Contamination of biologics with mycoplasmas may represent a considerable risk to human health. While mycoplasmas are generally considered to be host-specific bacteria, a few sporadic cases of mycoplasmal infections of humans caused by animal and avian mycoplasmas have been reported (35
). Severe cases of human infections with human and nonhuman mycoplasmas can occur, especially in immunocompromised individuals (19
). To ensure the purity and safety of parenterally administrated drugs and vaccines manufactured using different cell substrates, including embryonated chicken eggs, current FDA regulations (21 CFR 610.30 for viral vaccines) and FDA guidance in 1993 (PTC) and 2006 (54
) recommend that a manufacturer show that cell substrates and unprocessed virus harvests are free of mycoplasma contamination and other adventitious agents. The use of embryonated eggs from healthy (but not SPF-grade) flocks for manufacture of virus vaccines can pose some risk of inadvertent mycoplasma contamination of vaccine products. Therefore, a risk assessment approach is used to ensure the safety of the egg-based vaccine products. To ensure that there is no mycoplasma contamination, the manufacturers perform a thorough validation showing that the downstream manufacturing processes (virus concentration, inactivation, and purification procedures) can completely inactivate mycoplasmas, if such contamination inadvertently occurs (27
). The influenza virus concentration, inactivation, and purification procedures include the use of several chemical reagents (beta-propiolactone, formalin, cetyltrimethylammonium bromide, Triton X-100, and sodium deoxycholate) which exhibit germicidal activity and efficiently inactivate any mycoplasmal or other bacterial contaminants.
The main objective of our study was to evaluate the efficiency of mycoplasma inactivation under conditions generally used for the production of inactivated virus vaccines, including inactivated influenza virus (split-virion) vaccine. To assess the contribution of each chemical agent used in the mycoplasma inactivation process, the agents were tested in separate experiments at concentrations that are commonly used or represent “worst-case scenarios” for flu vaccine production.
The first group of reagents included formaldehyde and BPL, which are commonly used for inactivation of vaccine viruses via chemical reaction with viral capsid proteins and nucleic acids (3
). The second group consisted of surfactants, including cetyltrimethylammonium bromide, Triton X-100, and sodium deoxycholate, which are usually used to split virus particles into separate structural proteins.
The results obtained for BPL and formaldehyde demonstrated that all 22 mycoplasma species examined could be completely inactivated (no viable bacteria were detected by the microtiter and macrodilution methods) within 3 to 24 h at room temperature in mycoplasma media containing 0.2% (≥66.60 mM) formaldehyde and 0.1% (≥13.87 mM) BPL.
The results of our study using formaldehyde and BPL were consistent with previous data obtained for these reagents using only three mycoplasma species (M. gallisepticum
, M. canis
, and A. laidlawii
) spiked into Vero and DK cell culture suspensions (30
). Similar to the results described in the previous study, we observed incomplete inactivation of many of the species tested when the concentration of formaldehyde was 0.02% or lower. A significant improvement in inactivation of mycoplasmas was observed when the formaldehyde concentration was increased to 0.2% (30
). Our data also showed that A. laidlawii
exhibited enhanced stability in the presence of formaldehyde (30
) and resistance to inactivation for the first 5 h of incubation with 0.2% formaldehyde. However, A laidlawii
was not viable after 24 h of incubation with formaldehyde. A. laidlawii
was also found to be more resistant to BPL than the other species examined. A. laidlawii
remained viable for 2 h of incubation with 0.1% BPL; this result is consistent with the ability of this species to resist inactivation by BPL described previously (30
In a majority of the protocols used for production of inactivated virus vaccines (2
), the concentration of BPL and formaldehyde exceeds the critical concentration (0.1% to 0.2%) required for complete inactivation of mycoplasmas. Our data showed that these conditions resulted in complete inactivation of many of the mycoplasmas evaluated. However, the results can change dramatically if a lower incubation temperature is used for inactivation by formaldehyde or BPL. We compared the inactivation profiles obtained at different temperatures (e.g., RT and 4°C) for selected species and found that a reduction in the temperature from RT to 4°C resulted in significantly increased survival of all mycoplasmas at all concentrations of formaldehyde and BPL tested, including 0.2% and 0.1%, respectively.
