Fry those mollicutes!

By Dr. Ray Nims

It is not only viruses that may be introduced into biologics manufactured in mammalian cells using bovine sera in upstream cell growth processes. The other real concern is the introduction of mollicutes (mycoplasmas and acholeplasmas). Mollicutes, like viruses, are able to pass through the filters (including 0.2 micron pore size) used to sterilize process solutions. Because of this, filter sterilization will not assure mitigation of the risk of introducing a mollicute through use of contaminated bovine or other animal sera in upstream manufacturing processes.

Does mycoplasma contamination of biologics occur as a result of use of contaminated sera? The answer is yes. Most episodes are not reported to the public domain, but occasionally we hear of such occurrences. Dehghani and coworkers reported the occurrence of a contamination with M. mycoides mycoides bovine group 7 that was proven to have originated in the specific bovine serum used in the upstream process (Case studies of mycoplasma contamination in CHO cell cultures. Proceedings from the PDA Workshop on Mycoplasma Contamination by Plant Peptones. Pharmaceutical Drug Association, Bethesda, MD. 2007, pp. 53-59). Contamination with M. arginini and Acholeplasma laidlawii attributed to use of specific contaminated lots of bovine serum have also occurred.

Fortunately, the risk of introducing an adventitious mollicute into a biologics manufacturing process utilizing a mammalian cell substrate may be mitigated by gamma-irradiating the animal serum prior to use. This may be done in the original containers while the serum is frozen. Unlike the case for viruses, in which the efficacy of irradiation for inactivation may depend upon the size of the virus, mollicute inactivation by gamma irradatiion has been found to be highly effective (essentially complete), regardless of the species of molicute. The radiation doses required for inactivation are relatively low compared to those required for viruses (e.g., 10 kGy or less, compared to 25-45 kGy for viruses). The gamma irradiation that is performed by serum vendors is typically in the range of 25-40 kGy. This level of radiation is more than adequate to assure complete inactivation of any mollicutes that may be present in the serum. For instance, irradiation of calf serum at 26-34 kGy resulted in ≥6 log10 inactivation of M. orale, M. pneumoniae, and M. hyorhinis. In the table below I have assembled the data available on inactivation of mollicutes in frozen serum by gamma-irradiation.

So, the good news is that gamma irradiation of animal serum that is performed to mitigate the risk of introducing a viral contaminant will also mitigate the risk of introducing a mollicute contaminant. If the upstream manufacturing process cannot be engineered to avoid use of animal serum, the next best option is to validate the use of gamma irradiated serum in the process.  In fact, the EMEA Note for guidance on the use of bovine serum in the manufacture of human biological medicinal products strongly recommends the inactivation of serum using a validated and efficacious treatment, and states that the use of non-inactivated serum must be justified.


References: Gauvin and Nims, 2010; Wyatt et al. BioPharm 1993;6(4):34-40; Purtle et al., 2006

Is Clarence calculating clearance correctly?

by Dr. Ray Nims

As pointed out by Dr. Rudge in a recent posting “Do we have clearance, Clarence?”, spiking studies conducted for the purpose of validating impurity clearance are often done at only one spiking level (indeed often at the highest possible impurity load attainable). This is especially true for validation of adventitious agent (virus and mycoplasma) clearance in downstream processes. The studies are done in this way in order to determine the upper limit of agent clearance (in terms of log10 reduction) by the process. Such log10 reduction factors from individual process steps are then summed in order to determine the overall capability of the downstream processes to clear adventitious agents. The regulatory agencies have fairly clear expectations around such clearance capabilities which must generally be met by biologics manufacturers.

The limiting factor in such clearance studies is typically the amount or titer of the agent that is able to be spiked into the process solution, which is determined by: 1) the titer of the stock used for spiking, and 2) the maximum dilution of the process solution allowed during spiking (typically 10%). Under these circumstances, as Scott points out, there is a possibility that the determined clearance efficiency (i.e., the percentage of the load which is cleared during the step) is an underestimate of the actual clearance that might be obtained at lower impurity loading levels.

Adventitious agent clearance is comprised of two possible modalities, removal and inactivation. Removal refers to physical processes designed to eliminate the agent from the process solution, usually through filtration or chromatography. Removal efficiency through filtration would not be expected to display variability based on impurity loading. On the other hand, chromatographic separation of agents (by, for example, ion-exchange columns) may display saturation at the highest loadings, and therefore use of the highest possible loading levels may result in underestimates of removal efficiency at lower (i.e., more typical) impurity levels.

