Mycoplasma release test with low volume protocol: From sample to results in less than 1 hour

Introduction

Testing for mycoplasma contamination is a required release test for Cell and Gene Therapy (CGT) products as specified in the USP, EP and JP. Current compendial methods require 28 days of testing to generate results and are not suitable for short shelf-life products. Alternative nucleic acid test (NAT) methods are available and can reduce the time to results to hours instead of days.

The BIOFIRE® Mycoplasma test is a closed system sample-to-answer nucleic acid test that is designed to report the presence/absence of over 130 mycoplasma species in less than an hour and thereby suitable as a release test for short shelf-life CGT products. A validated protocol involving pre-processing of 10 mL cell samples allows release testing of CGT products at 1x10⁵ cells/mL, following regulatory guidelines with a limit of detection (LOD) of ≤ 10 CFU/mL. As the availability of sample for testing is limited in CGT manufacturing, a protocol which minimizes the sample volume while retaining cells to comply with pharmacopeial guidance is needed.

The objective of to present a new Low Volume protocol that allows the inclusion of mammalian cells from high cell density products thus aligning with the upcoming EP guideline whilst providing the required level of detection (≤ 10 CFU/mL) as a mycoplasma release test.