Among the chemical agents tested, cetyltrimethylammonium bromide at concentrations higher than 0.08% was the most efficient mycoplasma-inactivating agent. Complete inactivation of mycoplasmas by CTAB was observed in the first 30 min of incubation, regardless of the temperature and the protein composition of biological matrices (allantoic fluid or mycoplasma broth). The working concentration of CTAB during typical virus vaccine production varies from 0.075 to 0.15%. This concentration allows efficient disruption of virion structure and release of individual structural proteins. Our data showed that this concentration of CTAB is highly likely to kill any inadvertent mycoplasmas in the first 30 min of exposure.
The risk assessment approach used to ensure that mycoplasma contaminants are not present in the production of egg-derived viral biologics relies on data provided by special evaluation experiments aimed at assessing the efficiency of mycoplasma inactivation during the manufacturing processes (inactivation and split manufacturing steps). There are no standardized model mycoplasmas for this validation procedure, but M. gallisepticum
, M. synoviae
, M. orale
, M. pneumoniae
, and A. laidlawii
are commonly used. These species were selected for testing based on the probability of contamination of eggs with avian species (M. synoviae
, M. gallisepticum
), differences in fermentation activity (M. orale
, M. pneumoniae
), and a wide distribution in natural environments (A. laidlawii
). For example, A. laidlawii
has been found as a mycoplasma contaminant in plant-derived peptones (26
). To evaluate whether this set of mycoplasma species can be used as appropriate model species for evaluating mycoplasma clearance obtained with different chemical agents at different concentrations, we compared the sensitivities of 22 mycoplasmas (including the species mentioned above) that were of human, animal, and avian origin (see Materials and Methods). The results of this study did not reveal any significant intraspecies difference in sensitivity to the chemicals tested. However, the analysis of inactivation profiles obtained for different species showed that species could differ substantially in their sensitivities to formaldehyde, BPL, and CTAB, particularly when these reagents were used at the lowest concentrations tested. These differences were not apparent when the chemical concentrations were increased to the concentrations used during virus vaccine production.
A detailed evaluation of the inactivation profiles of the five mycoplasmas currently recommended for validation of mycoplasma inactivation during virus vaccine production, M. synoviae, M. gallisepticum, M. orale, M. pneumoniae, and A. laidlawii, showed that these species are appropriate for validation purposes. Thus, inactivation of M. synoviae and A. laidlawii usually required longer incubation times with lower and borderline inactivation doses of formaldehyde, beta-propiolactone, and CTAB than inactivation of other mycoplasmas, including M. gallisepticum, M. orale, and M. pneumoniae. However, differences were observed with formaldehyde, BPL, and CTAB only at concentrations less than 0.2%, 0.1%, and 0.04%, respectively. No differences in inactivation of these mycoplasmas were observed when Triton X-100 and DOC were used at a concentration of 0.5%, which is the concentration currently used for manufacture of split-virus influenza vaccines. Thus, M. synoviae, M. gallisepticum, M. orale, M. pneumoniae, and A. laidlawii represent a set of organisms acceptable for performing clearance validation procedures to ensure that there is no mycoplasma contamination in influenza vaccine products.
Moreover, a combination of different chemicals, such as formaldehyde or beta-propiolactone with surfactants (Triton X-100, sodium deoxycholate, and cetyltrimethylammonium bromide), during production provides additive safety, increasing the probability that any inadvertently introduced mycoplasmas are removed from egg-derived biologic products utilizing virus inactivation and detergent-based virion disruption procedures. However, it is important to emphasize that biologic manufacturing processes that utilize chemicals and conditions other than those included in our study should be carefully evaluated on a case-by-case basis to determine the efficiency of clearance of mycoplasmas by the specific production processes using appropriately selected strains.