BET
The Bacterial Endotoxins Test – Back to the Future
Karen Zink McCullough MMI Associates
The fifteenth anniversary of American Pharmaceutical Review prompted my reflection on the 40+ years of evolution of the Limulus Amebocyte Lysate (LAL) test in our industry. This article will reflect on where the Bacterial Endotoxins Test (BET) was 15 years ago and predict what the future holds for the next 15.
The Past 15 Years
15 years ago, the test was referred to as the LAL test. Today, the compendial name for the assay is the Bacterial Endotoxins Test, or BET.
15 years ago, the gel clot test was the most commonly used test, although endpoint and kinetic assays were becoming widely accepted for routine release tests. Today, quantitative tests are used more often than gel clot across the industry, but the gel clot limits test still remains the referee test in the harmonized compendial chapter.
15 years ago, we had the choice of running quantitative assays on microtiter plate readers and tube readers. Today, these instruments have become more sophisticated and are supported by Part 11 compliant software that interfaces with laboratory information management systems (LIMS) and tracks and trends data. In addition, we have the option of a cartridge system, which is a convenient choice for many applications.
15 years ago, all reagents used for testing were derived directly from the circulating blood cells of the horseshoe crab. Today, we have alternatives. We can choose a reagent formulated with recombinant coagulogen, the clotting protein in the LAL reaction. This reagent, and future reagents that do not rely on bleeding horseshoe crabs will increase consistency in testing and will help ease concerns regarding pressure on the horseshoe crab population. In addition to Limulus-based reagents, we have the Monocyte Activation Test, an assay-based reaction of human blood to endotoxin that more closely mimics the action of endotoxin in humans.
Karen Zink McCullough is owner and
principal consultant at MMI Associates. She is widely published on the topic of the BET. Ms.
McCullough is Founder and Steering Committee Member for the LAL Users’ Group. She is an
elected member of the USP Expert Microbiology Committee, General Chapters and is a US
delegate to ISO TC 209, WG02, Ms. McCullough received her BA in Bacteriology from Rutgers
University and her MS in Molecular Biology from the University of Oregon.
15 years ago, the FDA’s 1987 “Guideline on Validation of the Limulus Amebocyte Lysate Test as an End- Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices” provided an Out of Specification (OOS) investigation scheme that included an immediate retest of twice the number of original replicates and a second retest of 10 units tested individually. Since then, FDA released its 2006, “Guidance for Industry: Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production”, which requires an investigation to justify a retest. However, footnote 3 in that Guidance indicates that the document is not intended to address biological assays. So, analysts and managers are left to decide: is the BET an analytical assay or a biological assay? There is still some debate, but the Agency’s philosophy is clear – a retest without justification will be questioned.
15 years ago, we saw a different pattern of reagent consumption than we do today. Interestingly, when the LAL test was first introduced in the early 1970s, the focus was on raw material and in process testing, largely because it unclear that FDA would ever abandon the compendial Rabbit Pyrogen Test for release of finished pharmaceuticals and devices. After an exemplary collaboration between industry and the Agency, and the subsequent publication of the 1987 Guideline, the focus shifted to end product testing and replacement of the rabbit test. As test methods and data
18 American Pharmaceutical Review | Endotoxin Supplement 2013
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128 |
Page 129 |
Page 130 |
Page 131 |
Page 132 |
Page 133 |
Page 134 |
Page 135 |
Page 136 |
Page 137 |
Page 138 |
Page 139 |
Page 140 |
Page 141 |
Page 142 |
Page 143 |
Page 144 |
Page 145 |
Page 146 |
Page 147 |
Page 148 |
Page 149 |
Page 150 |
Page 151 |
Page 152 |
Page 153 |
Page 154 |
Page 155 |
Page 156