Note: Descriptions are shown in the official language in which they were submitted.
~ 21~0834
h~l~O~ FOR REMOVING THE PRIONS IN COLLAGENS
AND COT.T~G~NS THEREBY OBTAINED
The invention relates to removal of the riskæ of
contamination of biological extraction products with
unconventional transmissible agents (UTA), also known as
"prions". It relates more especially to a method which
ensures removal of the prions for the preparation of
collagens intended, in particular, for the manufacture of
biomaterials.
~ The risks of cont~m;n~tion of biological extrac-
tion products with unconventional transmissible agents
(UTA) currently form the subject of detailed studies.
These risks have been confirmed in several ~n;m~l species
and in man.
As regards ~n;~-l species, scrapie of sheep has
existed on farms for several hundred years. Since 1986,
in the United Ringdom, an epidemic of bovine spongiform
encephalopathy (BSE) has affected cattle, and more than
70,000 cases have been reported to date. The hypothesis
adopted today to explain this catastrophic epidemic of
BSE appears to be the use of meat meals of cont~m; n~ ted
sheep, originating from British knackeries, in the feed
of young calves. This hypothesis seems to indicate the
pos~ibility of transfer from one species to another, even
orally, which obviously has repercussions, at least in
people's minds and attracting media coverage, as regards
the risks of transmission to man.
In man, a comparable and fatal degenerative
disease has been known for some decades. It is
Creutzfeldt-Jakob disease (CJD), the prevalence of which
in the world is approximately 0.6 cases per million
inhabitants. CJD has been stable at this level ever since
it was identified and subject to census; there appears to
be no relationship to the cases of scrapie of sheep or
BSE epidemic. However, the very long incubation of these
diseases, which can extend to se~eral decades, does not
facilitate correlation studies or isolated observations,
and strengthens doubts and the importance of preventive
- - 2 - .214083~
measures.
Cases of accidental transmission of CJD to man
have been reported. ~uru disease, a form of ~ Lology ~n
to CJD and restricted to certain man-eating tribes of New
Guinea, disappeared once the rites of removal and con-
sumption of the brains of deceased ancestors were aban-
doned, thanks to the discoveries and action of Dr Gad-
jusek. Cases of transmission of CJD have been reported in
neurosurgery in patients who have been in contact with
poorly sterilized, cont~m;n~ted instruments (BERNOUILLI
et al., 1977; FONCIN et al., 1980; WIL~ et al., 1982), or
who have received corneal or dura mater grafts removed
from cadavers (DUFFY et al., 1974; PRICHARD et al., 1992;
MASULO et al., 1989; MIYASHITA et al., 1991; NISBET et
al., 1989; POC~T~RT et al., 1992). Transmissions to
neurosurgeons or to their collaborators have also been
described (SCHOENE et al., 1981; MTrT~R, 1988; SITWELL et
al., 1988; GORMAN et al., 1992).
Moreover, the use of hormones extracted from
human pituitaries and associated nervous tissues ha~ led
to several tens of cases of contamination of children
treated for dwarfism with growth hormone (POWELL-JACRSON
et al., 1985; ~OCX et al., 1985; GIBBS et al., 1985;
TINTNER et al., 1986; CROXSON et al., 1988; MARZEWSRI et
al., 1988; NEW et al., 1988; MACARIO et al., 1991;
FRADgIN et al., 1991; BUCHANAN et al., 1991; BROWN et
al., 1992; BILLETTE DE VTTT~MT~UR et al., 1992), and of
two women treated for sterility with gonadotrophins
(COCHIUS et al., 1990).
To date, only nervous tissue is recognized unani-
mously by experts as the major, if not exclusive, source
of UTA. Many other human or ~n;m~l tissues are used in
the biological industry and, for the time being, no non-
nervous tissue has been the source of a documented and
confirmed transmission of ~TA. Doubts exist about the
risks of transmission of UTA from organs rich in lymphoid
cells, and a classification of tissues in terms of these
risks has been proposed by the W~O: WHO 1991.
Using particular ~nim~l models, various
214083~
-- 3
publications report the presence of transmissible prions
in the placenta of infected ewes (PATTISON et al., 1972
and 1974), as well as in the placenta, plasma and
lymphocytes of a patient suffering from CJD (TAMAI et
al., 1992), and in the leukocyte concentrates or whole
blood of patients suffering from CJD (MANUELIDIS et al.,
1985) or of persons in good health (MANUELIDIS et al.,
1993). These various studies and results have not been
confirmed by other authors (BROWN et al., 1984), and the
criticisms voiced regarding the experimental conditions,
suspected of laboratory contamination, demand con-
firmation before any definitive conclusions can be drawn.
