Note: Descriptions are shown in the official language in which they were submitted.
1059C~Z9
STABILI~ATION OF INTERFERON
AGAINST MECHANICAL STRESS
The present invention encompasses a method for
stabilizing interreron during purification comprising
treating an interferon preparation with an effective
stabilizing amount of reduced glutathione, thiodiglycol,
thioethanolamine, thioalkanoic acid having 1-7 carbon
atoms, monothioglycerol, dithiotreitol, thiotic acid,
N-acetylcysteine, or N-acetylhomocysteine as a sulfur
containing reducing agent for selectively retaining
interferon sulfhydryl in the reduced state without reduc-
tion of essential disulfide linkages. The metho,d of the
present invention preserves the activity of interferon
by selectively retaining interferon sulfhydryl (-SH)
functions in the reduced state without reducing inter-
feron disulfide linkages (-S-S-) which are essential for
activity. Methods for producing and isolating interferon
are extensively taught, Ciba Foundation Symposium-Inter-
feron-edited by G. E. W. Wolstanholme and Maeve O'Connor;
Little, Brown and Co., Boston, 1967~
It is well known that interferon may be derived
from cells grown in tissue culture and that cultures of
non-transformed cells are preferred as a source of inter-
feron. However, the,interferon derived from such,cells
is highly susceptible to inactivation by mechanical stress,
and other procedures involved in conventional purification
of the material, and the yields of purified material
obtained by these procedures are low.
"For the purpose of the present invention inactivation
by mechanical stress is also considered to encompass the in-
activation of interferon which can occur on standing."
~.
~059~Z9
It is known that interferon contains disulfide
bonds which must remain intact if activity is to be main-
tained and reduction of these disulfide bonds with strong
reducing agents destroys the actiYity of the interferon.
However, it has now been shown that interferon also con-
tains sulfhydryl groups and that these also are essential
to the activity of the material. The process of inacti-
vation, for example by mechanical stress described above
probably involves either the linking together of the
sulfhydryl groups or disulfide interchange reactions, to
form inter or intra-molecular disulfide bridges, thereby
causing the interferon to adopt a biologically inactive
configuration.
Certain water soluble sulfur-containing mild
reducing agents retain interferon sulfhydryl groups in
a reduced state without affecting disulfide linkages which
are essential for interferon activity. The preferred
reagents are: thioacetlc acid, reduced glutathione (~-L-
glutamyl-L-cysteinylglyc$ne), monothioglycerol (3-mercapto-
1,2-propanediol), dithiothreitol (1,4-dithiothreitol),
thiotic acid (1,2-dithiolane-3-valerlc acid), N-acetyl-
cysteine, N-acetylhomocysteine, thioethanolamine (2-
aminoethanethiol) and thiodiglycol (2,2~-thiodiethanol).
These compounds are represented by the following structures.
O ' . ' .
CH3-C-SH thioacetic acid, thioalkanoic acids
havlng 1-7 carbon atoms are likewise suitable.
S - S thiotic acid(lj2-
/ \ dithiolane-3-valeric
(CH2)4- C02H acid)
--3--
~os9oz~
IH2- SH
CHOH monothloglycerol
¦ (3-mercapto-1,2-propanediol)
CH2- OH
CH2- SH thioethanolamine
¦ (2-aminoethanethiol)
CH2 H2
. (CH2) - OH
1 2 thiodiglycol
(2,2'-thiodlethanol)
(CH2)2--OH
H-CI-SH
H-C-OH dithiothreitol
¦ ~1,4-dithiothreitol)
HO-CI-H
H-CI-SH
H
HS-CH2-CH-C02H
¦ N-acetylcysteine
NH-CO-CH3
HS-CH -CH -CH-CO2H N-acetylhomocysteine
2 2 1 (2-acetamido-4-mercapto-
NH-CQ-CH3 butyric acid)
CH2-SH
H02C-CIH-CH2-CH2-CO-NH-CH-CO-NH-CH2-C02H
NH2
reduced glutathione
(y-L-glutamyl-L-cystein-
ylglycine)
For purposes of the present lnvention an effec-
tive stabilizing amount is 0.1 to 10-g molar concentration
of the reducing agent in the ~olutlon containlng interferon.
I Preferred embodiment of the present invention.
are represented by the method of stabilizing fibroblast
lOS9OZ9
interferon against mechanical stress by treating an inter-
feron preparation with at least l x 10-4 moles per liter
of thiotic acid, at least 1 x 10 1.5 moies per liter of
N-acetylcysteine, or at least 1 x 10 2 moles per liter of
dithiothreitol.
Interferon derived from fibroblast cells is
most preferably stabilized by treating the crude inter-
feron preparation with at least 1 x 10-4 moles per liter
of thiotic acid before sub~ecting the interferon to
mechanical stress such as ultra filtration, shaking, or
precipitation.
Itis to be particularly noted that related
structures not having the requisite sulfur moiety are
ineffective. For example, thiotic acid and thioalkanoic
acids having 1-7 carbon atoms such as thioacetic acid are
; effective interferon stabilizing agents while octanoic
acid is not. N-acetylcysteine is effective and N-acetyl
valine is not.
The reagent may be added to the crude prepara-
tlon of interferon at the appropriate concentration (aneffective stabilizing amount) and the interferon subse-
quently purified and concentrated by conventional tech-
nlques such as chromatography, ultrafiltration or centri-
fugation. Upon completion of the purification the
stabllizing agent may be removed by dialysis against a
buffer solution ~hich does not contain the agent.
The following exampl0s are illustrations of the
present lnvention and should not be construed as limiting
the invention in spirit or scope.
