conventional microbiological methods in terms of analytical throughput, simplicity, and turnaround time. However, a challenge to the application of alternative methods for detection of mycoplasmas remains whether these alternative methods can provide a limit of detection comparable or superior to those of the culture methods. An additional challenge is that nucleic acid amplification technique (NAT) methods do not allow for accurate discrimination between viable and non-viable mycoplasma contaminants, which might lead to false-positive results (e.g. from inactivated raw materials, etc.). Our review provides an overview of these alternative methods and discusses the pros and cons of their application for the testing of mycoplasmas in biologics and cell substrates.

Introduction

Mycoplasmas (commonly used trivial name for microorganism of the class Mollicutes) are known to be one of the smallest free-living forms of bacteria that are broadly distributed in nature [1]. Based on their genetic background and molecular features, mycoplasmas were hypothesized to originate evolutionarily from gram-positive eubacteria [1], [2]. As a result of degenerative evolution, mycoplasmas irreversibly lost the bacterial cell wall and some metabolic pathways present in phylogenetically closely related bacteria (Lactobacilli, Clostridia, and Streptococci) [2]. These structural and biochemical deficiencies make mycoplasma replication largely dependent on the nutritional compounds provided by their natural hosts or in the environment they inhabit [1], [3]. Thus, Ureaplasma species colonizing the genitourinary tract are able to efficiently utilize urea as a source of energy [4], whereas the hemotrophic mycoplasmas (hemoplasmas) are able to replicate only in the bloodstream of their hosts [5]. The majority of known mycoplasmas are host-specific commensals colonizing a wide range of plant, insect, reptilian, avian, mammalian, and humans [1]. Nevertheless, a few mycoplasma species are pathogenic for their hosts and can, under certain conditions, cause diseases in the hosts [1], [6].

Due to their broad distribution in the nature, mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are found to frequently be the cause of contaminations of cell substrates as well as raw materials commonly used for manufacture of a variety of biological and therapeutic products [7]. Mycoplasma contamination of cell substrates inevitably results in a dramatic alteration of biological characteristics of the contaminated cells [7]. This in turn causes manufacturing and safety problems for biologics produced from the contaminated cells. Most mycoplasmas from warm-blooded animals have a potential to efficiently grow in cell substrates. Mycoplasma contamination, even at very low levels, at the initial steps of manufacture, generally results in the loss of the whole batch of the product due to poor cell growth or regulatory safety concerns. Regardless of an observable impact during manufacturing, the detection of mycoplasma would necessitate the time and expense of the impacted batches being discarded and the facility being appropriately cleaned. The detection of mycoplasma contamination during manufacture of even one batch of product, therefore, represents a serious risk to the facility and other products manufactured in that facility. To mitigate the risk of mycoplasma contamination of cell substrates and to ensure the safety of cell-derived biologics, all current regulation documents do not permit the use of mycoplasma contaminated cell substrates, starting materials or crude harvests for manufacture of biological and therapeutic products [8], [9], [10], [11]. According to key regulatory guidance documents, the purity of cell substrates should be assured by using well-defined detection methods recommended by regulatory agencies in guidances or in compendia. It is necessary to note that in general the recommended protocols for mycoplasma testing rely on the use of approaches based on culturing of viable mycoplasmas in broth or indicator cell line. Needless to say, these testing protocols are laborious and usually necessitate a long time (up to 28 days) for the test completion. The latter limitation significantly complicates the application of recommended methods for testing of some novel biological and therapeutic products, which possess short shelf-lives not exceeding the overall testing time (e.g., products used for tissue regenerative therapy). This time frame is also inconvenient for the mycoplasma testing of intermediate products such as bioreactor harvests, which in some cases need to be rapidly processed. Thus, development and application of alternative methods based on latest achievements in molecular technologies are important and urgent needs for safety testing of novel cell-derived biologics. The purpose of the current review is to analyze advantages that alternative molecular technologies offer for improving mycoplasma testing and describe their limitations in terms of critical issues that impact their adequate evaluation, validation, and comparison to conventional mycoplasma testing methods.