Regardless of whether or not these risks are
real, and in the absence of prior diagnosis of these UTA,
the most reliable safety factor for the future lies in
the quality of the purification and/or inactivation
methods used in the preparation of biological extraction
products. Thus, it becomes necessary, under pressure from
the regulatory bodies and bearing in mind the safety
stAn~Ards introduced, to be able to guarantee the effi-
cacy of these methods as regards their capacity to remove
UTA. In addition, the safety requirements for a product
obviously depend on the risks associated with this
product, but also on the benefits provided to the
patients. Thus, in all cases where the benefit to the
patient is not a major feature, or in cases where equally
effective alternative products exist, biological extrac-
tion products will have to provide ~-~;mll~ guarantees.
The collagens, of human or An;~l origin, which
are used nowadays in surgery in many biomaterials, are
among these biological products for which it is sought to
ensure removal of the prions. The properties of collagens
enable them to be used as hemostatic agents, tissue
repair guides, filling products, adhesives, corneal
lenses and tissues reconstituted by crossl;nk;ng methods.
The value of collagens is their excellent biocompat-
ibility, which enables them to exert the desired function
and then to disappear by absorption in a few days, a few
weeks or a few months, dep~n~;ng upon their mode of
2140834
-- 4
CrO881; nk; ng.
It is generally accepted that ~n;m~l collagens
lack the risks of tran~mission of UTA, in particular for
the following reasons:
- the hide or t~n~on~ of young calves which are used
for the preparation of bovine collagen (mainly type
I) have never been considered to be carriers of UTA,
even when they come from sick ~n;~ls (WHO 1991);
- the ~n;~-ls which are the sources of these tissues
come from controlled farms unaffected by BSE, and
are subject to strict health controls.
In addition, the ~n;~-l tissues used are some-
times subjected to prior alkali treatments intended to
remove the hairs from the hide and some soluble im-
purities under these conditions, especially keratinous
and elastic proteinaceous substances (French Patent
No. 1,568,829, Nov. 1967). The authors point out that the
collagenous substances are relatively intact after
separation.
For some preparations, young ox hide is sub-
jected, before t~nn;ng~ to dipping in an aqueous solution
comprising 0.3 to 1.0 N sodium hydroxide with 10 - 25%
(w/w) of sodium sulfate and a 0.05 - 0.3 M concentration
of an amino compound at a temperature of 15 - 25C; the
action time varies from a few hours to several days
(Japanese Patent No. 140,582 of 1976, NIPPI In-
corporated). Under these conditions, the authors assert
that products intended for medical applications may be
prepared from the collagen obtained, 80 as to display a
very weak antigenic power, by prolonging the alkali
treatment and promoting the decomposition of telopep-
tides.
Similarly, US Patent No. 4, 511,653 describes the
preparation of human collagens by treatment of placental
tissues with 0.5 M sodium hydroxide for 48 hours at a
temperature below 10C. One of the advantages put forward
by the authors is the removal of ~iruses such as that of
hepatitis B under these alkaline conditions.
French Patent Application No. 92/00,739 describes
21~0834
-- 5
a method of preparation of collagens by alkali treatment
of ~n;~-l hides with sodium hydroxide (or potassium
hydroxide) at a concentration of 1 N for 0.5 to 1.5 hours
at a temperature not exceeding 30 to 32C, before ex-
traction of the collagen. The authors seem surprised thatthere i8 no modification of the helical structure of the
collagen or of its molecular structure. They also assert
that they obtain collagen fibers which display a hemo-
static power 1.5 to 2.5 times as great as that of
collagen fibers obt~;ne~ by a method differing only in
the absence of the Al k~l; treatment step.
It is generally accepted that alkali treatments
are effective for inactivating UTA. Treatment with 1 N
sodium hydroxide for 1 hour at 20C is nowadays acknow-
ledged to be one of the few possible approaches fordecontaminating biological products. This treatment is,
moreover, recomm~n~ed by the WHO (WHO 1991) whenever it
is possible.
However, the efficacy of this sodium hydroxide
treatment depends on the experimental conditions and on
the UTA strains (extracted from brains of infected
An;m~lg) uged in the ~n;m-l models.
