Assessing rapid viral enumeration/detection systems

By Dr. Ray Nims

In a previous posting, we alluded to the recent availability of rapid methods for identification of viruses. These technologies, together with rapid methods for enumerating viruses, should greatly expedite the quantification and identification of viruses (and bacteriophage) as compared with the existing cell culture-based approaches.

Rapid enumeration technologies are intended to replace the cell-based infectivity endpoints such as plaque assays or tissue-culture infectious dose assays, which typically require 7-10 days for completion. The use of the rapid methods may be appropriate in cases where it is not necessary to determine the infectious titer of a virus stock. An example of this might be for monitoring the amplification of viruses for preparation of live or subunit vaccines. The particle enumeration technologies include those that specifically measure viral particles and those that measure particles in general. As shown in Table 1, the particle enumeration technologies are not specific to any given virus. These are not generally useful, therefore, for viral identification, although the particle detection method associated with the NanoSight system does allow for sizing of the particles. Viral particle size is a key attribute to be aware of when, for instance, attempting to identify an unknown viral contaminant.

Table 1. Characteristics of rapid viral enumeration/identification technologies

The quantitative polymerase chain reaction (Q-PCR) and universal biosensor (Ibis T5000) technologies represent approaches that are capable of providing information both on the relative quantity of a virus in a sample and its identity. The important difference between the two is that in the former case (Q-PCR), the user is typically evaluating the identity and or quantity of a virus which is reactive with the specific primers and probes used in the assay. From an identification standpoint then, the Q-PCR technique has typically been used to confirm whether an unknown virus is related to the virus for which the assay primers and probes was designed. The degree of relatedness required is determined by the specific primers and probes used in the assay, and may be either to the genus level or the species level. Efforts are being made to incorporate primers for more highly conserved sequences to allow for more broad coverage in Q-PCR assays intended for viral screening. In the case of the universal biosensor (Ibis T5000), an unknown virus in a sample may be simultaneously identified and quantified, as long as the virus is or is closely related to one for which mass spectrometry information is present in the software used for assay analysis. Quantification in either case is in genomic units, and as with the particle enumeration methods, the readout of the quantitative nucleic acid methods does not indicate whether the virus detected is infectious. An additional nucleic acid-based method that may prove useful, in cases where relatively rapid identification of an unknown viral contaminant is needed, is deep (massively parallel) sequencing. This method is more labor intensive (and perhaps costly) then the other quantitative nucleic acid methods described above, but has the advantage that it can provide information regarding the completeness (partial vs. full-length) of the viral genomic sequences detected. This approach has displayed utility in identifying a novel picornavirus in harbor seal samples, porcine circovirus in rotavirus vaccines, and a new parvovirus in bovine serum.

Microarray screening is a technology that may be used to rapidly identify (but not enumerate) an unknown virus in a sample, provided that a probe for the virus is part of the microarray chip. Some microarray chips intended for viral identification also contain probes for conserved viral genomic sequences. In this case, the microarray may identify a novel unknown virus, at least to the genus level. As with the other rapid methods that are based on presence of specific genomic material, the assay cannot discriminate between infectious and non-infectious virus.

See Table 1 for some of the important characteristics and limitations of each method. The use of the rapid methods discussed above and in Table 1 should reduce the time needed for viral quantitation from weeks to hours, and for identification of an unknown contaminant in a sample from months (or years) to one or more days. This should greatly facilitate the monitoring of viral proliferation in manufacturing processes and the investigation of viral contamination events.

Porcine circoviruses, vaccines, and trypsin

By Dr. Ray Nims

It has now been more than a year since the announcements by GlaxoSmithKline (GSK) and Merck of the presence of porcine circovirus (PCV) genomic material in their rotavirus vaccines.

The presence of the PCV viral sequences was, in both cases, provisionally attributed to the use of porcine trypsin during the culture of the cell substrates used in the manufacture of the vaccines. It has been reported that the genomic sequences were associated with low levels of infectious PCV in the GSK vaccine.     

