a limitation to the use of radiation sterilization is the adverse effects that ionizing radiation may have on the product being irradiated . similarly steam sterilization cannot be applied to heat - labile product . heat may also be a limitation for ethylene oxide as well as effects produced from reaction with the gas itself . with modern technology , heat labile or otherwise process - sensitive product may be produced by aseptic processing to a level where a less than one - in - a - thousand items are contaminated . such product cannot realistically achieve a 10 − 6 sal through aseptic processing , but could , provided that product could stand the necessary radiation dose , be brought to a 10 − 6 sal by a relatively low radiation dose . the microbial world contains a vast number of species varying widely in their resistance to ionizing radiation . although many are extremely sensitive there are some that are very radiation resistant , thus an essential element of any acceptable dose setting method is the inclusion in the process of a step that measures the radiation response of the product bioburden . the present invention improves upon prior methods to determine 10 − 6 sal product - specific sterilization dose that has an appropriate level of conservatism . it includes a determination of the bioburden to more accurately calculate sterilization doses in the presence of a low bioburden . to determine the sterilization dose according to the present invention it is preferred to select at least 270 product items from each of three independent production batches of a product to be sterilized . the bioburden is determined for each of ten nonsterile selected product items from each batch including the average bioburden per item for each of the three batches and the average bioburden per item for all selected product items . it is preferred to determine the bioburden on individual product items but if the bioburden is too low it is possible to pull ten items from a single batch for the purpose of determining a batch average bioburden . preferably , bioburden is determined following iso 11737 . the average bioburden level for each of the three batches are compared to the overall average bioburden to determine whether any one of the batch averages is two or more time greater than the overall average bioburden . if one or more of the batch averages are two or more times higher than the overall average bioburden then for subsequent calculations the bioburden used should be the highest batch average , otherwise for subsequent calculations the overall average bioburden will be used . an ide is performed by irradiating twenty product items from one of the three production batches at one of a series of not less than eight doses increasing in nominal increments of 0 . 25 kgy starting at not less than 0 . 25 kgy . each of these is monitored with dosimeters . preferably , the dosages should be within a tolerance of + 0 . 05 kgy or + 10 %, whichever is greater . the irradiated product items are then tested for sterility and the number of positive tests is noted . preferably , such testing follows iso 11737 - 2 . the fnp dosage is then determined . for each of the three batches the fnp dose is the lower of two consecutive doses at which all the tests of sterility are negative followed by no more than one further positive test in any of the remaining tests in the incremental dose series . alternatively , fnp can be determined by finding the lowest dose at which one positive in 20 tests of sterility occurs immediately preceded by one and only one incremental dose at which all tests were negative and followed by incremental doses at which all tests are negative . this information is used to determine a “ verification dose ” ( vd ) which is equal to the highest of the three fnp doses . the vd is used to perform a vde in which 100 products from each of the three batches are irradiated at the vd . the tolerances for the vde should be similar to those in the ide . the irradiated product items are individually tested for sterility and the number of positive tests recorded no more than two positive tests of sterility should appear for each of the batches . this data is then used to calculate a primary d 10 value ( pd 10 ) using the formula pd 10 = vd /( 2 + log ( bb )), where bb is the batch average bioburden in colony - forming units ( cfu ). the present inventors have examined a large number of actual populations and a large series of simulated populations of microorganisms to determine a relationship of the d 10 required to reduce each population over a wide range of bioburden numbers between that yielding a 10 − 2 sal ( pd 10 ) and that necessary to reduce the population remaining at the 10 − 2 dose to a 10 − 6 sal . the second d 10 is termed the terminal d 10 or td 10 . populations a through f comprising distributions of resistance of six different microbial populations , and selected modifications thereof , have found use as challenges in computer evaluations of various dose setting and substantiation methods . population c is the standard employed in dose setting method 1 and , as such , was designed to represent a highly severe challenge to the radiation sterilization process ; in developing the distribution , measurements of the resistance of constituent microorganisms giving d 10 values ranging from 1 . 