Note: Descriptions are shown in the official language in which they were submitted.
078767
~CKGROUND 0~ THE INVENTION
This invention relates to an improved process for producing
a defatted heparin tissue and the product therefrom. More
particularly, this invention relates to an improved process for
producing a defatted heparin tissue wherein prior to desiccating
and defatting with an azeotropic solvent, the frozen heparin-
i~ bearing animal tissue is tempered under controlled conditionsof temperature and time. The tissue is first particulated as
by grinding, chopping or other means while in a frozen or
partially frozen state, thawed and warmed in a heat exchanger,
fermented at 45-95 F. for 2 to 15 hours but not substantially
,~ beyond the time severe foaming and gassing begins and thereafter
1:
~ subjected to the boiling action of an azeotropic solvent to
j~:
i~ substantially remove tissue water and fat. The desiccated and~ ~ defatted tissue is especially suited for use in heparin recovery
j~ 15 processes.
!~ Heretofore, prior to desiccating and defatting heparin
tissues with an azeotropic solvent, frozen heparin-bearing
. animal tissue was tempered by allowing solidly frozen blocks of
~, ~ ; the tissue in bags or boxes to gradually thaw and warm up over
period~ of 2 to 8 days at ambiant tempeiature of 60 -120 F.
while in these containers, during which time it had been thought
optimal conditioning of the tissue for heparin release in
subsequent heparin recovery was occurring. However, the tissue
- is very sensitive to enzymatic action and subject to decomposition
by undesirable bacterial growth and rotting of tissue. The
outside of a frozen block of tissue subjected to these older
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` 1078~67
temperin~ method~ could actually putrefy before the interior
had reached a thawed state. Thi9 re~ulted in generation of
obnoxious undesirable odors which, durin~ the 2-8 day period,
w~uld ~pread 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 bio-
t chemically 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 is
known to cause high pyrogen content and increased effort was
,,
required for pyrogen removal. Tissues tempered by this prior
method which were then subiected to azeotropic processing to
desiccate and defat normally contained at least 0.5 weight ~
fat even under the most favorable circumstance and more generally
the fat content ranged from 1.0 to 2.0%. In addition, the
I 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. The defatted-dehydrated tissue prepared according to
tbe present invention is of unusual high quality and contains
only about 0.1 to 0.2 weight % fat and is readily wetted in the
above described heparin recovery process and mixtures are more
easily filtered. Other indi~ations of improved quality of the
i,
~ ~ 1078767
product of th is invention are light color, little odor, good
texture, homogeneity and low residual solvent. In addition,
more fat i9 recoverable from the solvent for a given amount of
heparin-bearing tissue which i8 an advantage. Apparently, the
novel combination of steps in the present process of particulating
frozen tissue, rapidly thawing and warming the tissue, fermenting
under controlled conditions and azeotropic processing is
responsible for the increased wettability. Further and equally
important due to the control of conditions during tempering,
the heparin yield obtainable therefrom and content of the defatted
tissue of this invention ca~ be as much as about 10-12% higher
than for the above-described prior art defatted tissue.
U. S. Patent 2,539,544 discloses comminution of frozen
tissue in a hammermill prior to azeotropic extraction and
immediately after grinding, suspending the ground tissue in a
; solvent. In the present invention the tissue is thawed and
warmed by passing it through a heat exchanger and thereafter
fermented.
. 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 .r
about 1/4 inch mesh, or the act of dividing larger pieces which
.
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1078767
have been prebroken or flaked. The term "fermentation" refers to the
combined action of enzymes present in the tissue and enzymes generated
by growth of bacteria or micro-organisms 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 a solvent which forms an azeotrope with water to substantially
remove it and which extracts tissue fat into the solvent stream and there-
after collecting and washing the dehydrated tissue on a filter with solvent.
SUMMARY OF THE INVENTION
The present invention therefore resides principally in the
discovery that improved tempering of frozen or partially frozen heparin-
bearing animal tissue can be effected by particulating and pumping to a
heat exchanger where, under controlled conditions, it is thawed to obtain
tissues which, after fermentation at controlled temperature for a period
of time to substantially effect enzymatic conditioning of tissue to improve
heparin availability and azeotropic processing, are exceptionally low in
fat content and exceptionally high in available heparin due to control of
time and temperature throughout the processing of each lot and are very low
in pyrogen content.
According to the present invention, there is provided a process for
producing a defatted heparin tissue from frozen heparin-bearing animal tissue
in preparation for isolation of heparin which comprises the steps of
1) particulating the frozen tissue,
2) thawing and warming the particulated tissue to a temperature within the
range of 45 to 95F. in a heat exchanger.