Inactivation refers to physical or chemical means of rendering the agent non-infectious. Agent inactivation is not always a simple, first-order reaction. It may be more complex, with a fast phase 1 stage of inactivation followed by a slow phase 2 stage of inactivation. An inactivation study is planned in such a way that samples are taken at different times so that an inactivation time curve can be constructed. As with removal studies, the highest possible impurity levels are typically used to determine inactivation kinetics.

Source: Omar et al. Transfusion 36:866-872, 1996

While the information obtained through clearance studies of this type may be incomplete from the point of view of understanding the relationships between impurity loading levels and clearance efficiency, the results obtained are consistent with the regulatory expectation that the clearance modalities be evaluated under worst-case conditions. Therefore, at least in the case of adventitious agent clearance validation, I would say that Clarence is calculating clearance correctly!

Assessing rapid mycoplasma detection systems

by Dr. Ray Nims

The European Pharmacopoeia chapter 2.6.7 Mycoplasmas, begining with version 5.8, has provided a mechanism for replacement of the current 28-day culture method for detection of mollicute (mycoplasma and acholeplasma) contaminants in biopharmaceutical bulk harvest samples with more rapid, nucleic acid-based, methods. The US FDA has yet to provide formal guidance on this topic, although it has become clear that the agency is willing to consider such methods, provided that they are shown to be equivalent to or superior to the current approved methods.

For biopharmaceuticals, a satisfactory outcome in a mycoplasma detection assay which is compliant with European Pharmacopoeia 2.6.7 or the 1993 FDA Points to Consider guidance is required on a lot-by-lot basis. Of the various lot-release assays performed on each given lot of a biopharmaceutical, this particular test is typically the most lengthy. Expediting the lot-release process through replacement of the 28-day approved culture test with a rapid mycoplasma detection test is therefore a strong motivating factor for the biopharmaceutical industry.

Figure. The MicroSEQ Mycoplasma assay provides a level of detection less

than 10 CFU/ML.

What options are now available to the industry? Several contract testing laboratories have recently announced the availability of validated rapid mycoplasma assays suitable for biopharmaceutical lot-release. For instance, BioReliance offers a hybrid culture/quantitative polymerase chain reaction (qPCR) assay, Charles River Laboratories offers a reverse transcriptase (RT)-PCR assay (BioProcess Int. April 2009, 30-42), Vitrology offers a qPCR assay, and WuXi AppTec offers a “touchdown” PCR assay.

In addition, several vendors are now offering mycoplasma detection kits which will allow biopharmaceutical entities to perform rapid mycoplasma testing in-house. For example, Life Technologies offers the MicroSEQ® Mycoplasma Detection Assay, Roche Applied Science offers the MycoTool™ PCR test and Millipore offers the MilliPROBE® mycoplasma detection system.


It is incumbent upon the biopharmaceutical company to demonstrate comparability between the rapid mycoplasma method and the current approved culture method for each product matrix for which a rapid method is proposed. Guidance on such comparability testing is provided in the European Pharmacopoeia chapter 2.6.7. Comparability studies for rapid methods intended to satisfy the US FDA should be discussed with that agency, as no formal guidance has been published.

What attributes should be considered when selecting a rapid mycoplasma detection method?

1. Sample volume. The current approved culture methods test at least 10 mL of sample. A rapid method intended to replace the current methods should ideally be able to test an equivalent volume of sample. It may be difficult to gain FDA approval for nucleic acid-based methods which can test only microliter amounts of sample.
2. Duration. Hybrid culture/PCR systems may take as long as 14 days to complete, while direct nucleic acid-based methods should be completed within a week or less.
3. Specificity. European Pharmacopoeia 2.6.7 specifies that the nucleic acid test must be able to exclude closely-related bacterial species.
4. Sensitivity. FDA indicates that the rapid method should be equivalent to or better than the approved culture method in terms of sensitivity (limit of detection), based on comparability studies using viable mycoplasma organisms.
5. Orthogonal endpoints. Having two or more orthogonal endpoints is desirable to allow one to discriminate between low level positive and negative signals.
6. Validation status. For contract methods, has the method been validated per European Pharmacopoeia 2.6.7? For kit methods, has the vendor validated the method per European Pharmacopoeia 2.6.7?
7. Drug Master File. For kit methods, has the vendor submitted a drug master file to the FDA for the method?

