In this issue:Endotoxin LimitsTechnical Services DepartmentCalendar

Volume 13, No.2

pfiJLALUPDATE

June 1995

Dear LAL User,

There are now definite signs that summer will soon arrive onCape Cod. The horseshoe crabs have appeared once again in theshallow bays in preparation for spawning and our laboratory isbursting with activity. Although the amount ofour harvest fromseason to season is difficult to predict, the last two years have seenrecord yields ofLAL and we hope for a repeat this year. Even withincreased sales, these good harvests have resulted in an excellentinventory ofPyrotell'" (all sensitivities), Pyrotell'"-T, andPyrochrome"

For our European customers, the changeoverfrom afragmenteddistribution system to direct supply and service from Associates ofCape Cod International, Inc. is complete. Our two European officesare located in Walldorf (near Frankfurt) Germany, and Liverpool,England. Our general managers, Dr. Peter Weidner (ACCI-Europein Germany), and Tony Coyle (ACCI-UK) have extensive experiencewith all aspects ofLAL and endotoxin testing. In addition to ourexpanded technical services, ACCI-Europe can provide multilingualhelp for our clients in France, Italy, and Spain.

The Tyros for Windows" software for the LAL-5000 is nowfirmly established in the marketplace. Ifyou are using the LAL-5000and have not yet switched to Tyros, I strongly urge you toconsider doing so. It is much easier andfaster to use and has a hostofadvancedfeatures.

As you can see, it is not all vacation and retirement on Cape Cod.Even so, please feel free to make our summer even busier! Ourtechnical service team, Laurie, Keith, and Carmen are ready toanswer your technical questions, as are other personnel, includingDrs. Dawson and Gould, and ofcourse, myself.

Sincerely,

Thomas 1. Novitsky, Ph.D.President/CEO

o 1995, Associates of Cape Cod, Inc., Woods Hole, Massachusetts

EndotoxinLimits

Michael E. Dawson, Ph.D.

It has been established that thepyrogenic threshold for humans isapproximately five endotoxin units(EU) per kilogram body weight (1,2).This means that administration ofendotoxin concentrations of 5 EUlkgor greater can be expected to elicit apyrogenic response. Consequently,an endotoxin limit of 5 EUlkglhr hasbeen set for most injectable(parenteral) products. An injectableproduct for which this limit appliescannot be sold if the endotoxinconcentration is such that a recipientof the maximum dose would receivethis amount of endotoxin or more.

The endotoxin limit is representedby the term K. The value of K is 5EUlkglhr for injectables, other thanthose administered intrathecally. Forthese, the endotoxin limit is morestringent and K= 0.2 EUlkglhr.

Given that the average humanweighs 70 kg, the maximum amountofendotoxin that can be received by apatient being given a non-intrathecalproduct should not exceed 350 EUper hour (70 x 5 EUlkglhr).

However, the limit does notaccount for a patient being givenseveral drugs and/or solutions at

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once, which is a common occurrence.Fortunately, most drug products andsolutions contain considerably lessendotoxin than the allowable limit.Many manufacturers set specifica-tions that are tighter than the requiredlimit.

The calculation of endotoxinlimits for injectable drugs adminis-tered per square meter is based on theassumption that a typical adult has asurface area of 1.8 square meters.For drugs with a dose expressed perkg, the whole body limit is 350 ED.Thus, the endotoxin limit per squaremeter is 350 EU/1.8 m2= 194EU/m2This is an absolute and is equivalentto a value of 5 EU/kg for K forproducts administered per kg. Tocalculate the endotoxin limit of aproduct with a dose expressed persquare meter, divide 194 EU/ m? bythe dose to give an endotoxin limit perunit of product.

Endotoxin limits for radio-pharmaceuticals are addressed in afootnote at the end of Appendix E tothe FDA Guideline on the LAL Test(3). For these productsK = 175 (EU)/V (or 14(EU)N for intrathecals),where V is the maximum (wholebody) dose in ml at expiration of theproduct. The units (the EU given inparentheses) are not specified in theGuideline but must be included if theunits are to be expressed in EU/m!.

The USP limit for large volumeparenterals (LVPs) is 0.5 EU/ml andis derived from a rabbit dose of 10 ml/kg for Pyrogen tests. A typical LVPadministered at 700 ml/hr also gives alimit of 0.5 EU/m!. The USPendotoxin limit for WFI and SWFI is0.25 EU/m!. For medical devices,the USP limit is 20 EU/device, and2.15 EU/device for devices thatcontact the cerebrospinal fluid. Theendotoxin limits for devices werediscussed in the September 1994

LAL UPDATE Vol. 13, Number 2, page 2

issue of the LAL UPDATEGenerally, the aim of testing is to

insure that the sample under testcould not deliver more than 5 (or 0.2)EU/kg/hr of endotoxin to a patient.Thus, the endotoxin limit for aspecific product depends on themaximum dose of product that apatient might receive. The smallerthe dose of the product per kg, thehigher the endotoxin limit per unitdose of the product.

This discussion of limits onlyapplies to release testing of finishedproducts. However, endotoxin limitsmay be set for raw materials and in-process samples.

Determining the Endotoxin Limitofa Product

The first step is to look up theproduct in the FDA Guideline to seeif an endotoxin limit is listed in theAppendix E. (The most recentrevision to Appendix E at this time isdated July, 1994). If the product islisted, check that the maximum doseof the product is correct for yourproduct. If the dose is greater thanthat given in the table, calculate thelimit as described below. It is a goodidea to calculate the limit as describedbelow anyway. There have beenmistakes in the Appendix in the past.