0 to 4 . 2 kgy , were carried out with the organisms dried in the presence of organic material , thereby deliberately creating highly effective radiation protective conditions . population b represents the resistances of the same microorganisms as in population c but with resistances measured in the absence of organic solutes ; overall , it &# 39 ; s response to radiation is somewhat greater than that of population c , the d 10 range being 0 . 8 to 3 . 3 kgy . theoretical population a was postulated from population b to represent a minimal microbial challenge to the radiation sterilization process — it was derived by reducing each of the 9 classes of d 10 values by around 30 % while retaining frequencies of occurrence found for population b . clearly , the initial selection of population a is in keeping with the envisaged microbiological status of candidate products to which a new method of dose setting would be applied . d , e and f are populations which possess resistance distributions that exhibit responses to radiation much less than that of population c and thus are not relevant in the present context . the notion of dividing a radiation dose - response curve of a heterogeneous microbial population into two distinct parts was first utilized in the development of method vd max . in taking this activity forward , it was recognized that , subject to passing the vde at a specified sal ( generally 10 − 2 ), it is solely the response of the microbial population on product surviving at this sal that sets the sterilization dose to achieve a target sal somewhat below 10 − 2 ( generally 10 − 6 ). this terminal response is definable quantitatively by the d 10 value derived from the linear line joining the two points ( log 10 − 2 , dose at 10 − 2 ), ( log 10 − 6 , dose at 10 − 6 ); its value has been symbolized by the term td 10 . the upper part of the dose - response curve , occurring above an sal of 10 − 2 , is similarly definable through a d 10 derived from the line joining the points ( log bioburden , dose = 0 ), ( log 10 − 2 , dose at 10 − 2 ), its value being symbolized by pd 10 . this analysis of dose - response curves of population c is the foundation of method vd max that is now being applied to the substantiation of a range of sterilization doses extending from 15 to 35 kgy ; in this context , it has proved to be both a valuable and valid approach ( note , with method vd max , verification is performed at an sal of 10 − 1 and , hence , this sal is the transition point on the dose - response curve for the calculation of values of pd 10 and td 10 for use with this method ). heterogeneous microbial populations with an associated heterogeneous radiation response will generally constitute the bioburden present on product prior to sterilization . such populations inevitably yield values of td 10 that exceed those of pd 10 and , hence , give td 10 / pd 10 ratios that are greater than 1 . 0 . exceptionally , bioburden may comprise microorganisms of single type exhibiting a homogeneous response to radiation . homogeneous populations give ratios of 1 . 0 or , in circumstances where the microorganism &# 39 ; s dose - response curve exhibits a shoulder , less than 1 . 0 . for “ population a ” ( popa ), which is heterogeneous , the expectation is for td 10 / pd 10 ratios to be greater than 1 . 0 irrespective of the level of bioburden . this was found to be so . td 10 / pd 10 ratios vary systematically over the bioburden range , 0 . 02 to 1000 , taking a value of 2 . 01 at the lower limit and 1 . 67 at the upper while passing through a maximum of 2 . 18 at a bioburden of 0 . 50 . the existence of a maximum is due to the different rates at which values of td 10 and pd 10 increase with increasing bioburden . rounding up of this maximum to a value of 2 . 2 provides an initial choice of the pd 10 coefficient against which values of td 10 / pd 10 ratios derived from populations possessing distributions of resistance other than that of popa can be compared . values of td 10 / pd 10 ratios for “ population c ” ( popc ), another heterogeneous microbial population , behave in a manner similar to that found for popa with increasing bioburden . for bioburden levels extending from 0 . 02 to 1000 , ratios range from 1 . 78 to 1 . 58 with a maximum of 1 . 92 at a bioburden of around 0 . 30 . popc is the resistance distribution ( so - called standard distribution of resistances ) on which dose setting method 1 is founded and its td 10 / pd 10 ratios are , at comparable bioburden levels , universally less than those of popa , a finding indicating a degree of conservativeness associated with a choice of 2 . 