3) fermenting the thawed tissue from step 2 at a controlled temperature
within the range of 45 to 95F for a period of time of 2 to 15 hours to
substantially effect enzymatic conditioning of said tissue to improve heparin
availability,
30 4) subjecting the fermenting tissue from step 3 to azeotropic extraction
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78767
with a organic solvent to substantially dehydrate and defat said fermented
tissue.
The process of the invention may be represented diagrammatically
as follows: ;
~ - 5a -
.~
AHR-320~
i078767
¦ Flaked or Prebroken
Frozen Tissue
.
Particulate
. .
,Thaw and Warm
in Heat
Exchanger .
__ .
,
Ferment
45-95 F-
.
, Azeotr~ ~pically
Des iccate &
Defat
~ To Heparin Isolation
~ /
TEMPERING A~D DEFATTING PROCESS
FOR HEPARI~ TISSUE
5~ ~
, ,, , .,, .~ .
.
1078767
The preferred process comprises grinding frozen or partially frozen
tissue and rapidly thawing and warming the thawed tissue in a heat exchanger,
fermenting the tissue at 45 to 95 F. for a period of time to optimize hepa-
rin availability and thereafter subjecting the tissue to azeotropic processing
to remove moisture and fat. Generally speaking, the higher the temperature
within thisrange during fermentation the shorter will be the time required. `
For some unexplained reason, severe evolution of gas and foaming signals the
end of the desirable fermentation phase.
The heparin in the tissue tempered as in this invention may be
recovered by a number of techniques including the methods of United States
Patents 2~797,184 and 2~954,321 with easy wettability and processing advanta-
ges being evidencedO
The present invention, generally stated, provides an improved
process for preparing desiccated and defatted heparin tissue from frozen
heparin-bearing animal tissue, particularly wherein the process eliminates
the unsanitary conditions of rotting and obnoxious odor which are attendant
in prior art methods and wherein more of the fat is extracted.
It also provides an improved desiccated and defatted heparin tissue
especially adaptable to heparin recovery because of its ready extractability
in heparin recovery.
Furthermore the present invention provides a process for preparing
a desiccated and defatted heparin tissue wherein prior to azeotropic processing,
frozen heparin-bearing animal tissue is tempered under controlled conditions
as to precise temperature at all times including a controlled fermentation
step wherein the microbiological population is evenly distributed in the subs-
trate and their growth promoted, allowing action of both endogenous and exoge-
nous enzymes on the substrate and which produces a uniformly conditioned product
1078767
having a more consistent bioche~ical content which is lower in pyrogen
content and high in available heparin content and is exceptionally low
in fat content.
There now follows a more detailed description of the best mode
of carrying out the invention.
DETAILED DESCRIPTION OF THE INVENTION
The preferred process of the present invention for producing
desiccated and defatted heparin tissue in preparation for isolation and
recovery of heparin comprises the steps of
1) particulating the frozen tissue to any size up to about 1/4 inch
mesh size, preferably 1/8 to 1/4 inch mesh size, preferably using a
grinder,
2) thawing and warming the particulated tissue from step 1 to a
6~ o
temperature within the range of 45 to 95 F., preferably ~ -85 F.
using a heat exchanger, preferably a shell and tube heat exchanger,
3) fermenting the warmed tissue from step 2 while maintaining
it at a temperature range of 45 to 95 Fo for 2 to 15 hours, preferably
75 to 85 F. for a period of 6 to 8 hours time but not substantially
beyond the time foam1ng starts due to gas liberation, and
4) subjecting the fermented tissue from step 3 to azeotropic
processing with a suitable solvent, preferably ethylene dichloride,
to substantially dehydrate and defat the fermented tissue.
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~078767
- Th~ heparin-b~aring animal tissue for the proce~s of this
invention oriyinates at the meat packing plant where it is
cut rom animal carcasses and handlcd according to specified
procedur~s for presorving and enhancing heparin values, boxed
or bagged and dcep frozen. As a con~equence, the animal parts
arrive at the heparin recovery plant as blocks of one kind of
frozen, agylomerated animal part such as, for example, lung
in the siæe and shape of the containers. Usually, the size
of the blocks of frozen animal parts is too large for direct .*
particulation in the size equipment feasible for this art and
it i8 therefore necessary to reduce the size of the blocks by
~; some prior means. To accomplish this, the deep frozen blocks
; of tissue may be cracked or prebroken mechanically by some
means. Preferably, however, the blocks of deep frozen tissue
- 15 are partially defrosted for about 8 hours at ambient temperatures
of 80 -100 F. and thereby brought from their deep-frozen state
to a softer state by raising the temperature of the blocks to
about 20 -~2 F. after which time the soft-frozen tissue may be
chipped or flaked in preparation for particulation. When the
flaking or chipping procedures are followed the preferable
,,~,; .
temperature to which the blocks of tissue are raised is about
26 F. as the tissue is rigid enough to be flaked, yet in a
somewhat softened condition feasible for the flaking operation.