As mentioned in a previous posting, an expected outcome of these disclosures was heightened regulatory expectations, going forward, for PCV screening of porcine raw materials and of Master and Working cell banks which were exposed to porcine ingredients (e.g., trypsin) at some point in their development. In January of 2011, the European Pharmacopoeia (Ph. Eur.) chapter 5.2.3 Cell substrates for production of vaccines for human use was revised to include the following instruction: “Trypsin used for the preparation of cell cultures is examined by suitable methods and shown to be sterile and free from mycoplasmas and viruses, notably pestiviruses, <circoviruses> and parvoviruses.” The addition of circoviruses to the list of viruses of concern (previously, mainly bovine viral diarrhea virus and porcine parvovirus) in Ph. Eur. 7.2 was not unexpected, based on the rotavirus vaccine experience.

A more broad expectation going forward may also be that vaccine and biologics production cell banks be proactively screened for unexpected, perhaps previously undetectable, viruses using detection techniques such as the deep sequencing used initially to detect the PCV in the GSK rotavirus vaccine. A related technique referred to as massively parallel sequencing (Massively Parallel Sequencing (MP-Seq), a New Tool For Adventitious Agent Detection and Virus Discovery) has been adopted for detection of viral contaminants in cells and viral seed stocks and for evaluating vaccine cell substrates by the contract testing organization BioReliance.

The more important sequella of the porcine circovirus disclosures may therefore be the proactive use of these new and powerful virus detection techniques for ensuring the viral safety of production cell banks, going forward.

Rapid Identification of Viral Contaminants, Finally

By Ray Nims, Ph.D.


There was a time, not long ago, when it might take months to years to identify a viral contaminant isolated from a biological production process or from an animal or patient tissue sample. The identification process took this long because it involved what I have referred to as the “shotgun approach”, or it involved luck.

Let’s start with luck. That is probably the wrong term. What I mean by this is that there have been instances where an informed guess has led to a fairly rapid (i.e., weeks to months) identification of a contaminant. For instance, our group at BioReliance was able to rapidly identify contamination with REO virus (REO type 2 actually) and Cache Valley virus  because we had observed these viruses in culture previously and because these viruses had unique properties (a unique cytopathic effect in the case of REO and a unique growth pattern in the case of Cache Valley virus). The time required to identify these viruses consisted of the time required to submit and obtain results from confirmatory PCR testing for the specific agents.

The first time we ran into Cache Valley virus, however, it was a different story. This was, it turns out, the first time that this particular virus had been detected in a biopharmaceutical bulk harvest sample. In this case, we participated in the “shotgun approach” that was applied to the identification of the isolate. The “shotgun approach” consisted of utilizing any detection technique available at the lab, namely, in vitro screening, bovine screening, application of any immunofluorescent stains available, and transmission electron microscopy (TEM). The TEM was helpful, as it indicated a 80-100 nm virus with 7-9 nm spikes. A bunyavirus-specific stain showed positive, and eventually (after months of work), sequencing and BLAST alignment was used to confirm the identity of the virus as Cache Valley virus.

The “shotgun approach” was subsequently applied to a virus isolated from harbor seal tissues, with no identity established as a result. After approximately a year of floundering using the old methods, the virus was eventually found to be a new picornavirus (Seal Picornavirus 1).  How was this accomplished? During the time between the identification of the Cache Valley virus and the seal virus, a new technology called deep sequencing became available. Eric Delwart’s group used the technique to rapidly identify the virus to the species level. As this was the first time this particular picornavirus had ever been detected, deep sequencing is likely the only method that would have been able to make the identification.

Deep (massively parallel) sequencing is one of a few new technologies that will make virus isolate identification routine and rapid in the future. It has been adopted for detection of viral contaminants in cells and viral seed stocks and for evaluating vaccine cell substrates by BioReliance.The other is referred to as the T5000 universal biosensor. Houman Dehghani’s group at Amgen has been characterizing this methodology as a rapid identification platform for adventitious agent contaminations.  Each technology has its advantages. Deep sequencing is more labor intensive, but has the ability to indicate (as described above) a new species. The universal biosensor can both serve as a detection method and as an identification method. Both can identify multiple contaminants within a sample.

Since identification of an adventitious viral contaminant of a biopharmaceutical manufacturing process is required for establishment of root cause, for evaluating effectiveness of facility cleaning procedures and viral purification procedures, and for assuring safety of both workers and patients, it is critical that the identification of a viral isolate is completed accurately and rapidly. Happily, we now have the tools at hand to accomplish this.