2 as the pd 10 coefficient . these microbial populations comprise a family developed by the modification of popa . modification involved shifting systematically to the right the frequency of the highest resistance occurring in popa while reversing the displaced frequencies in ascending order within the eight populations . this gave populations with responses to radiation that progressively decrease with increasing md designator . as all eight mds are heterogeneous , they gave values of td 10 / pd 10 ratios greater than 1 . 0 . for this group of populations with widely varying resistance distributions , no value of the td 10 / pd 10 ratio was greater than 2 . 05 for the bioburden range 0 . 02 to 1000 with the vast majority well below the comparator value of 2 . 2 . in fact , as the overall resistance of the populations increased ( in other words , the response to radiation decreased ), the ratios approached a value of 1 . 0 , again pointing to the conservative nature of a coefficient of 2 . 2 . another means of developing distributions of resistance varying overall from that of the parent distribution is to progressively sum the resistance probabilities , starting with that which takes the lowest value and proceeding to the highest . this provides populations for which responses to radiation are , to varying degrees , either more than or less than that of the parent population . popa_md6 and md9 is one such pair of populations that respond in this opposite way , popa_md10 and md14 is another and popa_md15 and 20 is a third , the population with the higher numerical designator of the pair showing the lesser response to radiation and the difference in response for the paired populations becoming greater as the designator increases . for those modified populations whose response to radiation is greater than that of popa ( md6 , 9 and 15 ), calculated values of td 10 / pd 10 ratios across the bioburden range 0 . 02 - 1000 are all below corresponding values for popa and thus are amply covered by a value of 2 . 2 . in contrast , the populations , which have been modified in a way that provides a proportion of resistant microorganisms in excess of that of popa resulting in a lessened response to radiation , give values of ratios at the low end of bioburden range that are greater than 2 . 2 . for example , a maximum ratio of 2 . 52 is seen for popa_md9 at a bioburden of 0 . 3 , 2 . 76 for md14 at 0 . 2 and 3 . 04 for md20 at 0 . 08 . clearly , these findings require consideration in making the final choice for the value of the pd 10 coefficient . seven further populations have been studied . two , created from each of popa and popc , were developed so that the same probability occurred in each resistance class making up the distribution ; they were designated popa_even and popc_even , respectively . two more populations were modifications of popa and popc . they comprised equal probabilities of each of solely the most sensitive and most resistant classes of the populations and were given designates popa — 50s — 50r and popc — 50s — 50r . three populations were homogeneous in nature , each comprising a single type of microorganism having a resistance defined by a d 10 value of 0 . 5 , 2 . 5 or 4 . 2 kgy ; they were designated pop_mono 0 . 5 , 2 . 5 and 4 . 5 , respectively . all seven populations gave td 10 / pd 10 ratios according to the general expectation noted above . the heterogeneous populations provided a range of values of ratios that varied systematically with changing bioburden level and exhibited a maximum . furthermore , with the exception of popa — 50s — 50r at a bioburden of 0 . 02 , ratios took values of less than 2 . 2 . the exception resulted from the presence of an unduly low pd 10 value at this bioburden level . the homogeneous ‘ mono ’ populations all showed td 10 / pd 10 ratios of 1 . 0 . simulated experiments , employing mixed suspensions of b . pumilus spores and s . marcescens cells irradiated at doses ranging from 0 . 25 to 1 . 0 kgy , gave the necessary assurance that detection is technically feasible with these radiation doses . they also demonstrated that an incremental dose experiment employing this dose range could yield an array of fraction positive results from which an estimate of the dose to achieve an sal of 10 − 2 could be obtained . the simulated ide and vde have demonstrated that associated procedures , as modified , give successful outcomes . thus , there is good reason to believe that ‘ low dose ’ ides and vdes carried out on product will be technically - feasible , practical procedures from which meaningful and necessary doses can be identified . the td 10 / pd 10 analysis of popa produced a maximal rounded - up ratio of 2 . 2 and this value has been set against values of ratios derived from analyses done on a wide variety of populations having substantially different , but often allied , resistance distributions . the general outcome from this comparison is that popc , which has a greater radiation resistance than popa , and most populations with modifications to the resistance distribution of popa provide values of ratios appreciably less than 2 . 