Karen Zink McCullough MMI Associates BET scientists have known for years that product matrices can significantly interfere with the detection of endotoxin in drug products. In fact, FDA and
Robert Westney President, Cryologics and Westney Associates On June 25, 2007, the Food and Drug Administration (FDA) published its final rule, 21 CFR 111: Current Good Manufacturing Practice
Scott Sutton, Ph.D. Microbiology Network We are familiar with the many problems surrounding “compounding” pharmacies that are functioning as de facto pharma manufacturers without any concern for GMP (Lolas, Sutton
Scott Sutton, Ph.D. Microbiology Network, Inc. There is a good deal of interest from FDA in the cosmetics industry and the Quality systems in place at manufacturing facilities. The
by Karen Z. McCullough
What’s new at this year’s BES? EVERYTHING!
Last year our BES highlighted test interference, but our topic for this year is LER, a very specific form of interference, which is a significantly more focused topic than in years past. We’re fortunate that each of our speakers is a published expert on the topic. This is the ONLY conference to be held this year that focuses on this very timely and important topic.
Have you wondered if your products could exhibit LER? Have you wondered how to resolve LER in your product? Have you wondered if LER is a public health problem? THIS IS THE CONFERENCE for your questions to be answered!
The conference will be held in Iselin, NJ on September 14-15, 2015.
by Gemma Verma
Many pharmaceutical companies perform their research and manufacturing operations across international borders, and ship their goods to people around the world. However, operating between different countries and continents poses significant challenges, not least when it comes to obeying different sets of regulations for safety and GMP.
Growing Regulatory Pressure
Companies of all sizes are facing increasing pressure from a growing number of increasingly complex regulatory systems, put in place at global, regional and national levels. The rising number of cGMP violations being handled by the FDA is, at least in part, a symptom of this tangled regulatory maze and the growing importance that is being placed on GMP compliance. The FDA is paying more attention to procedural flaws that could result in future safety issues, with a large proportion of the 67 warning letters sent out between 2010 and 2013 citing lapses in procedure or problems with data integrity as the reasons for the warning. The added pressures that individual regulators like the FDA are increasingly placing on manufacturers is being compounded by the fact that they are often dealing with a larger number of regulators too. The risk of confusing the requirements of different regulators, and the additional pressure placed on manufacturers, could make compliance problems more likely. Crossing between the main markets has always added pressure, as companies have needed to meet slightly different demands from each regulator, but health authorities in the enticing emerging markets are also adding their own regulations to the load.
The Global Picture
Drug companies are working with increasing numbers of regulators, each with their own particular demands for GMP. A global company might need to work with the regulations of the FDA, the EU, individual countries in Europe and the rest of the Western world, and with individual developing countries that are busy creating their own regulations. Some, like Brazil, are gradually matching the standards set in the US and EU, and signing regional agreements to coordinate their requirements. Others, like China and India, two major producers of APIs for the global market, do not yet set the same GMP standards for their manufacturers. Companies can seek confirmation from their national regulators that their products meet international requirements, but it is a complicated business.
Dealing With Complexity
Succeeding within this complicated world of regulation takes great care. Companies need to devote sufficient time to understanding and keeping up to date with the different sets of regulations they need to follow. Sometimes they will need to adapt to a new regulatory culture or to a shift in a regulator’s perspective, like the FDA’s increasing demands for GMP. Action needs to be taken to make sure the company adapts to the differences and changes that are detected, and companies also need to invest in proper training to ensure that employees understand the regulations that they are supposed to be following. A well trained employee will not only know what they should be doing, but will also understand why it is important to complete procedures in the right way.
It is also important that regulators recognize how difficult it can be for companies to process the vast amounts of constantly updated information that they are being presented with. A successful system is not just one with excellent regulations and effective enforcement, but one in which the regulator is at pains to state their requirements as clearly as possible and to quickly disseminate details of any changes being made.
Simplifying and Saving Lives
Although complying with GMP regulations is likely to become more complicated for many companies in the near future, there is some light at the end of the tunnel. Efforts are underway that could help to harmonize regulatory systems across borders. The WHO has been developing standard GMPs that are already used in more than 100 countries, mainly in the developing world, while networks such as the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Cooperation Scheme are encouraging thecoordination of GMP regulations across international borders.
Such efforts could make life much easier for companies operating internationally, but they will also help to ensure that all of the new regulations being put in place in the emerging markets will give consumers the right level of protection. The current standards are shockingly low in some countries, such as Nigeria, where recent testing revealed potentially dangerous pathogens lurking in many pharmaceutical products. Products manufactured in such an under-regulated system are barely safer than the contaminated substances that so frequently result in illness, infection, or even death, when taken recreationally, or the counterfeit medicines that are manufactured with no intention of meeting adequate safety standards.