FDA to viral vaccine makers: it's time to update viral testing methods

By Dr. Ray Nims

If you have been following the recent (2010) unfolding of the discovery of porcine circovirus DNA contamination in rotavirus vaccines from GSK and Merck, you may not be surprised to hear that the FDA has asked viral vaccine manufacturers to outline, by October, their plans to update their testing methodologies to prevent future revelations of this type.
 
I had predicted earlier that biologics manufacturers would be asked to provide evidence, going forward, that their porcine raw materials (trypsin being the most common) are free of porcine circovirus. This testing has not been manditory in the past, but adding this to the porcine raw material virus screening battery moving forward is a prudent action in light of the recent rotavirus vaccine experience.

The FDA has appropriately gone a little farther in it's request to the viral vaccine manufacturers. The regulators would like to assure that the future will not bring additional discoveries of viral contaminants in licensed vaccines, and the best way to accomplish this at the moment appears to be to request implementation of updated viral screening methodologies. Does this mean that viral vaccine makers will need to employ deep sequencing on a lot-by-lot basis? Most likely not. It appears that reliance on the in vivo and in vitro virus screening methods which have been the gold standards since the 1980s will, however, no longer be sufficient. So what does this leave us with? What FDA appears to be asking for is a relatively sensitive universal viral screening method.

The in vivo and in vitro methods were, until now, the best option for this purpose. These methods detect infectious virus only and depend upon the ability of the virus to cause an endpoint response in the system (cytopathic effect, hemagglutination, hemadsorption, or pathology in the laboratory animal species used). So viral genomic material would not be detected, and the methods have had to be supplemented with specific nucleic acid-based tests for viruses which could not otherwise be detected (e.g., HIV, hepatitis B, human parvovirus B9, porcine circovirus).

Some options for sensitive and universal viral screening methods which might fit the requirements include DNA microarrays and universal sequencing methods performed on cell and viral stocks. The latter technology may be preferable, as microarrays are constructed to detect known viruses, while the desire is that the technology be universal in the sense that it detect both known and unknown viruses. Such a test will provide additional assurance that the virus and cell banks used to manufacture viral vaccines do not harbor a viral contaminant.

Other universal viral screening methods which are less labor intensive than the sequencing technologies may be developed in the near future and addition of one of these to the release testing battery for viral vaccine lots may need to be considered in satisfying the FDA's goals.

Assessing rapid microbial detection systems

by Dr. Ray Nims

With each passing year, it seems that there are more options available for rapid microbial detection. These rapid systems come in a variety of “flavors”, that is - they differ with respect to a set of key attributes. For instance, how rapid is rapid? What is the sensitivity? What is the maximum sample volume that may be tested? Is it quantitative or qualitative? What units are the results given in? Is it destructive or non-destructive (i.e., can the organism, once detected, be identified)? When one considers the variety of applications for which rapid methods may potentially replace existing culture methods, it rapidly becomes clear that there may not be “one shoe that fits all”.

In order to select an appropriate rapid method for use in one of the many microbial detection applications, one must first assess the available rapid systems for the key attributes mentioned above. This then provides the opportunity to rule out systems which for one reason or the other will not suit the application. There may be some applications for which no rapid system currently meets all requirements. Those rapid systems which do appear to possess the attributes required may be further evaluated for cost and for performance capabilities using specific sample matrices.

In the table below, we have listed some of the currently available rapid microbial detection systems. These include only systems which are 48 hours in duration or less, and therefore some of the sterility replacement assays involving reduced incubation durations (e.g., BacT/ALERT®, Growth Direct™) are not listed.

The key attributes of these rapid systems are displayed in the table below. The systems are arranged by principle of detection, as in the table above. For certain methods (e.g., Micro Pro™) increased sensitivity can be gained through increasing the duration of the incubation time. For non-destructive methods, the ability to identify the organism(s) detected is facilitated by an additional incubation post-detection.

What is the regulatory position on rapid microbial detection methods? The U.S. FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing states that other suitable microbiological tests (e.g., rapid methods) may be considered for environmental monitoring, in-process control testing, and finished product release testing after it has been demonstrated that these new methods are equivalent or better than conventional (e.g., USP) methods. Additionally, the FDA Process Analytical Technology (PAT) initiative encourages the voluntary development and implementation of innovative approaches in pharmaceutical development, manufacturing, and quality assurance (from MJ Miller, PDA Journal 45: 1-5, 2002).

