IE44542B1 - Proces for tempering tissui for heparin production - Google Patents

Abstract

IMPROVED PROCESS FOR TEMPERING TISSUE FOR HEPARIN PRODUCTION An improved method of tempering frozen heparin-bearing animal tissue is disclosed wherein the frozen tissue is particulated, thawed and warmed in a heat exchanger and fermented at temperatures ranging from 45.degree.-95.degree.F. for 2 to 15 hours. Putrefaction and odor are avoided, the tissue has improved uniformity in biochemical content and is capable of high heparin yields when processed according to known procedures. When the tempered tissue is defatted with an azeotropic solvent, more of the fat is removed and the defatted tissue is more permeable to heparin recovery solutions and heparin isolation is facilitated.

Description

This invention relates to a process for .tempering animal tissue for heparin production.

As used herein, the term ’'tempering refers to raising the temperature of frozen heparin-bearing animal tissue and conditioning, it for further heparin recovery processing.

The term heparin-bearing animal tissue refers to those animal tissues rich in heparin and suitable for heparin production, such as lung, brain, liver, intestines or inexpensive fleshy parts of animals.

The term particulate or derivatives thereof pertains to the divided state of the tissue: i.e. size up to about 1/4 inch mesh, or the act of dividing larger pieces which have been pre-broken or flaked.

The terms fermenting and fermentation refer to the combined action of enzymes present in the tissue and enzymes generated by growth of bacteria or microorganisms either present in the animal tissue or added enzymes or bacteria to speed the liberation of heparin.

The term azeotropic processing refers to the subjection of the tissue to the boiling action of an organic solvent which forms an azeotrope with water to remove substantially all the water and which extracts tissue fat into the solvent stream, and thereafter collecting and washing the dehydrated tissue on a filter with solvent.

Heretofore, frozen heparin-bearing animal tissue was tempered by allowing solidly frozen blocks of the tissue in bags or boxes to thaw gradually and warm up over periods of from 2 to 8 days at ambient temperature of 60° - 120°F -2I4S42 win 1<· in t-licKi* cnnlniiipi·», (luring which time, It was thought, optimul conditioning of the tissue for hepnriti release in subsequent heparin recovery was occurring. However, the tissue is very sensitive to enzymatic action and subject to ilei umpun I I I uu l>v utiilew 1 ml· 1 e haetpl'lnl growth anti rotting ot tissue. The outside of a frozen block of tissue subjected to such tempering treatment could putrefy before the interior had reached a thawed state. This resulted in generation of obnoxious odours which, during the 2-8 day period, would spread throughout the community surrounding the tempering plant.

This long period of time required for tempering resulted in poor utilization of space with consequent high overhead expense, bloody fluid and sewage disposal problems, and tempered tissue which was not sufficiently uniform biochemically from one lot to another due to non-uniform tissue breakdown, resulting in heparin unavailability and consequent need for continual adjustment during later processing in heparin isolation. In addition, unwanted bacterial decomposition causes high pyrogen content, necessitating procedures for pyrogen removal. Moreover, when tissues tempered by the above described prior art procedures were particulated and subjected to azeotropic solvent processing to remove fat and water, the fat content was not lowered below about 0.5# by weight even under the most favourable circumstances and more generally ranged from 1.0 to 2.0#. In addition, the defatted-dehydrated tissue particles were difficult to wet and floated for long periods of time in conventional solutions used in the initial step of the heparin recovery process, and the mixtures were difficult to handle and filter subsequently in the process. 45 4-2 According to the present invention a process for tempering frozen heparin-hearing animal tissue in preparation for isolation of heparin comprises the steps of: 1) particulating the frozen tissue; 2) thawing and warming the particulate tissue in a heat exchanger to a temperature in the range from 45° to 95°F; and 3) fermenting the thawed tissue from step 2 at a temperature in the range from 45° to 95°F for a period of from 2 to 15 hours tc improve heparin availability.

Tissue tempered according to the present invention is capable of conversion to desiccated and defatted tissue of unusually high quality by azeotropic processing wherein the fat content is generally reduced to about 0.1 to 0.3 weight the product is readily wetted in the above described heparin recovery process and mixtures are more easily filtered. Other indications of improved quality of such defatted tissues are lighter colour, less odour, good texture and homogeneity as . . well as consistent low levels of fat and residual solvent. In addition, more fat is recoverable from the solvent for a given amount of heparin-bearing tissue, which is an advantage. Apparently the combination of steps in the present process Of particulating frozen tissue, rapidly thawing and warming the tissue, and fermenting it under controlled conditions, is responsible for the increased wettability. Further, and 2? -4•14 5 4 3 ι equally important, due to the control of conditions during tempering the heparin yield and content of the defatted tissue having been first tempered by the process of this invention can be as much as 10 to 12% higher than for the above-described prior art defatted tissue.