A suitable flaking machine is the Hydrauflaker produced by
the General Machinery Corp., Sheboygan, Wis. In any case, the
tissue should not be so cold that the particulated tissue in
the next step refreezes into balls and clumps which prevent
1078767
pumping. In general, the particle size of prebroken or flaked
frozen tissue can vary from 1/8 inch to 2 inches.
In step 1 of the process, grinders operate to reduce
the size of the prebroken or flaked, frozen, or partially froæen
tissue to that ranging from that present in a puree up to a
maximum dimensional mesh size of about 1/4 inch, preferably
up to a mesh size of 1/8". Grinders which are suitable are the
Comitrol produced by Urschel Laboratories of Valparaiso, Ind.
and the Autio grinder produced 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 a 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 45 -
95 F. using a heat exchange surface temperature not to exceed
140 F. Above about 30 minutes too much variation is introduced
in later processing. Heat exchange surfaces having a higher
¦ temperature than 140 F. cause fouling of the surfacesJ denaturing
3j; . of protein and microbiological kill-off. Shell and tube heat
exchangers with tissue passing through the tube are highly
satisfactory and preferred but wiped surface exchangers may be
also used. The preferred shell and tube heat exchangers will
range in tube size of about 3/4 inch diameter to about one inch
in diameter and will conse~uently have surface to volume ratios
of about 50 to 75 ft.2 per ft.3. 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
1078'767
~- inert surface such as a stainless steel tank at a temperature
of 45 to 95 F., preferably 75 to 85 F., for a period of
time sufficient to condition the heparin tissues as a result
of a fenmentation involving enzymes already present and enzymes
produced by growing microorganisms. Above about 95F. heparin
values are rapidly lost and below about 45 F. the fermentation
step is ineffective. Two to 1~ hours fermentation time is
required at 45 to 95 F. and for some unknown reason the
completion of the beneficial fermentation is signalled by
severe gas liberation and rising in the holding tanks and
further fermentation decrea$es 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 coordinates obtained by trial
and error at which the gassing phase had begun.
~ o
Time, hr. Temp., F.
9-10 75
~4: 8 80
6 85
. ~ .
. 20 It is not necessary to wait until gassing occurs to obtain the
~ superior product of this invention. Generally, there is some
;~l variation of microorganisms in lung tissue among individual
animals; however, grinding and mixing of many lung lobes assures
that the bacterial population will be such that fermentation
will eventually proceed. When it is desirable to speed the
fermentation microorganism starters may be added as, for example,
by seeding the tissue at the beginning of the holding period
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~078767
with tissue which has already been fermented. Liver and other
tissue psually lack the high levels of microorganism and may
require such starting or seeding.
In step 4, the fermented tisque from step 3 is subjected
to azeotropic processing procedures such as are disclosed in
U. S. Patents 2,619,425 and 2,539,544. Preferably, the
azeotropic processing is conducted at atmospheric pressure
using ethylene dichloride at a temperature not exceeding 180 F.
;~ However, other solvents such as propylene dichloride, tri-
chloroethylene, hexane and the like may be used.
~` Defatted heparin tissue obtained by the foregoing process
~; is characterized by its low fat content of about 0.1 to 0.3
and by its excellent permeability as measured by wettability
and suspendability.
~15 ~he following are specific examples of the process of
this invention. Units heparin are USP units.
.~ ~ ExamPle 1
Partially defrosted frozen beef lung at 25-30F. in
amount of 15,190 lbs. was flaked using a Hydrauflaker (Model
FS-6) to a size range of 1/8 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 size openings.
The ground frozen or partially frozen lung was pumped with a
TM
Moyno pump through 3/4 inch diameter tubes of a shell and
tube heat exchanger to thaw and warm the 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 70-75F. No external heat was needed to maintain
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107~76~7
tl~e tcmpernture during the fermentation and a slight rise in
tcmperatur~ due to heat of reaction was al~o noted. The tissue
was then subjected to boiling action at atmospheric pressure of
ethylene dichloride until water had been substantially removed.
me dehydrated tissue was then collected on a filter and washed
once with redistilled ethylene dichloride. Residual solvent
was removed by conventional desolventizing methods. There
was obtained 2, 804 lbs. of desiccated and defatted tissue
containing 0.18,¢ fat and 1.6% moisture. Processing of the
desiccated and defatted tissue to recover heparin by the procedure
of U. S. Patent 2,954,321 yielded 158 x 103 units heparin/kg.
of desiccated and defatted lung. Heparin potency was 79 units
per milligram.
Example 2
Partially defrosted frozen beef lung at 26 ~. in amount of
16,200 lbs. was flaked and ground as in Example 1, using .120
inch size openings on the grinder. The ground lung was thawed
and warmed to 83CJF. in a heat exchanger as in Example 1,
residence time being about 4 minutes. The warm ground lung was
then held in a stainless steel tank for 6 hours at 83-85 F.