It took the deaths of almost 300 people, killed by tainted sulfathiazole tablets, in the 1940s for the FDA to start creating strict quality controls and to write the first examples of what would come to be known as GMPs. Emerging markets face the prospect of similar tragedies if they do not implement adequate regulation, but they also risk losing out on valuable international trade if they cannot satisfy the requirements of the US and European markets. The importance of GMP, quality control and data integrity were key topics at the first Indian Pharmaceutical Association Pharmexcil Technical Conference in November 2014, and according to Ajit Singh, Chairman of ACG Worldwide, the need to work with the FDA to gain access to the US market is a key driving factor in the development of India’s own regulations. Whatever the motivation,better safety measures can only be a good thing for consumers all around the world, even if the multiplication of regulators poses a challenge for manufacturers.
1. Drug Regulatory Scrutiny and the Pharmaceutical Industry, Dilip Shah, CPhI Annual Report 2013
2. The Global Implementation of GMP, Michael Glessner, Specialty Chemicals Magazine
3. WHO Good Manufacturing Practices, WHO
4. Role of the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Cooperation Scheme, PIC/S
5. Microbiological Contamination in Counterfeit and Unapproved Drugs, Dieter Pullirsch, Julie Bellemare, Andreas Hackl, Yvon-Louis Trottier, Andreas Mayrhofer, Heidemarie Schindl, Christine Taillon, Christian Gartner, Brigitte Hottowy, Gerhard Beck, Jacques Gagnon, BMC Pharmacology and Toxicology, 2014, 15:34
6. IPA, Pharmexcil Organises Technical Conference, November 11 2014, Express Pharma
Michael E. Dawson, Ph.D., RAC
Director of Regulatory Affairs
Associates of Cape Cod, Inc.
The question is often asked, how many bacteria does it take to make one Endotoxin Unit (EU)? Although it sounds like the beginning of a joke, unfortunately, there is neither a punch line nor a simple answer. In the case of sterile solutions, which may contain high concentrations of endotoxin, the answer is none, regardless of how much endotoxin is present. In the case of non-sterile solutions, endotoxin concentration may or may not correlate with bacterial number. When there is a correlation, the relationship between the two parameters can vary substantially. Great caution should be exercised in any attempt to relate endotoxin concentrations to numbers of organisms because of the variability of this relationship. With those caveats in mind, this article reviews some aspects of this relationship and the measurements involved.
As endotoxin is derived from the outer membrane of gram negative bacteria, it is not unreasonable to suppose there might be a relationship between bacterial number and endotoxin concentration. Watson and coworkers  demonstrated strong positive correlations between the two parameters both in the laboratory for an Escherichia coli culture in log phase of growth and in seawater samples from the open ocean. For E. coli in log phase the authors reported 49.4 fg of bound lipopolysaccharide (LPS) per cell; this declined to 28.9 fg/cell (i.e. 2.89 10-16 g/cell) in stationary phase. By contrast the average concentration of LPS for the seawater samples was 2.78 fg/cell. Thus, the relationships were quite different for the two data sets. For natural freshwaters, Evans et al.  have reported strong correlations between endotoxin concentration and both coliform and heterotrophic bacterial counts.
A significant correlation between endotoxin concentration and bacterial number has also been demonstrated in a pure water system . In this case, the concentration of LPS per cell varied throughout the system from a high of 15.7 fg/cell to a low of 2.1 fg/cell. The high value was recorded early in the treatment train and the low at the beginning of the distribution loop, which was the cleanest (highly oligotrophic) part of the system. In this study, as in the study by Watson et al. , bacterial number was determined by epifluorescence direct counting (EDC), not by culture. It should be noted that the numbers obtained by EDC can be an order of magnitude, or more, greater than those obtained by culture (for example, see Armisen and Servais ), particularly in the cases of natural and oligotrophic systems. Consequently, the amounts of endotoxin per cell that are determined using EDC values are lower than if culture techniques are used.
Another major caveat surrounding these determinations is that the values for mass of LPS reported are not based on an absolute determination of the mass of LPS. The amounts of LPS per cell are obtained in bacterial endotoxin tests using Limulus amebocyte lysate (LAL) reagent. The results represent the mass of standard endotoxin that has the same activity as that in the sample, not the amount of endotoxin present in the sample. Thus, a value of 2.1 fg/cell means that the activity of the endotoxin from a single cell is equal to that of 2.1 fg of the standard endotoxin preparation; we do not know the actual mass of endotoxin in the cell which gives that activity.
In the work of Watson et al.  and Dawson et al. , LPS preparations from of E. coli were used as endotoxin standards and results are expressed in units of mass of that standard. The problem is that different endotoxins can differ markedly in activity (or potency) per unit mass. This is true regardless of whether activity is determined in pyrogen tests or in LAL tests (,,, and ). Consequently, results expressed in units of mass of one endotoxin cannot be readily compared with another study in which a different standard endotoxin was used. The results within any one such study can be meaningfully compared with other results in the same study. The standard endotoxins used in such studies are usually derived from a single organism (commonly an E. coli strain) and consist of a purified preparation of lipopolysaccharide. In contrast, the endotoxin being measured may come from a variety of bacteria, which may not include the organism from which the standard endotoxin was obtained. In the two studies discussed at the beginning of this paragraph the bacteria in waters studied probably did not include E. coli. This problem of the different relative potencies of different endotoxin standards has now largely been addressed by the development of a reference standard endotoxin (RSE) and the establishment of the Endotoxin Unit (EU).