In effect, P. BROWN et al. (1984) describe a
reduction in infectivity o 5.5 log1O LD50 of a CJD strain
after treatment with 0.1 N or 1 N sodium hydroxide for 1
hour. With this CJD strain, no residual infectivity is
detectable.
P. BROWN et al. (1986) also describe reductions
in infectivity of more than 5 loglO LD50 for a CJD strain,
and more than 6.8 log1O LD50 for a sheep scrapie strain,
after treatment with 1 N sodium hydroxide for 1 hour. No
residual infectivity is detectable in either case. A
residual infectivity is observed in the case of treatment
with 0.1 N sodium hydroxide.
While DI MARTINO et al. (1992) describe a reduc-
tion in infectivity of 6 log1O LD50 for a scrapie strain
after treatment with 1 N sodium hydroxide for 1 hour at
room temperature, with, however, a detectable residual
infectivity in the sodium hydroxide-treated contAm;n~ted
2140834
-- 6
sample injected undiluted.
In view of this state of the art, the Applicant
wanted to test the relative efficacy of treatment with
1 N NaOH for 1 hour at a temperature in the region of
20C, especially for collagens of placental origin.
This treatment was applied to a ground prepara-
tion of placental tissues cont~m;n~ted with a ground
preparation of mouse brain infected with sheep scrapie
strain NIH C 506/M3 at the sixth passage. The experi-
mental ~n;~-l was the C57B16 mouse. After treatment of
the tissues with 5 volumes of 1.2 N NaOH for one hour,
the collagens were precipitated by ~;ng HCl to a pH in
the region of 3 at +8C. The precipitate obtained was
collected by centrifugation, and then subjected to
several washes at room temperature with an 80:20 v/v
acetone/water mixture and lastly w~h;ng with pure
acetone to obtain, after drying under l~m;n~ flow, a
collagenous powder. This collagenous powder was digested
with collagenase 80 as to obtain a fluid solution, and
injected in its entirety in 20 ~1 portions into mice
intracerebrally in the right hippocampus.
Tests were carried out in parallel on con-
t~m;n~ted tissues not subjected to the alkali treatment,
serving as a positive control, or other tests carried out
using uncont~ ; n~ ted placental tissues, serving as a
negative control.
It was possible in this way to conclude that 1 N
sodium hydroxide treatment applied for 1 hour at a
temperature in the region of 20C does not enable the
scrapie strain used to be inactivated completely. From an
initial infectious titer of 108 LDs0/g of ground brain
preparation, only an inactivation of approximately 4 logl0
LD50 was observed.
Hence, contrary to the previous, more optimistic,
published results, it is not obvious that collagen
preparations completely freed from the risks of residual
presence of UTA can be obtained from tissues inten-
tionally cont~m;n~ted beforehand with UTA.
These results unquestionably leave very
_ 7 _ 21 4 083~
considerable doubt hanging over the possibility of
validating the methods of purification of collagens, even
when an alkali treatment step i8 present.
The object of the present invention is to provide
a method for the preparation of collagens, of ~n;m~l or
human origin, which ensures a complete and reliable
removal of UTA.
The object of the invention is also to carry out
a complete and reliable inactivation of UTA while pre-
serving the structure and properties of the solublecollagen molecules.
Another object of the invention is, lastly, to
define the optimal conditions of the alkali treatment
applied to collagens in solution.
To thiæ end, the subject of the invention is a
method for the preparation of collagens, according to
which collagenous tissues are extracted and the collagen
is solubilized, the collagen being subjected to an alkali
treatment, ~ld~ e~ in that, for the ~l~e of r3~n~1 of
the UTA, it ~ the steps ~ sisting in :
- extracting collagenous tissues;
- solubilizing the collagen;
- removing the tissue or cell debris present in the
collagen solution obtained;5 - subjecting the collagen in solution to an alkali
treatment;
- isolating the collagen free from risks of trans-
mission of UTA.
The subject of the invention is also the composi-
tions based on collagen or its derivati~es which areobtained by the abovementioned method, free from risks of
transmission of UTA, as well as the biomaterials produced
from the collagens obtained.
The inventors discovered that, surprisingly, all
risk of residual cont~m; n~ tion with UTA could be elimi-
nated during the preparation of collagens, whether of
~n; ~-1 or h~ n origin, on the one hand by removing the
tissue or cell debris present with the collagen prior to
its alkali treatment, and on the other hand by carrying
214083~
-- 8
out said alkali treatment under specific conditions.
