Quality control is a part of good manufacturing practice (GMP) regulations that is concerned with the sampling, specifications, and testing of materials, as well as the organization, documentation, and issuing procedures that ensure the necessary and relevant tests are carried out, and that starting materials are not released for use, nor products released for sale or supply, until their quality has been judged satisfactory. It is an important aspect of pharmaceutical production, because the tests (or analytical procedures) have to confirm that pharmaceutical environments, raw materials, packaging materials, and intermediate and finished products are in compliance with their specifications.
To help ensure the correctness of analytical procedures, the GMPs require validation to provide documented evidence that every method, instrument, and material/reagent produces expected results. While validation does not improve the analytical process, it does provide confidence regarding its output. Usually validation mainly tests the technological and instrumental aspects of a process, although human factors might still affect the final result. Since 2003, the International Conference on Harmonization (ICH), “Quality Risk Management” (QRM), has been suggesting using risk analysis in every phase of the life cycle of a drug. Therefore, risk analysis should not be limited to development, production, and distribution, but also should be extended to quality control in order to improve the assurance, quality, and cost savings of analytical procedures.
ONE BAD ACTOR
Endotoxins are lipopolysaccharides (LPS) derived from cell membrane of Gram-negative bacteria and are responsible for its organization and stability. In pharmaceutical industries it is possible to find endotoxins during production processes or in the final product. Although endotoxins are linked within the bacterial cell wall, they are continuously liberated into the environment. A single Escherichia coli contains about 2 million LPS molecules per cell. Endotoxin elicits a wide variety of pathophysiological effects. In conditions where the body is exposed to LPS excessively or systemically (as when small concentrations of LPS enter the blood stream), a systemic inflammatory reaction can occur, leading to multiple pathophysiological effects, such as endotoxin shock, tissue injury, and death. The maximum level of endotoxin for intravenous applications of pharmaceutical and biologic product is set to 5 endotoxin units (EU) per kg of body weight per hour by all pharmacopoeias. The term EU describes the biological activity of an endotoxin. For example, 100 pg of the standard endotoxin EC-5 and 120 pg of endotoxin from Escherichia coli O111:B4 have activity of 1 EU. Meeting this threshold level has always been a challenge in biological research and pharmaceutical industry.
In the biotechnology industry, Gram-negative bacteria are widely used to produce recombinant DNA products such as peptides and proteins. Many recombinant proteins are produced by the Gram-negative bacteria Escherichia coli. These products are always contaminated with endotoxins. For this reason, proteins prepared from Gram-negative bacteria must be as free as possible of endotoxin in order not to induce side effects when administered to animals or humans. However, endotoxins are very stable molecules, resisting to extreme temperatures and pH values in comparison to proteins. Many different processes have been developed for the removal of LPS from proteins based on the unique molecular properties of the endotoxin molecules. These include LPS affinity resins, two-phase extractions, ultrafiltration, hydrophobic interaction chromatography, ion exchange chromatography, and membrane adsorbers. These procedures provide different degrees of success in the separation of LPS from proteins, which is highly dependent on the properties of the protein of interest.
Endotoxin elicits a wide variety of pathophysiological effects, such as endotoxin shock, tissue injury, and death. Endotoxins do not act directly against cells or organs but through activation of immune system, especially the monocytes and macrophages, thereby enhancing immune responses. These cells release mediators, such as tumor necrosis factor, several interleukins, prostaglandins, colony stimulating factor, platelet activating factor and free radicals. The mediators have potent biological activity and are responsible for the side effects upon endotoxin exposure. Finally, it should be mentioned that endotoxins may also have beneficial effects. They have been used in artificial fever therapy, to destroy tumors and to improve, non-specifically, the immune defense. On the other hand, any superfluous endotoxin exposure must be strictly avoided to prevent complications. This is especially true for intravenously-administered medicines.
RABBIT TEST FADING FAST
The commonly used FDA-approved techniques for endotoxin detection are the rabbit pyrogen test and Limulus Amoebocyte Lysate (LAL) assay. The rabbit pyrogen test, developed in the 1920s, involves measuring the rise in temperature of rabbits after intravenous injection of a test solution. Due to its high cost and long turnaround time, the use of the rabbit pyrogen test has diminished, and is now only applied in combination with the LAL test to analyze biological compounds in the earlier development phase of parenteral devices. Today the most popular endotoxin detection systems are based on LAL, which is derived from the blood of horseshoe crab, Limulus polyphemus, and clots upon exposure to endotoxin. The simplest form of LAL assay is the LAL gel-clot assay. When LAL assay is combined with a dilution of the sample containing endotoxin, a gel will be formed proportionally to the endotoxin sensitivity of the given assay. The endotoxin concentration is approximated by continuing to use an assay of less sensitivity until a negative reaction (no observable clot) is obtained. This procedure can require several hours. The concentration of 0.5 EU/mL was defined as the threshold between pyrogenic and non-pyrogenic samples.
In addition to the gel-clot technique, manufacturers have also developed two other techniques: turbidimetric LAL technique and the chromogenic LAL technique. These newer techniques are kinetic based, which means they can provide the concentration of endotoxin by extracting the real-time responses of the LAL assay. Turbidimetric LAL assay contains enough coagulogen to form turbidity when cleaved by the clotting enzyme, but not enough to form a clot.
The LAL turbidimetric assay, when compared to the LAL gel-clot assay, gives a more quantitative measurement of endotoxin over a range of concentrations (0.01 EU/mL to 100.0 EU/mL.). This assay is based on the turbidity increase due to protein coagulation related to endotoxin concentration in the sample. The optical densities of various test-sample dilutions are measured and correlated to endotoxin concentration helped by a standard curve obtained from samples with known amounts of endotoxin. A kinetic chromogenic substrate assay differs from gel-clot and turbidimetric reactions because the coagulogen is partially or completely replaced by a chromogenic substrate. When hydrolyzed by the pre-clotting enzyme, the chromogenic substrate releases a yellow-colored substance known as p-nitroaniline. The time required to attain the yellow substance is related to the endotoxin concentration.
When implementing Quality by Design concepts, the strategy for endotoxins testing should be based upon product and process understanding in combination with risk management to ensure consistent final product quality. The appropriate in-process testing should be used to evaluate the production process areas at risk of endotoxins formation or incursion. Many firms already have programs for monitoring incoming ingredients and components, including the processing water, for endotoxins contamination. The finished product release specification should be considered when determining in-process limits for each phase of manufacturing tested. For purposes of evaluating the relative risk of product contamination, quantitative testing may be preferable to limit testing to facilitate product quality trending and to identify and correct excursions before they exceed the specification and cause product failure. An endotoxins limit should be justified on a case-by-case basis, and will be evaluated as a part of each relevant marketing application or supplement.
Efficient removal of endotoxins from biopharm solutions is challenging and a lot of research is being focused on this area. Despite the development of chromatography and membrane adsorbers in recent years, more research is needed to ensure the removal of endotoxin is a cost-effective process. All of the methods devised for endotoxin removal possess operational limitations and result in protein loss when the endotoxin level in the therapeutic solution is high, thus, increasing operational costs. Endotoxin removal from pharmaceutical and biotechnology solutions requires strict adherence to cGMP guidelines, as well as the selection, optimization and validation of the endotoxin removal method.
Published in the January 2014 edition of Pharmaceutical Manufacturing magazine