Relatively early in the application of LAL tests for the detection of endotoxin, FDA recognized the problems cause by the use of different endotoxin standards in different studies and the difficulty in comparing results. To address these problems, FDA decided to establish a standard endotoxin preparation expressed in units of activity as opposed to mass. The first significant batch RSE was designated EC-2. It comprised vials containing 1.0 µg of LPS derived from E. coli O113:H10 K negative lyophilized with 0.1% human serum albumin. A potency (a measure of biological activity) of 5,000 Endotoxin Units (EU) per vial was assigned to the standard. Given the mass of 1.0 µg of LPS per vial, this equates to 5 EU/ng. Batch EC-2 was followed by other reference preparations, including those provided by the current World Health Organization, the United States Pharmacopeia (USP) and European Pharmacopoeia, all of which are essentially the same material but with different labeling (and expressed in different Units in the case of the USP endotoxin reference standard).
For standard E. coli LPS preparations, it is common to think of a potency of approximately 10 EU/ng, which is a common value reported on certificates of analysis for control standard endotoxins (CSEs). CSEs are secondary standard endotoxin preparations (such as those provided by LAL reagent manufacturers) that have been standardized against RSE. Sometimes values of 20 EU/ng or higher are obtained for CSEs; in other cases values down to about 5 EU/ng are obtained. The potency of a given endotoxin preparation relative to RSE can vary depending upon the lot of lysate reagent used to make the determination. Consequently, it is important to use the potency stated on the appropriate certificate of analysis for the specific lots of CSE and of lysate reagent used.
Bearing in mind the caveats given above, the reported amounts of endotoxin per cell range between about 2 and 50 fg/cell. If we assume that 1 EU unit represents about 0.1 ng of endotoxin (i.e. 105 fg), then 1 EU would be equivalent to between 2,000 and 50,000 cells. However, given all the assumptions and caveats (including the fact that sterile solutions may contain high endotoxin concentrations), any predictions about the numbers of viable organisms that are derived from endotoxin concentrations should be treated with a healthy dose of skepticism.
 Watson, S. W., T. J. Novitsky, H. L. Quinby, and F. W. Valois. 1977. Determination of bacterial number and biomass in the marine environment. Appl.Environ.Microbiol. 33: 940-946.
 Evans, T. M., J. E. Schillinger, and D. G. Stuart. 1978. Rapid determination of bacteriological water quality by using Limulus lysate. Appl.Environ.Microbiol. 35: 376-382.
 Dawson, M. E., T. J. Novitsky, and M. J. Gould. 1988. Microbes, endotoxins and water. Pharm.Engineering. 8(2): 9-12.
 Armisen ,T.G. and P. Servais. 2004. Combining direct viable count (DVC) and fluorescent in situ hybridisation (FISH) to enumerate viable E. coil in rivers and wastewaters. Water Sci Technol. 50(1): 271-275.
 Greisman, S.E. and R. B. Hornick, R.B. 1969. Comparative pyrogenic reactivity of rabbit and man to bacterial endotoxin. Proc. Soc. Exp. Biol. Med. 131: 1154-1158.
 Pearson, F.C., M. E. Weary, J. Bohon. 1982. Relative potency of “environmental” endotoxin as measured by the Limulus amebocyte lysate test and the USP rabbit pyrogen test. In Watson S. W., J. Levin, T. J. Novitsky, eds. Endotoxins and their detection with the Limulus amebocyte lysate test. Alan R. Liss, Inc. New York. pp. 65-77.
 Pearson, F. C. III, M. E. Weary, H. E. Sargent, T. J. Novitsky, M. P. Winegar, H. Lin, G. Lindsay, R. N. Berzofsky, A. L. Lane, J. D. Wilson, J. F. Cooper, E. J. Helme, C. W. Twohy, H. I. Basch, M. Rech, and J. W. Slade. 1985. Comparison of several control standard endotoxins to the national reference standard endotoxin – an HIMA collaborative study. Appl. Environ. Microbiol. 50: 91-93.
 Weary, M. E., G. Donohue, F. C. Pearson, and K Story. 1980. Relative potencies of four reference endotoxin standards as measured by the Limulus amebocyte lysate and USP rabbit pyrogen tests. Appl. Environ. Microbiol. 40: 1148-1151.
 Weary, M. E., F. C. Pearson, III, J. Bohon, and G. Donohue. 1982. The activity of various endotoxins in the USP rabbit test and in three different LAL tests. In S. W. Watson, J. Levin. and T. J. Novitsky (eds.), Endotoxins and their Detection with the Limulus Amebocyte Lysate Test. Alan R. Liss, New York, 1982, pp. 365-379.