Evaluation of the capability of a real-time PCR assay to detect mycoplasma in advanced therapy medicinal products

Abstract

Background Advanced therapy medicinal products (ATMPs) are new medical products based on genes (gene therapy), cells (cell therapy) and tissues (tissue engineering). Mycoplasmas are among the world’s smallest bacteria capable of independent reproduction. They belong to the class of Mollicutes, have a very slow and parasitic growth and can cause human infections. Mycoplasma contaminations of ATMPs can arise from unsterile source material of different human origin, starting materials or the complex manufacturing process. The traditional growth-based detection method requires a cultivation time of at least 28 days before a contamination can be ruled out with certainty [1]. But shelf lives of final ATMPs are often extremely short compared to classical drugs (24 48 h, sometimes only a few hours). That is why the official culture method or indicator cell method are not suitable. Furthermore ATMP sample amounts are usually limited and of great “value”. Nucleic Acid Amplification Techniques (NAT) allow reducing time to result to just hours and only small sample amount are necessary to generate highly sensitive results. A Mycoplasma Real-time PCR kit was designed especially for the detection of Mollicutes (Mycoplasma, Acholeplasma, Spiroplasma) contamination in ATMPs and cell cultures by using the cells itself, cell culture supernatant or a defined mixture as test material. An acceptable sample volume in respect of the expensive, unique and limited sample is processed for Mycoplasma detection. In this studies the robustness of the kit was demonstrated by testing 20 randomly selected Advanced Therapy Medicinal Products from samples submitted by customers for Mycoplasma detection. These tests were done as part of the kit validation. All selected ATMPs were tested negative for Mycoplasma and could therefore be used as spiking matrices for the intended studies. These 20 different products were spiked with 10 CFU/ml of Mycoplasma fermentans. Each spiked ATMP matrix was then subjected to a DNA isolation process and tested by Real-time PCR. All 20 randomly selected ATMPs out of 20 in total were tested positive in duplicate and fulfilled the acceptance criterion. These results confirmed the suitability and the robustness of the designed Mycoplasma Real-time PCR kit for ATMP testing.