Wednesday, June 23, 2010

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.

Thursday, June 17, 2010

Informing the FMEA

By Dr. Scott Rudge
Risk reduction tools are all the rage in pharmaceutical, biotech and medical device process/product development and manufacturing. The International Conference on Harmonization has enshrined some of the techniques common in risk management in their Q9 guidance, “Quality Risk Management”. The Failure Modes and Effects Analysis, or FMEA, is one of the most useful and popular tools described. The FMEA stems from a military procedure, published in 1949 as MIL-P-1629, and has been applied in many different ways. The most used method in the health care involves making a list of potential ways in which a process can “fail”, or produce out of specification results. After this list has been generated, each failure mode in the list is assessed for its proclivity to “Occur”, “Be Severe” and “Be Detected”. Typically, these are scored from 1 to 10, with 10 being the worst case for each category, and 1 being the best case. The scores are multiplied together, and the product (mathematically speaking) is called the “Risk Priority Number”, or RPN. Then, typically, development work is directed towards the failure modes with the highest RPN.

The problem is, it’s very hard to assign a ranking from 1 to 10 for each of these categories in a scientific manner. More often, a diverse group of experts from process and product development, quality, manufacturing, regulatory, analytical and other stake holding departments, convene a meeting and assign rankings based on their experience. This is done once in the product life-cycle, and never revisited as actual manufacturing data start to accumulate. And, while large companies with mature products have become more sophisticated, and can pull data from other similar or “platform” products, small companies and development companies can really only rely on the opinion of experts, either from internal or external sources. The same considerations apply to small market or orphan drugs.

Each of these categories can probably be informed by data, but by far the easiest to assign a numerical value to is the “Occurrence” ranking. A typical Occurrence ranking chart might look something like this:

These rankings come from “piece” manufacturing, where thousands to millions of widgets might be manufactured in a short period of time. This kind of manufacturing rarely applies in the health care industry. However, this evaluation fits very nicely with the Capability Index analysis.

The Capability Index is calculated by dividing the variability of a process into its allowable variable range. Or, said less obtusely, dividing the specification range by the standard deviation of the process performance. The capability index is directly related to the probability that a process will operate out of range or out of specification. This table, found on Wikipedia (my source for truth), gives an example of the correlation between the calculated capability index to the probability of failure:

As a reminder, the capability index is the upper specification limit minus the lower specification limit divided by six times the standard deviation of the process. The two tables can be combined to be approximately:
How many process data points are required to calculate a capability index? Of course, the larger the number of points, the better the estimate of average and standard deviation, but technically, two or three data points will get you started. Is it better than guessing?

Wednesday, June 9, 2010

Riboflavin plus UVA irradiation: another inactivation approach to consider

by Dr. Ray Nims

Short-wavelength ultraviolet irradiation (UVC) has been used for years to disinfect air, surfaces, and thin liquid films because it is effective in inactivating a variety of bacteria, protozoa, phage, and viruses. More recently, UVC (100-280 nm) irradiation has been shown to be useful for viral risk mitigation in biologics manufacturing. UVC-treatment of culture media and other liquid reagents has been demonstrated to inactivate potential adventitious viral contaminants, including those which are resistant to inactivation by other physical means (e.g., murine minute viruscalicivirus; and porcine parvovirus and SV-40 [Wang et al., Vox Sanguinis 86: 230-238, 2004]).

Another approach that has been used recently, especially in the ophthalmologic and blood products communities, is UVA (315-400 nm) in the presence of the photosensitizer riboflavin. Riboflavin interacts with nucleic acid and photosensitizes to damage by UVA leading to direct electron transfer, production of singlet oxygen, and generation of hydrogen peroxide. The treatment results in oxidation and ring-opening of purines and in DNA strand breakage. The advantage of riboflavin over other photosensitizers (e.g., methylene blue, psoralens, etc.) is that riboflavin (vitamin B2) is an endogenous physiological substrate.

The photosensitizer interacts with nucleic acids.
Upon irradiation, the results may include cross-linking, mutation, or strand breakage.
Source: Bryant and Klein

Riboflavin/UVA treatment has been explored in ophthalmology applications such as infectious keratitis and keratomycosis. The typical treatment paradigm involves application of a solution of 0.1% riboflavin (as riboflavin-5-phosphate) followed by irradiation using 365 nm light (5 to 10 J/ml). The approach has shown effectiveness against a variety of pathogenic bacteria, including drug-resistant strains. Effectiveness against fungal pathogens requires combination therapy with amphotericin B.

In the blood products community, photosensitizer/UV treatment is being explored for pathogen reduction. For instance, riboflavin/UV treatment is being evaluated for platelet and plasma pathogen reduction, for prevention of graft versus host reactions, and for pathogen reduction in whole blood products. In the proprietary application (Mirasol PRT®), blood product pools are combined with riboflavin (final concentration 50 µM) and the solutions are irradiated with 6.24 J/ml broadband (265-370 nm) UV light. The technology has been shown to be effective for a variety of pathogenic bacteria and viruses (see Table 3 in the review by Bryant and Klein).

Will this approach be useful in the biopharma industry? It appears so. Recently, irradiation with UVA (365 nm) light in the presence of 50 µM riboflavin has been evaluated for controlled inactivation of gene transfer (adenovirus, adeno-associated virus, lentivirus) virus preparations. Complete inactivation was obtained in each case within 90 minutes.