Thus, according to the invention, the problem of
the efficacy and reliability of the UTA-inactivation
treatment is solved, in particular, by carrying out,
after extraction of collagenous tissues according to
st~n~rd methods, removal of the tissue or cell debris by
filtration through a membrane of porosity less than or
equal to 1.2 ~, or by any suitable means for removal of
the debris, for example centrifugation, and by then
carrying out the alkali treatment of the collagen in
~olution.
This filtration step makes it obligatory, how-
ever, to use only collagens which have been solubilized
beforehand, either by enzymatic digestion of the covalent
bonds link;ng the collagen ch~; n~ to one another, or by
alkaline cleavage of these same bonds.
It is essential to note here that the methods of
alkali treatment of collagens known hitherto were always
applied to solid tissues, since their object was, at
best, to solubilize these collagens partially and to
remove some impurities.
In addition, the conditions of sodium hydroxide
treatment enabling the UTA to be inactivated and, con-
comitantly, the properties of collagen solutions to be
preserved were not yet known.
An important objective of the invention is hence
to ensure the effective inactivation of UTA while pre-
serving as far as possible the structure and properties
of collagen.
Thus, according to the invention, only the alkali
treatment applied to a previously solubilized and fil-
tered collagen solution enables all trace of UTA to be
removed when there has been intentional cont~m;n~tion of
the initial collagenous tissue.
The alkali treatment according to the invention
consists in ~;ng to a collagen solution sodium
hydroxide whose concentration is between 0.1 N and 2 N,
and in allowing the action to proceed for approximately
1 hour with stirring at a temperature of the order of
2190834
g
20C
According to the invention, the treatment condi-
tions are advantageously defined in accordance with the
collagen type.
5For type I or III collagens in solution and
previously filtered in the dilute state through membranes
of porosity less than or equal to 1.2 microns, the alkali
treatment conditions are advantageously a time of 60 to
70 minutes, a temperature of 20C +3C and a sodium
hydroxide concentration of between 0.1 N and 2 N, and
p-eferably 1 N. If the sodium hydroxide concentration is
increased above 2 N, or if the contact time is increased
beyond 2 to 3 hours, the isoelectric point and the
electrophoretic migration of the collagen molecules
become distinctly modified. The modified properties of
the collagens then make some applications more difficult.
For type IV collagen in solution and previously
filtered in the dilute state through m~mhranes of poro-
sity less than or equal to 1.2 microns, the alkali
treatment conditions are advantageouæly a time of 60 to
70 minutes, a temperature of 20C +3C and a sodium
hydroxide concentration in the region of 0.1 N. If the
alkali treatment time i8 increased or the sodium
hydroxide concentration is increased above 0.1 N, the
viscosity of the collagen IV solutions decreases greatly
and no longer enables gels which are of sufficient
viscosity in some applications to be obtained. For
applications independent of viscosity, treatment with 1 N
sodium hydroxide is possible and provides a further
formal guarantee of the removal of UTA.
The inactivated collagen is then precipitated and
isolated in the desired form, such as, for example,
powder or gel.
Collagen is thereby obtained according to a
preparation method which can be officially validated by
the health regulatory bodies.
The collagen thereby obtained no longer presents
a risk of transmission of UTA, e~en in cases of acci-
dental cont~m; n~ tion of the collagenous tissues. Its
, 2140834
- 10 --
helical and molecular structure is prefierved. Its
properties such as isoelectric point or alternatively
viscosity can also be preserved.
The collagen according to the in~ention may be
used for the preparation of compositions of biomaterials
for medical or surgical use, such as injectable products,
hemostatic products, biological glues for bonding tissues
to one another or to an implanted biomaterial, filling
products, cicatrizing products, and the like.
A better underst~n~;ng of the invention will be
gained on reading the examples given below by way of
illustration and without implied limitation.
EXAMPLES
I - PREPARATION OF INTE~MEDIATE COLLAGENS
Exampl e 1 .
Human type I, III or IV collagens are extracted
according to the methods described in French Patent
Application No. 85/1~,004, by digestion of placental
tissu~ with pepsin, then separation and purification of
the three collagen types by salt precipitations at acid
and neutral pH values.
Example 2.
~ovine collagen i8 prepared from dermis or
tendons of young calves, by the method described in
French Patent Application No. 81/22,691, according to
which the collagen is solubilized by the action of sodium
hydroxide.