2 over a wide range of bioburden levels . clearly , this outcome supports strongly the choice of 2 . 2 as the pd 10 coefficient and underlines the conservativeness of the value . the exceptions to this finding are certain populations possessing distributions in which there are present higher proportions of microorganisms of high radiation resistance than that in popa . their presence produces principally increases in the values of td 10 which , in turn , give high td 10 / pd 10 ratios . given the above exceptions , what has to be considered is the relevance of such distributions to the ‘ real world ’. at present this is a judgment decision , although it has to be said that , in the light of the stipulated manufacturing conditions and the controls to be imposed on them , the occurrence on product prior to sterilization of microbial populations having a significant number of microorganisms of high radiation resistance is highly unlikely . moreover , the results of the vdes will act as a check for their absence . if such microorganisms do occur , they will have to present at a specific bioburden in a proportion in excess of that of popa level for the pd 10 coefficient of 2 . 2 to be invalid . td 10 is thus assumed to be 2 . 2 times pd 10 . the sterilization dose to achieve a 10 − 6 sal is then calculated by adding four td 10 doses to the vd , in other words sterilization dose equals vd plus ( 4 * td 10 ). the sterilization dose is thus the dose used to sterilize the devices in question and provides a sterility assurance level of 10 − 6 . the method is recommended for extremely low average bioburdens of five cfu or below . it is useful for sterilization of delicate drugs and radiation sensitive devices . proteins are particularly difficult to sterilize without damage . one particular area of concern for the inventors is the sterilization of blood proteins and plasma proteins . the source of the proteins may be natural ( i . e . human , animal ), synthetic or recombinant . blood protein / plasma protein serves as a transport molecule for lipids , hormones , vitamins and metals . they also serve as enzymes , complement components , protease inhibitors , and kinin precursors . blood protein / plasma protein includes , but is not limited to , albumin , ancrod , batroxobin , collagen , ecarin , elastin , epinephrine , factor x / xa , factor vii / viia , factor ix / ixa , factor xi / xia , factor xii / xiia , fibrin , ficolin , fibrinogen , fibronectin , gelatin , globin , haptoglobin , hemoglobin , heparinase , inhibin , insulin , interleukin , lamininthrombin , platelet surface glycoproteins , prothrombin , selectin , thrombin , transferin , von willebrand factor , vasopressin , vasopressin analogs , procoagulant venom , platelet activating agents and synthetic peptides having hemostatic activity . the present inventors are also concerned with sterilization of polymers , and in particular polymers useful in preparing the fabric substrates in wound dressings , which include , without limitation , collagen , calcium alginate , chitin , polyester , polypropylene , polysaccharides , polyacrylic acids , polymethacrylic acids , polyamines , polyimines , polyamides , polyesters , polyethers , polynucleotides , polynucleic acids , polypeptides , proteins , poly ( alkylene oxide ), polyalkylenes , polythioesters , polythioethers , polyvinyls , polymers comprising lipids , and mixtures thereof . preferred fibers comprise oxidized regenerated polysaccharides , in particular oxidized regenerated cellulose . the methods of the present invention are expected to be quite useful with the preceding polymers and proteins . while the invention has been particularly described in connection with specific embodiments thereof , it is to be understood that this is by way of illustration and not of limitation , and that the scope of the appended claims should be construed as broadly as the prior art will permit .
a method provides for sterilizing with radiation objects which have a low bioburden and which are sensitive to radiation . a dosage of radiation sufficient to ensure sterilization without damaging the object is determined by determining the bioburden upon one or more samples of the objects , determining an estimate of the dose that results in a probability of 0 . 01 of a surviving microorganism by testing a quantity of samples of the objects at varying dosage levels of radiation , confirming the estimate by testing a quantity of samples of the objects at the dose that was estimated ; and calculating a dosage for the sterility assurance level of 10 - 6 by adding a factor to the dose that was confirmed to result in a probability of 0 . 01 of a surviving microorganism and wherein the factor is proportional to the dose that yields a probability of 0 . 01 of a surviving microorganism and inversely proportional to a log of the bioburden .