Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins

Abstract

Chinese hamster ovary (CHO) cell cultures used to produce biopharmaceuticals are tested for mycoplasma contamination as part of the ensurance of a safe and pure product. The current U.S. Food and Drug Administration (FDA) regulatory guideline recommends using two procedures: broth/agar cultures and DNA staining of indicator cell cultures. Although these culture methods are relatively sensitive to most species, theoretically capable of detecting as few as 1–10 cfu/ml of most species, the overall procedure is lengthy (28 d), costly and less sensitive to noncultivable species. The detection of mycoplasma using the polymerase chain reaction (PCR) method has been considered an alternative method because it is relatively fast (1–2 d), inexpensive, and independent of culture conditions, however, limitations in sensitivity (limit of detection ≥ 1000 cfu/ml) and the risk of false positive and false negative results have prevented PCR from replacing the traditional culture methods in the industrial setting. In this report, we describe a new PCR assay for mycoplasma detection that appears to resolve these issues while being sufficiently simple and inexpensive for routine use. This assay applies readily available techniques in DNA extraction together with a modified single-step PCR using a previously characterized primer pair that is homologous to a broad spectrum of mycoplasma species known to infect mammalian cell cultures. Analysis is made easy by the detection of only a single amplification product within a narrow size range, 438–470 bp. A high sensitivity and specificity for mycoplasma detection in CHO cell production cultures is made possible through the combination of three key techniques: 8-methoxypsoralen and UV light treatment to decontaminate PCR reagents of DNA; hot-start Taq DNA polymerase to reduce nonspecific priming events; and touchdown- (TD-) PCR to increase sensitivity while also reducing nonspecific priming events. In extracts of mycoplasma DNA, the limit of detection for eight different mycoplasma species is 10 genomic copies. In CHO cell production cultures containing gentamicin, the limit of detection for a model organism, gentamicin-resistant M. hyorhinis, is 1 cfu/ml. The sensitivity and specificity of this PCR assay for mycoplasma detection in CHO cell production cultures appear similar to the currently used culture methods and thus should be considered as an alternative method by the biopharmaceutical industry.

Introduction

Mycoplasma species, including Acholeplasma, are common contaminants of mammalian cell cultures that can disrupt cell function [1], [2], [3] and are undesirable in both research and industrial environments. Mammalian cell cultures used as a substrate in the production of biological products are required to be tested to verify the absence of a viable mycoplasma in part to ensure product purity and safety. The current industry standard in the U.S. for mycoplasma testing is specified in the Food and Drug Administration (FDA) guideline “Points to Consider in the Characterization of Cell Lines used to Produce Biologicals” [4] and involves two tests. The broth/agar culture test is conducted by inoculating cell samples directly on an agar medium as well as into enriched broth to amplify low level contaminants for subsequent detection on agar. In order to detect species not cultivable under these conditions, the sample must also be inoculated into an indicator cell culture for subsequent DNA staining. This latter test is less sensitive and nonspecific for mycoplasma DNA. The broth/agar culture test and the indicator cell culture test are valid only if two species of mycoplasma used as positive controls are detected when inoculated at 10 cfu/ml and 100 cfu/ml, respectively. These culture methods are lengthy (28 d) and costly. Furthermore, some products, such as those used in cell therapy, have a shelf life shorter than the time required to obtain results.

Polymerase chain reaction (PCR) technology is faster and less expensive than the current standard culture methods for mycoplasma detection, thus it is an attractive alternative. Several PCR primers have been designed to amplify evolutionarily conserved rRNA genes and are capable of detecting a broad spectrum of Mycoplasma as well as members of the genera Acholeplasma, Ureaplasma and Spiroplasma [5], [6]. PCR detection of mycoplasmas has been performed routinely in clinical and research laboratories to screen for cell and tissue contaminants. However, limitations in sensitivity and specificity need to be addressed before a PCR assay can be accepted as an alternative method for lot release testing in the biopharmaceutical industry. Although mycoplasma PCR is quite sensitive, detecting 1–10 genomic copies of purified mycoplasma DNA [7], [8], [9], [10], PCR detection of mycoplasmal infections in mammalian cell cultures reported to date is quite insensitive, at approximately 1000 cfu/ml [11], [12], [13]. Such a detection limit is well above the theoretical limit (1–10 cfu/ml) of the standard culture method [14] and also well above the amount (10–100 cfu/ml) in the positive control inoculum recommended by FDA for the culture tests [4]. Even though infection levels of mycoplasmas in cell cultures without antibiotic are typically 10⁷–10⁸ cfu/ml [1], false negative results would be obtained in the event of a low level contamination using an insensitive PCR detection method. This report is the first to describe a highly sensitive and specific PCR assay for the detection of mycoplasmas in CHO cell cultures used in the biopharmaceutical manufacture of recombinant therapeutic proteins.