Calf hides originating from freshly slaughtered
~n~m~l S are washed with water by stirring for 1 hour in
a vat. The hairs and the subcutaneous tissue are separ-
ated from the dermis using a rotating-strip splitting
machine. The recovered dermis is chopped and ground. The
ground preparation is washed in three successive baths of
pH 7.8 phosphate buffer. Between each bath, the ground
35 preparation is separated from the solution by continuous
centrifugation at between 1,000 and 4,000 rpm. The
residue i8 then rinsed in two successive baths of
softened water, and the liguid is separated from the
ground preparation by centrifugation. Those first washes
214083~
11
serve to remove non-collagenous substances. The tissue is
then placed in a vat cont~;n;ng 1 N sodium hydroxide
solution at a temperature in the region of +4C for a
period of 1 to 10 days. The medium is then acidified with
hydrochloric acid to a pH below 3, adding sodium chloride
so as to achieve a concentration of 100 g/l. The precipi-
tated collagen is dialyzed against softened water.
This product which has undergone a prolonged
alkali attack is not preferred when the electrical
properties of the collagen molecule have to be as close
as possible to the initial state, and taking into account
the heterogeneity of the molecules obtained (some being
monomers, others polymers, others insoluble aggregates,
modified to a greater or lesser extent in accordance with
the conditions of greater or lesser intensity of action
of sodium hydroxide on the tissue).
Exampl e 3 .
Bo~ine collagen is prepared from dermis of young
calves, washed as abo~e and subjected to pepsin digestion
in 0.05 M citric acid buffer, pH 2.4. The dose of pepsin
is approximately 40 g per kg of collagenous tissue. The
digestion time is approximately 60 hours at 17C +2C.
As in the previous examples, the solubilized
collagen is then purified by salt precipitation at acid
pH and at neutral pH. Separations of precipitate are
accomplished by continuous centrifugation.
II - INACTIVATION OF COLLAGENS
Example 4.
The precipitates of h~ n or bo~ine type I or III
collagen obtained according to ~Y~mple 1, 2 or 3 are
solubilized in 0.05 M citric acid at a concentration of
1 to 2 g/l. After dissolution for at lea~t 8 hours, the
solution obtained is centrifuged to remove insoluble
aggregates, and then filtered through membranes having a
porosity of up to approximately 1.2 microns (a porosity
of 0.45 micron is preferable whenever bacteriological
sterility is sought).
The filtered acid solution i8 treated with sodium
chloride at a concentration of 41 g/l (the sodium
2140835
- 12 -
chloride content should be increased to 100 g/l for
collagens pretreated for a long time with sodium hydrox-
ide). After the mixture has stood overnight at a tem-
perature of approximately 10C, the collagen precipitate
i8 recovered by continuous centrifugation. The precipi-
tate is redissolved with 0.01 N hydrochloric acid at a
concentration of approximately 8 g/l. The clear solution
obtA;ne~ is neutralized to pH 7 by adding 2 N sodium
hydroxide.
1 volume of 2 N sodium hydroxide is added to 1
volume of the above collagenous solution, and the mixture
is kept stirring for 60 to 70 minutes at 20C +3C,
giving a final sodium hydroxide concentration of 1 N.
The mixture is then diluted 5 times with
demineralized water and immediately neutralized to pH 7.5
with 1 M citric acid. The collagen is precipitated by
adjustment to 100 g/l of NaCl at 20C and at neutral pH,
or by adjustment to 41 g/l of NaCl at pH 2.8 and at 10C.
The collagen precipitate is washed with acetone
to obtain a powder enabling various known biomaterials to
be prepared according to st~n~rd methods.
Exampl e 5 .
For type IV collagen, the procedure is as in
Example 4, except that the final sodium hydroxide concen-
tration is reduced to a value of 0.1 N.
III - TEST OF EFFICACY OF THE METHOD ACCORDING TO THE
lN v~l-lON
To test the efficacy of the method, collagens may
be prepared from dermis or from placentas previously
mixed with 1/lOth the weight of brain of mouse infected
with scrapie strain NIH C 506/M3 at the sixth passage and
assaying at approximately 108 LD50/g of ground brain
preparation.
The collagen acetone powders obt~;ne~ according
to the above examples are digested with collagenase 80 as
to obtain a fluid solution which is nontoxic to the
brain, and injected in their entirety in 20 ~1 portions
into mice intracerebrally, in the right hippocampus.
The absence of symptoms of acute degenerative
- 2l40834
- 13 -
encephalopathy after an interval of 18 months will be
established, whereas the control mice have all died.
211l~8~
- 14 -
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21gO834
- 15 -
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2140834
- 16 -
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"Further observations on the production of scrapie in
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