Company insight
Filtration for preventing mycoplasma contamination
Without effective filtration and sterilisation, mycoplasma contamination of bioreactors is a very real problem that can have serious consequences for the production of pharmaceutical products. However, the PES membrane filters manufactured by BEA Technologies are here to ensure that contamination risk is reduced.
T
he mycoplasma contamination of a cell culture can be difficult to resolve and may cause serious consequences for the preparation of pharmaceutical products. This can lead to a negative impact on production costs and supply schedule. Bacterial contaminations of cell culture in a bioreactor is an important issue which can potentially trigger a serious loss of culture cells. In order to prevent this, it is necessary to take all measures to maintain the sterility of the bioreactor. Initially, steam is passed through the bioreactor and piping to sterilise the vessel and the connected transfer lines. All gases, nitrogen, oxygen, compressed air, as well as all venting connections, must be treated with sterilising gas filters. All liquids, including additives, must pass through sterilising liquid filters before entering into the bioreactor. As liquid additives may contain some substances which are sensitive to heat, they cannot be sterilised using steam and need to be sterilised only through cold sterilisation using bacterial retentive filters.
Origin of mycoplasma contamination
Besides bacteria, however, there is even mycoplasma that may contaminate cell cultures. In this case, the usual 0.2-pm-rated sterilising-grade membrane filters are not able to fully retain mycoplasma. Mycoplasma are the smallest free-living, self-replicating organisms currently identified. They lack a true cell wall and are therefore deformable under pressure. They contain protein, RNA, DNA and enzymes. Various types of mycoplasma are able to contaminate cell cultures and other biological substances. Mycoplasma lacks a cell membrane, so antibiotics that interfere with the formation of cell membranes of the living microorganism are ineffective against mycoplasma. Mycoplasma are extracellular
and additives fed in bioreactor with 0.1 micron retentive membrane filters.
Mycoplasma contamination can be minimised through BEA’s PES membrane filters.
parasites which usually attack the external surface of a cell membrane. They can contaminate different cells in culture, leading to a decrease in growth and significative modifications in the morphology, metabolism and protein synthesis. Mycoplasma species can be of different origins: from human, bovine and porcine sources. The most common are Acholeplasma laidlanii, Mycoplasma aiginini, Mycoplasma fermentariSrMycoplasma hyorhinis, and Mycoplasma orale. All mycoplasma infections can be generally identified as coming from contaminated animal-derived materials and poor aseptic techniques. Animal-derived materials such as serum added in cell culture media always represent a risk for mycoplasma infection.
Mycoplasma contamination has no effect on the serum’s turbidity and it is not easily detectable using microscopes, even at a higher concentration of 107 cells/ ml. To reduce the presence of mycoplasma infections in the production-of cell culture, it is necessary to carefully sterilise all products
World Pharmaceutical Frontiers /
www.worldpharmaceuticals.net
The STERYKLEAR series As for the filtration of serum and all other products added to bioreactors, BEA Technologies is proposing the use of its STERYKLEAR series of PES membrane filters with a 0.1 micron filtration rating. Tests show that 0.1-micron rated membrane filtration of serum can consistently reduce the risk of mycoplasma infection during serum preparation. However, it is necessary to control how the performance of 0.1-micron filters have been tested against various concentrations of mycoplasma. STERYKLEAR, the line of PES membrane filters manufactured by BEA Technologies with a rating of 0.1 micron for mycoplasma filtration, has been carefully tested for mycoplasma retention using a concentration of 107 cells/cm2
of Acholeplasma laidlawii.
This type of mycoplasma has been selected because Acholeplasma laidlawii is not only known to be responsible for more than 50% of all cell culture mycoplasma contaminations but also because it is the mycoplasma that can be cultivated to prepare the requested challenge concentrations in microbiological laboratories.
Membrane Filter elements in size 10-inches were challenged with a suspension of Acholeplasma laidlawii at a challenge concentration of 107 cfu/cm2
. The
challenged mycoplasma was diluted in consistent volume of broth in order to be filtered in a single pass. The membrane filter elements were challenged and the filtered broth passed through following control membranes to define and calculate the retention rating. The membranes used for recovery are then incubated and counted to control full retention of mycoplasma. ●
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