The 1987 Guideline states that thedose used should be the rabbit dose(as used in the USP Pyrogen Test) orthe maximum human dose, which-ever is greater. Since 1987, the USPhas used only human doses tocalculate endotoxin limits and theFD A has incorporated this into theirmore recent revisions ofAppendix E.The term M is used for the maximumdose.

When calculating the limit fordrug products, if the dose isexpressed for whole (adult) body,

first divide it by 70 kg to give the doseper kg. Then divide K (5 EU/kg, 0.2EU/kg, 194 EU/m2 or 175 EU) by M(or V) to give the endotoxin limit perunit weight/volumelinternationalunit, etc.

Examples:

Calculation of the endotoxin limitfor a non-intrathecal, injectabledrug product:

Product A has a maximum human(whole body) dose of 1.0 g

Dose per kg =.Ls, = 0.0143 g/kg = 14.3 mg/kg70 kg

Endotoxin limit =K = 5 ED/kg = 0.35 ED/mgM 14.3 mg/kg

Use the concentration of the product(potency in final product) to convertthe endotoxin limit into EU/m!.

Thus, using the example above, ifthe concentration of product in the(presentation) container is 100 mg/ml, or if bulk product is dissolved togive 100 mg/m!:

Endotoxin limit =0.35 EU/mg x 100 mg/ml = 35 ED/ml

Convert the limit to EU/mlbecause these are the units of theendotoxin standards in the LAL test(as well as the limits for the sensitivityof an LAL gel-clot reagent). Thisgives an endotoxin limit for theproduct so that it can be determinedwhether the product passes or fails atthat limit. It is important to note thatthe limit in EU/ml only applies to thestated concentration ofproduct. Iftheconcentration changes, the limitchanges.

The limits calculated above arethe maximum allowed. It may bedecided that tighter limits are

From left to right: Laurie Fife, Keith Richardson and Carmen Barillas

Meet Associates of Cape Cod, Inc.' sTechnical Services Department

desirable. Usually the limit is greaterthan the sensitivity ofthe test method.If the gel-clot method is used as apass/fail test, the product must bediluted.

Calculation of the endotoxin limitfor a product with a doseexpressed per square meter:

For a product with a dose of I g/m?and a potency of 50 mg/ml:

Endotoxin Limit =194 ED/ m?= 194 ED/g = 0.194 ED/mg

1 g/m?

0.194 ED/mg x 50 mg/ml = 9.7 El.l/rnl

Calculation of an endotoxin limitfor a radio-pharmaceuticalproduct:

For a product with a (whole body)dose at expiration of 7 ml:

Endotoxin Limit =K 175 ED 25 ED/mlV 7ml

ConclusionA clear understanding of endotoxinlimits and how they are derived is ofcritical importance to the release ofproduct by any LAL test method.

References:

1. EIin, R. J., S. M. Wolff, K. P. W. J. McAdam, L.Chedid, and F. Oberling. 1981. Properties ofreference Escherichia coli endotoxin and itsphthalylated derivative in humans. J. Infect. Dis. 144:329-336.2. Hochstein, H. D., E. A. Fitzgerald, F. G.McMachon, and R. Vargas. 1994. Properties of USStandard Endotoxin (EC-5) in human male volunteers.Journal of Endotoxin Research. 1:52-56.3. Guideline on Validation of the LimulusAmebocyte Test as an End-product EndotoxinTest for Human and Animal Parenteral Drugs,Biological Products, and Medical Devices. U.S.Department of Health and Human Services, PublicHealth Services, Food and Drug Administration,December 1987.

LAL UPDATE Vol. 13, Number 2, page 3

With the continuing expansion ofourbusiness, we now have three TechnicalServices Representatives to answer yourLAL-related questions. As in the past,all calls are answered immediately byour Technical Services Department. Thedepartment includes: Laurie Fife, Se-nior Technical Services Specialist,Keith Richardson, Technical Analystand Carmen Barillas, Technical Ser-vice Representative.

Laurie joined ACC in 1993 afterdoing research for six years in the areasofcell/molecular biology and biochemis-try. For two years before moving to CapeCod, Laurie was a Scientific Liaison atOrganogenesis, Inc.

At ACC, Laurie provides technicalassistance and participates in the trainingcourses. She specializes in the chro-mogenic LAL method. She has a BSdegree in Animal Science from theUniversity of Vermont.

Keith has been an employee ofAssociates of Cape Cod, Inc. for morethan five years and has experience inmany facets of LAL technology, fromhemolymph collection to use of our finalproducts. He is responsible for all

aspects ofturbidimetric LAL testing, andis the technical support person for theLAL-5000 and Pyros software. Asour turbidimetric specialist, Keith par-ticipates in ACC training courses. Keithhas a BS degree in Biology and MarineSciences from Southeastern Massachu-setts University.

Carmen has an MS in Biology fromWorcester Polytechnic Institute and didher thesis work in the BiochemistryDepartment at the University of Massa-chusetts Medical Center. She was SeniorResearch Associate at Genica Pharma-ceuticals Corporation, where she didproduct development and research inmolecular biology for two years beforejoining ACC in 1994. Carmen is a nativeof Guatemala, Central America, and isfluent in both Spanish and English. Thecombination ofher technical backgroundand language skills makes Carmen awelcome addition to our technicaldepartment. She also participates in thetraining courses offered by ACe.

For expert technical help, regardless ofthe brand of LAL Limulus AmebocyteLysate you use, call 800-848-3248.