The process of the invention can provide improved biochemical uniformity from one lot to another, exceptionally high availability of heparin due to control of time and temperature throughout the processing of each lot, and conditioned tissues which are very low in pyrogen content.

It is believed that by the process of this invention the total microbiological population is evenly distributed in the substrate tissue and growth is promoted, allowing the action of both endogenous and exogenous enzymes on the substrate, which produces a uniformly conditioned product having a more consistent biochemical content, which is lower in pyrogen content and high in available heparin content, and which may be advantageously employed to prepare a dehydrated and defatted heparin tissue by azeotropic processing exceptionally low in fat content and eaeily wetted and easily suspended in liquid involved in heparin recovery processing. A preferred form of the process comprises grinding frozen or partially frozen tissue rapidly thawing and warming the thawed tissue in a heat exchanger at a temperature within the range from 45° to 95°^ and thereafter fermenting the tissue within the temperature range from 45° to 95°F for a period of time within the range from 2 to 15 hours to optimize heparin availability, the length -5f I . 445*3 ' . ' ..- ' - I of time required being dependent on temperature. Generally speaking, the higher the temperature within the range the shorter will be the time required. For some unexplained πιίΐΗοιι, .sevafe evolution of gas ami foaming signals tho end of . the desirable fermentation phase.

The heparin in the tissue tempered by the process of this invention can be recovered by a number of techniques including the method described in U.S. Patent 2,797,184. The tempered product may be dehydrated and defatted first by azeotropic processing procedures such as are described in U.S. Patent 2,539*544 to provide a solid heparin source which is exceptionally low in fat and readily wetted by solutions used in heparin separation such as are described in U.S. Patents 2,797*184 or 2,954,321, with superior recovery of heparin as compared with *5 that from conventionally tempered tissue which has also been defatted by azeotropic means.

One form of the process of the invention for tempering heparin-bearing tissue in preparation for isolation and recovery of heparin comprises the steps of: l) pnrticulating the frozen tissue to any size up to about 1/4 inch mesh size, preferably l/8 to 1/4 inch mesh size, preferably using a grinder j 2.) thawirig and warming the particulated tissue from step 1 to a temperature within the range from 45° to 95°F, preferably 650 to 85°F, using a heat exchanger, preferably a shell and tube heat exchanger; and -63) fermenting the warmed tissue from step 2 while maintaining it at a temperature within the range from 45° to 95°F« preferably 650 to 85°F, for a period of from 6 to 8 hours but not substantially beyond the time at which foaming starts due to gas liberation.

The heparin-bearing animal tissue for the process of this invention may originate at a meat packing plant where it is cut from animal carcasses and handled according to specified procedures for preserving and enhancing heparin values, boxed or bagged and deep frozen. As a consequence, the animal parts arrive at the heparin recovery plant as blocks of one kind of frozen, agglomerated animal parts for example lung, in the size and shape of the containers. Usually, the blocks of frozen animal parts are too large for direct grinding in the size of grinder feasible for this art, and it is therefore necessary tD reduce the size of the blocks by some means prior to feeding to a grinder. To accomplish this the deep-frozen blocks of tissue may be cracked or prebroken mechanically in a device known in U.S.A. as a Prebreaker. Preferably, however, the blocks of tissue are partially defrosted for about 8 hours at ambient temperatures of 80 - 100°P and thereby brought from their deepfrozen state to a softer state by raising the temperature of the blocks to about 20-32°P, after which the softened tissue may be chipped or flaked in preparation for grinding. When the flaking or chipping procedures are followed the preferable temperature to which the blocks of tissue are raised is about 26°P, as at that temperature the tissue is rigid enough to be -7445*2 flaked, yet in a somewhat softened condition feasible fob the flaking operation. A suitable flaking machine is the Hydrauflaker produced in U.S.A. by the General Machinery Corp., Sheboygan, Wis. In any case, the tissue should not be so cold that the ground tissue in the next step refreezes into balls and clumps which prevent pumping. In general, the particle size of prehroken or flaked frozen tissue can vary from l/8 inch to 2 inches in diameter.

In step 1 of the process, grinders operate to reduce the size of the pre-broken or flaked, frozen or partially frozen tissue to that ranging from the size present in a puree up to a maximum dimensional mesh size of about l/4 inch, preferably up to l/8 inch mesh size. Grinders which arc suitable are the Comitrol produced in U.S.A. by Urschel Laboratories of Valparaiso, Ind, and the Autio” grinder produced in U.S,A. by the Autio Company, Astoria, Oregon.