These considerations should help in deciding among the various options now available for implementing rapid nucleic acid-based mycoplasma testing for biopharmaceutical lot release applications.

USP 63 Mycoplasma Update

By Dr. Ray Nims

The United States Pharmacopeia’s (USP) new chapter <63> Mycoplasma Tests was planned to become effective on May 1, 2010 as part of USP 33. This new chapter was intended to fill a void in the USP for mycoplasma testing, which had been addressed previously within the FDA’s 1993 Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals and the European Pharmacopoeia (EP) chapter 2.6.7 Mycoplasmas (a separate document applying only to mycoplasma testing of live and inactivated viral vaccines, 21 CFR 610.30 will not be considered here). Since there were some methodological differences between the FDA guidance and the EP chapter, it was hoped by the industry that the USP guidance would serve to harmonize mycoplasma testing as much as practically possible.

Indeed, a quick look at the new USP chapter <63> indicates that the chapter was based in large part on EP chapter 2.6.7. A comparison of the three documents (USP, FDA, and EP) reveals methodological differences in only a few areas. These include the assessment of nutritive properties of the solid growth media (agar) used for mycoplasma testing, the assessment of inhibitory substances in the test material, the incubation temperature ranges to be used, and the number of positive controls to be used.

The EP chapter 2.6.7 states that “The solid medium complies with the test if adequate growth is found for each test micro-organism (growth obtained does not differ by a factor greater than 5 from the value calculated with respect to the inoculum)”. There is a different requirement within USP chapter <63>: “The solid medium complies with the test if a count within a 0.5-log unit range of the inoculate amount is found for each test microorganism”. Assuming an inoculate of 100 colony forming units (CFU), the acceptable ranges for the recovered organisms would be 32-316 CFU for the USP version vs 20-500 CFU for the EP version. The USP version is therefore more stringent in this respect.

Similarly, for the assessment of inhibitory substances, EP chapter 2.6.7 states that “…if plates directly inoculated with the product to be examined have fewer than 1/5 of the number of colonies of those inoculated without the product to be examined” there are inhibitory substances in the test material. The USP version indicates that there are inhibitory substances “…if plates directly inoculated with the test article/material are not within a 0.5-log unit range of the number of colonies of those inoculated without the test article/material.” So the USP version is again more stringent in this respect.

Minor differences in incubation temperature for test cultures exist between the documents (36 ± 1°C for the USP and PTC documents vs 35-38°C for the EP chapter). The USP and PTC documents specify the number and types of positive controls to be used in the assays: at least two known Mycoplasma species or strains should be included as positive controls (one a dextrose fermenter and one an arginine hydrolyzer). The EP chapter specifies that at least one of the six Mycoplasma species listed in the chapter be used as a positive control.

The USP chapter <63> differs from EP chapter 2.6.7 also in that the former does not provide requirements for validation of a nucleic acid-based test for mycoplasma. The USP chapter mentions the possibility of replacing the culture method with an alternative (nucleic-acid or enzymatic) method, stating that the alternative method must be validated and shown to be comparable to the agar/broth and cell culture methods. The EP chapter laid the foundation for validation of a nucleic acid-based mycoplasma detection test for the first time in version 5.8 (effective July 2007). This provided the industry with expectations for implementation of a rapid alternative test to the approved culture test for mycoplasma, which is 28 days in duration. Similar guidance is not yet forthcoming from the FDA or USP.

The issues of nutritive properties and inhibitory substances are not addressed within the FDA’s 1993 Points to Consider guidance. In order to now be compliant with the FDA and EP requirements as well as the new USP chapter, testing labs will have to make adjustment within their protocols to account for the stricter USP criteria for assessing nutritive properties and inhibitory substances. Due to errors within some of the monographs to appear in the issuance of the USP to become effective May 1, 2010, this issuance of USP 33, including chapter <63>, was retracted in January of 2010. However, it will be re-issued in March 2010 with an official date six months after reissue, and the methodological differences may need to be accounted for in testing protocols used by quality control laboratories for which USP compliance is applicable.

Once this chapter becomes effective, the mycoplasma test methods described will be considered compendial, meaning that labs following the methods outlined in the chapter will not be required to perform method validation. The labs will only be required to perform method verification for each test sample type (matrix qualification) per USP <1226>.