Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures

Abstract

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell‐based and indicator cell‐based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real‐time decision making during the biomanufacturing process. To support real‐time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell‐based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single‐use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read‐out from a traditional method.

Report of a clinical and laboratory management of cell therapy for knee cartilage in the face of mycoplasma contamination

“…(5) Therefore, mycoplasma detection and elimination are necessary for in vitro cell culture. (5)(6)(7)(8)(9)(10)(11)(12) Cell therapy laboratories are required to operate under strict good manufacturing practice conditions to minimize possible mycoplasma contamination. Nonetheless, occasional mycoplasma contamination of cell therapy products may also derive from the biological sample donor.…”

Sık Kullanılan Bazı Hücre Hatları için Kalite Kontrol: Mikoplazma Kontaminasyon Tespiti, Sitokrom B ve Sitokrom Oksidaz Alt Birim I Genlerinin DNA Dizi Analizlerinin Gerçekleştirilmesi

“…Bununla birlikte mikoplazma kontaminasyonu genellikle gözle kolayca tespit edilemez, ancak varlıkları hücrelerin sağlıklı bir şekilde büyümelerine engeldir (hücrelerde kromozom anomalileri de dâhil birçok sorun oluştururlar) 14,15 . Bu kontaminant hakkındaki bilginin artması ile son birkaç yılda mikoplazma kontaminasyonu nedeniyle sonuçların doğru yayımlanamaması üzerine bir dizi makale yayınlanmıştır 12,13,[16][17][18] . Bu nedenle hücre kültürlerinin sık ve gizli bir kontaminantı olan hücre duvarı bulunmayan mikoplazma hücre kültür çalışmaları için önemli bir tehdittir 14,15,19 .…”