~o undesirable odor was present at any time. There was obtained
15,800 lbs. of tempered lung. The tissue was then subjected
to boiling action of ethylene dichloride at atmospheric pressure
until water had been substantially removed. The defatted-dehydrated
tissue was then collected on a suction filter and washed once with
pure ethylene dichloride. Residual ethylene dichloride was removed
by conventional desolventizing techniques. There was obtained
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107~7b7
2816 lb. of defatted tissue containing 0.~ weight ~ fat and 2.2
weight % moisture. Proce~sing of the defatted ti~sue to recover
heparin by the procedure of U. S. Patent 2,954,321 yielded 152.7 x 10
103 units heparin per kg. of defatted-dehydrated tissue. Heparin
potency was 58.8 units/mg.
Example ~
Frozen beef lung (12,000 lb.) was ground as in Example 1,
thawed and warmed to 40 -50 F., residence time being about 2
minutes in the heat exchanger. The ground lung was then held
at 40 -50 F. for 6 hours. There was obtained about 11,900 lbs.
of tempered beef lung tissue. The tissue was then subjected
to azeotropic extraction w~th ethylene dichloride as in
Example 1. After removal of solvent there was obtained 2,046 lb.
of defatted-dehydrated tissue containing 0.12 weight % fat and
2.6 weight % moisture. Processing of the defatted tissue to
recover heparin by the procedure of U. S. Patent 2,954,~21
yielded 164 x 103 units heparin per kg. of defatted-dehydrated
tissue. Heparin potency was 48 units/mg.
Example 4
Frozen beef lung (12,000 lb.) was ground as in-Example 1,
thawed and warmed to 90 -110 F. in the heat exchanger, residence
time being about 6 minutes. The ground lung was held at
90-110 F. for 6 hours. There was obtained about 11,900 lb. of
tempered beef lung. ~he tissue was subjected to azetropic
extraction with ethylene dichloride as in Example 1. After
removal of solvent, there was obtained 1751 lbs. of defatted-
dehydrated tissue containing 0.08 weight % fat and 1.2 weight
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078~7~7
moisture. Proces8ing of the defattcd tissue to recover heparin
~` by the procedure of U. S. Patent 2,954,321 yielded 86 x 103
units heparin per kg. of defatted-dehydrated tissue. Heparin
potency was 74 units/mg.
S Example 5
Frozen beef lung (16,200 lb.) was ground, thawed and
warmed as in Example 1 to 790F., residence time in the heat
exchanger being about ~ minutes. The warm ground lung was then
held in a stainless steel tank for 8 hours at 79- 80F. No
undesirable odor was present at any time. There was obtained
15,850 lb. of tempered beef lung. The tissue was then subjected
to boi~ng action of ethylene dichloride at atmospheric pressure
until water had been substantially removed. The defatted-
dehydrated tissue was then collected on a filter and washed once
with ethylene dichloride. Residual ethylene dichloride was
removed by conventional desolventizing techniques. There was
obtained 2850 lb. of defatted tissue containing < 0.2 weight
fat and 2.2 weight ~ moisture. Processing of the defatted
tissue to recover heparin by the procedure of U. S. Patent
2,954,~21 yielded 155 x 103 units heparin per kg. of defatted-
dehydrated tissue. Heparin potency was 96 units/mg.
- Data for the foregoing examples are summarized in Table 1
for comparison~
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10787~;7
Table 1
Lung Fermentation, Extraction and Heparin Isolation
%
Yield ~f
Dried Fat in Crude
Fermentation Def~tted Dried Meparin Heparin
Ex.Conditions Lung Defatted Yield PotenCy
~Q~ Temp.F. Time.hr. (a) Lung(b) (c)
1 70-75 6 18.4 0.18158 79
2 85 6 17.4 0.30153 59
3 40-50 6 17.1 0.12164 48
4 90-llO 6 13.9 o.o8 86 74
8 18.0 <0.2 155 96
(a) Wt. ~ based on starting lung.
(b) Units heparin x 103/kg. desiccated and defatted lung.
(c) Units heparin/mg. in crude heparin.
COMPARISON OF WETTABILIT~ OF TEMPERED ~
DEFATTED TISSUE
Comparison was made of wettability of azeotropically
defatted tissue prepared by the new improved process of this
invention with that of the defatted tissue prepared from tissue
tempered by the old method, wherein the lung was tempered 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.
water until the particles appeared wet on the outside and the
stirring stopped. The time required for the bulk of the
particles to sink was then recorded. Data are in Table 2.
Table 2
Wettability Comparison
Time For Particles
Method of to Sink to Bottom,
Tempering Seconds
Old > 180
New < 10
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