In step 2, the ground frozen or partially frozen tissue is introduced by means of a pump such as Moyno pump to a - heat exchanger which operates to thaw and warm the tissue in 30 minutes or less, preferably within about 5 minutes, to within the 45° to 95°F temperature range, using a heat exchange surface temperature not exceeding l4o°F. Heat exchange for longer than about 3θ minutes may introduce too much variation in later processing. Heat exchange surfaces having a higher temperature than l40°F may cause fouling of the surfaces, denaturing of protein and microbiological kill-off. Shell and tube heat exchangers with tissue passing through the tube are -844542 highly satisfactory and preferred, but wiped surface heat exchangers may be also used. The preferred shell and tube beat exchangers may range in tube size from about 3/4 inch diameter to about one inch in diameter and consequently have 2 Π surface to volume ratios of about 50-75 ft per ft . Surfaces of tubes in this size range remain unfouled at normal pumping velocities.

In step 3» the warm tissue is held in a vessel having an inert surface such as a stainless steel tank at a temperature within the range from 45° to 95°F, preferably 650 to 85°F, for a period of time sufficient to condition the tissues as a result of a fermentation involving enzymes already present and enzymes produced by growing microorganisms. Above about 95°Ρ heparin values are rapidly lost, and below about 45°F the fermentation step is ineffective. From 2 to 15 hours fermentation time is required at 45° to 95°F and, as already mentioned, for some unknown reason the completion of the beneficial fermentation is signalled by severe gas liberation and rising in the holding tanks. Further fermentation decreases the yield of heparin. The holding period should be terminated then or just preceding this indicator according to previous experience as to time requirement for a particular temperature. Illustrative of the time/temperature relationship are the following results obtained by trial and error at which the frothing or gassing phase had begun. -9Time, hr Temp., °F 9-10 It is not necessary to wait until gassing occurs to obtain the superior products of this invention. Generally, there is some variation of microorganism in lung tissue taken from individual animals; however, grinding and mixing of hundreds of lung lobes assures that the fermentation will eventually proceed. When it is desirable to speed the fermentation, the necessary microorganism may be added, for example, by seeding the tissue at the beginning of the holding period with tissue which has already been fermented.

The conditioned product of this invention is well suited for use in azeotropic desiccating and defatting processes further to enhance heparin availability and separation such as are disclosed in U.S. Patent 2,619,425 and 2,539,544. Preferably, the azeotropic dehydrating-defatting operation is conducted at atmospheric pressure using ethylene dichloride at a temperature not exceeding 18O°F. Defatted tissue so obtained is characterized by its low fat content of about 0.1 to 0.3 weight % and by its excellent permeability as measured by wettability and suspendability.

Our Irish patent application No. 44543 filed . simultaneously herewith describes and claims a process for producing a defatted heparin tissue from frozen heparinbearing animal tissue in preparation for isolation of heparin which comprises the steps of; <148 4 2 1) particulating the frozen tissue; 2) thawing and warming the particulated tissue to within the temperature range from 4-5° to 95°F in a heat exchanger; 3) fermenting the thawed and warmed tissue from step 2 at a temperature in the range from 45° to 95°F for a period of from 2 to 15 hours to improve heparin availability! and 4) subjecting the fermented tissue from step 3 to azeotropic extraction with an organic solvent substantially to dehydrate and defat the fermented tissue.

The following Examples illustrate the invention.

Example 1, Partially defrosted frozen beef lung at 25-30°F in amount of 15,190 lbs was flaked using a Hydrauflaker (Model FS-6) to a size range of l/8 inch to 1/4 inch thick and up to 4 inches long. The flaked frozen lung was then ground with a Comitrol grinder (Model 2100) having 0.06 inch openings.

The ground frozen or partially frozen lung was pumped with a Moyno pump through 3/4 inch diameter tubes of a shell and tube heat exchanger to thaw and warm the particulated lung to 68° - 73°F, residence time in the heat exchanger being about 4 minutes. The warm ground lung was then held in a stainless steel tank for 6 hours at 70° - 75°F. No external heat was needed to maintain the temperature during the fermentation and a slight rise in temperature due to heat of reaction was also -11noted. There was obtained about 15,000 lbs of tempered lung suitable foi' heparin recovery processing. No undesirable odour was present during the processing.

Example 2.