Are rapid methods being used in the pharmaceutical industry? ScanRDI was approved by the FDA for water testing at GSK and for sterility testing at Alcon; Pallchek has been approved by the FDA for bioburden testing at GSK; and Wyeth received approval for use of Celsis for microbial limits testing.

Like all methods proposed to replace existing “gold standards”, these rapid microbial detection systems must be demonstrated through comparability protocols to be equivalent to or better than the existing methods. The effort required should pay dividends in terms of shortened turnaround times and reduced costs.

Oops, adventitious viral DNA fragments in a vaccine

by Dr. Ray Nims

On March 22, 2010, a press release from GlaxoSmithKline (GSK) announced that DNA from porcine circovirus had been detected in their rotavirus vaccine. According to GSK, the DNA ”was first detected following work done by a research team in the US using a novel technique for looking for viruses and then confirmed by additional tests conducted by GlaxoSmithKline”. As a result of this finding, the FDA “is recommending that US clinicians and public health professionals temporarily suspend the use of Rotarix as a precautionary measure. The FDA have also stated that they intend to convene an advisory committee, within approximately four to six weeks, to review the available data and make recommendations on rotavirus vaccines licensed in the USA. The FDA will also seek input on the use of new techniques for identifying viruses in vaccines.” The EMEA, on the other hand, does not appear to consider this finding to be a safety concern, citing the fact that porcine circovirus is not infectious for human cells and does not cause disease in humans.

Porcine circovirus

Source: Meat and Livestock Commission, UK

What is porcine circovirus? Porcine circovirus (PCV) is a member of the family Circoviridae, among the smallest of the known animal DNA viruses. It is approximately 17 nm in diameter, non-enveloped, with icosahedral symmetry. The virus was originally identified as a noncytopathic contaminant of the PK-15 porcine kidney cell line (Tischer et al., Zentralblatt Bakt Hyg A 226:153-167, 1974). Like many very small, non-enveloped viruses, PCV represents a challenge for removal and inactivation.

Why is this finding coming to light now, or stated another way, why wasn’t the PCV DNA detected when the vaccine was initially tested and approved for human use? The press release wasn’t specific as to method used. It has subsequently been revealed, however,  that these fragments were detected using sequence-independent amplification (deep sequencing; or as Eric Delwart calls it, metagenomics). The resulting library of amplified sequences is characterized by BLAST searching using identification algorithms. Confirmation in this case was obtained using microarray and PCR analyses. The sequencing techniques have been available for some time, and have proven useful for identification of viruses in an academic setting, though they have not been applied to safety testing of biopharmaceuticals until fairly recently due to the relatively high costs associated with the analyses.

The finding of viral DNA should not be equated with detecting the infectious virus in the product. The sequence-independent amplification, microarray, and virus-specific PCR assays used can detect viral nucleic acid, but as normally performed do not indicate whether infectious virus is present. Generally, with the possible exception of transforming viruses, it is the infectious virus that is of concern, not its DNA. Efforts to assess the presence of intact viral genomic material and of infectious porcine circovirus in this vaccine are most likely underway at this time.

The presence of the PCV viral sequences has provisionally been attributed to the use of porcine trypsin during the culture of the Vero cell substrate in which the vaccine is manufactured. The trypsin used had been gamma-irradiated to inactivate adventitious viruses prior to use. While it would be comforting to believe that the PCV DNA may simply have reflected carryover of non-infectious, lethally-irradiated PCV1 from the trypsin, the fact is that gamma-irradiation is not very effective at inactivating this small, non-enveloped virus (Plavsic and Bolin. Resistence of porcine circovirus to gamma irradiation. Biopharm Int. 14:32-36, 2001). In the case of porcine circovirus, there is little evidence to indicate that the virus is infectious or pathogenic for humans. So regardless of the outcome of the various ongoing studies, it is likely that the use of the GSK rotavirus vaccine will be re-instated after the FDA convenes and discusses the implications of this finding.

I predict that there will be more and more of this kind of revelation in the future as the sequencing techniques display stretches of viral or other contaminant DNA within samples of biopharmaceuticals. I would hate to see revelations like this impede the use of the sequencing technologies going forward, as these technologies are going to be very useful to the industry as rapid detection methods for contaminant identification.

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.