Partially defrosted frozen beef lung at 26°F in amount of 16,200 lbs was flaked and ground as in Example 1, but using 0.120 inch openings on the grinder. The ground lung was thawed, and -warmed to 8 3°F in a heat exchanger as in Example 1, residence time in the heat exchanger being about 4 minutes. The warm ground lung was then held in a stainless steel tank for 6 hours.at 83° - 85°F. No undesirable odour was present during the processing. There was obtained 15,800 lbs of tempered lung suitable for heparin recovery processing.

Example 3.

Frozen beef lung (12,000 lbs) at 25° - 3θ°Ρ was flaked and ground as in Example 1 and thawed and warmed to 40° - 50°F, residence time being about 2 minutes in the heat exchanger.

The ground lung was held at 40° - 52°F for 6 hours. There was obtained about 11,900 lbs of tempered beef lung.

Example 4.

Frozen beef lung (12,000 lbs) at 25° - 30°F was flaked and ground as in Example 1 and thawed and warmed to 9θ° - 110°F in the heat exchanger. The warm, ground lung was held at 90° 11O°F for 6 hours. There was obtained about 11,900 lbs of tempered beef lung. -1244542 Example 5.

Frozen lung (16,200 lbs) at 26°F was flaked and ground as in Example 1 and thawed and warmed to 79°F in the heat exchanger. The warm, ground lung was held at 79° - 80°F for 8 hours. There was obtained 15,850 lbs of tempered beef lung. AZEOTROPIC EXTRACTION OF TEMPERED TISSUE The tempered lung products prepared in Examples 1 to 5 in accordance with the invention were then separatelysubjected to azeotropic distillation and extraction with ethylene dichloride at atmospheric pressure, collected on a filter, washed with ethylene dichloride and dried.to removeresidual ethylene dichloride. The dried and defatted lung products were processed for their heparin contents by an identical procedure. Comparative data are in Table 1. TABLE 1 Lung Fermentation, : Extraction and Heparin Isolation Starting # Yield of # Material Fermentation Dried Fat in Crude from Conditions Defatted Dried Heparin Heparin Ex. Luna Defatted Yield Potency No, Temp, °F Time ,hr.(a) Lung (b) (c) 1 70-75 6 18.4 0.18 158 7? 2 85 6 17.4 0.30 153 59 3 40-50 6 17.1 0.12 164 48 4 90-110 6 13.9 0.08 86 74 5 80 8 18.0 <0.2 155 96 134 4 5 *2 (a) Wt. ?ό based on starting lung. (b) Units heparin x 10^/kg. desiccated and defatted lung. (c) Units per mg. in crude heparin.

COMPARISON OF WETTABILITY OF TEMPERED - DESICCATED AND DEFATTED TISSUE Comparison of wettability was made of azeotropically defatted tissue prepared from tissue tempered by the process of this invention with that of the defatted tissue prepared from tissue tempered by the old method, wherein the lung was tempered for about 4 days at ambient temperature of 80 - 100°F. For this comparison, a 20 gram sample of the defatted product was stirred in 200 ml. of water until the particles appeared wet on the outside and the stirring was then stopped. The time required for the bulk of the particles to sink was then recorded. Data are in Table 2.

TABLE 2.

Wettability Comparison Method of Tempering Time for Particles to Sink to Bottom,' Seconds Old New _ > 180 < 10 -l44 4 β Μ 3

Claims (5)

CLAIMS:1. A process for tempering frozen heparin-bearing animal tissue in preparation for isolation of heparin which comprises the steps of:

1. ) particulating the frozen tissue;

2. A process as claimed in Claim 1 wherein in step 3 the temperature is in the range from 65 0 to 85°F and the period is in the range from 6 to 8 hours. 2) thawing and warming the particulated tissue in a heat exchanger to a temperature within the range from 45° to 95°F; and

3. « A process as claimed in Claim 1 or Claim 2 wherein the heat exchanger used in step 2 is a shell and tube heat exchanger, the tissue being moved through the tube. 3) fermenting the thawed and warmed tissue from step 2 at a temperature in the range from 45° to 95° f a period of from 2 to 15 hours to improve heparin availability.

4. . 4. A process as claimed in any of the preceding Claims wherein in step 3 the fermentation is aided by seeding the warmed tissue with bacteria, 5. A process as claimed in any of the preceding Claims wherein in step 1 the starting tissue is in a flaked condition. 6. A process as claimed in any of the preceding Claime wherein the heparin-bearing animal tissue is beef lung. -154 4 S * 2 7. A process fqr tempering frozen heparin-bearing animal tissue, substantially as described in any of the Examples. 8. Heparin-bearing animal tissue which has been tempered by a process as claimed in any of the preceding

5. Claims. MACLACHLAN & DONALDSON,