~059C31;~9
EXAMPLE 1
The abllity of several:compounds to prevent
inactivation by mechanical stress of interferon derived
from human fibroblasts is shown in Table 1 below:
TABLE 1
_ CONCEN- .
COMPOUND TRATION
. ~mM) TIME OF AGITATION(HOURS)
. ._
. . 2 4 6 24
. . INTERFERON ACTIVITY
Control interferon 30 20 10 3
(no additives)
i Thiotic acid 1 100 100 100 95
3ctanoic acid 1 30 20 10 3
; N-acetylcysteine 100 90 85 85 80
N-acetylvaline 100 30 15 10 4
: Dithiothreitol 10 90 90 85 85
2-mercaptoethanol 14 10 10 5 10 _
Samples of interferon ln tissue culture medium
were agitated by shaking in a tube 50 times per minute at
a temperature of 4C. The percentage of the original
interferon activity remaining after increasing time inter-
vals is given in Table 1. An indication that the reactlve
species involved the sulfhydryl group is given by the fact
that octanoic acid and N-acetylvaline, which are closely
chemically related to thiotic acid and N-acetylcysteine
respectlvely, but lack the sulfhydryl moiety, are both
totally inactive.
. The strong reduclng agent 2-mercaptoethanol has .
no protective activity against the inactivation and itself
caused inactivation of samples which are not agitated.
1059029
2-mercaptoethanol ls shown separately to disrupt disulfide
bonds essential to the reactivlty of interferon.
It is, therefore, demonstrated that only those
preferred reagents which react with interferon sulfhydryl
groups without disrupting disulfide bonds are able to
stabilize interferon against mechanical inactivation. In
contrast potent reagents which do disrupt the disulflde
bonds of interferon cause a loss of activity of interferon
even ln the absence of physical stress.
EXAMPLE 2
Interferon from two types of fibroblast cells,
MRC5 and FSA were sub;ected to increasing physical stress
; by shearing in a rotational viscometer comprising two con-
centrlc cylinders rotating in relation to each other.
The interferon had initial activity of 1000 u/ml.
RESULTS
Activity of 1 Hour
unsheared control - 1000 u/ml
FSA/10-4 molar thiotlc acid - about 850 u/ml
MRC5/10-4 molar thiotic acld - about 700 u/ml
FSA sheared control - about 500 u/ml
MRC5 sheared control - about 50 u/ml
EXAMPLE 3
Samples of lnterferon 0.1 molar in N-acetylcys-
teine were shaken at the rate of 50 times per minute for
a total period of 24 hours at 4C. After 24 hours the
N-acetylcysteine treated interferon samples had essen-
tially unchanged activity and untreated controls had
1/10 the original activity after 24 hours of shaking.
1059(~Z9
EXAMPLE 4
Reagents of the present lnventlon functlon by
maintaining a reducing atmosphere around the critical
sulfhydryl groups. They do not bind to these groups and
their reaction is therefore reversible if, for example,
they are dialysed out of the interferon preparation.
Furthermore, interferon which is rendered unstable by
dialysing away a reducing agent stabilizer may be re-
stabilized by the addition of a fresh reducing agent.
The properties of reagents according to the
present invention are illustrated by the examples des-
cribed in Table 2. An aqueous preparation of interferon
was sub;ected to treatment with various reagents and then
agitated for 24 hours at 4C. The resulting loss of
total interferon activity as a result of this agitation
was measured.
TABLE ~
No. Treatment % Loss of Activity
(shaking for 24 hours at 4C.)
1 None 90
2 0.1 M N-acetylcysteine 0
3. As 2 above and dialysed 93
4. As 3 above then 0.1 M 0
N-acetyl cysteine added
.
EXAMPLE 5
A conventional procedure for purification of
interferon is ultra filtration on a membrane. ~lowever,
under conventional condltlons much of the activity of
the interferon is lost in carrying out this procedure,
1059029
when the interferon is that derived from fibroblasts,
It is shown in Table 3 below that interferon may be
stabilized against such inactivation by treatment wlth
0.1 molar N-acetylcysteine.
TABLE 3
Ultra- Concen- % Yield
filtration tration of
StabilizerMembrane Fao~or Activity
None UM 10 5 20
None UM 20 5 11
0.1 M N-acetylcysteine UM 10 5 100
0.1 M N-acetylcysteine UM 20 5 100
EXAMPLE 6
A further conventional purification procedure
for interferon is salt precipitation using for instance
increasing concentrations of ammonium sulfate or potassium
thiocyanate at ~C. at mildly acidic or neutral pH. Under
conventional conditions much of the activity of interferon
is lost in carrying out this procedure when the interferon
is derived from fibroblasts. In Table 4 it is shown that
interferon may be e~fectively stabilized in ammonium
sul~ate precipitation systems by addition of 1 mM thiotic
acid.
TABLE 4
USE OF SULFHYDRYL STABILIZER IN INTERFERON
PRECIPITATION BY AMMONIUM SULFATE AT pH 7.0 AT 4C.
Treatment Percent Yield
Unprotected interferon 30%
Interferon plus 1 mM Thiotic acid 100%
Reversible treatment with a reducing agent whose
reactivity is such that only those sulfhydryl groups which
play a part in interferon inactivation are reduced and not
_g_
1~590~9
the essential disulfide bonds stabilizes fibroblast interferon
against the inactivation which typically occurs while undergoing
conventional purification procedures.
EXAMPLE 7
.
Human fibroblast interferon is treated with 10 4
moles per liter of thiotic acid and stored at 4C for 14 weeks.
The stability of thiotic acid treated interferon is compared
to that of the control. The log10 of interferon titer remains
substantially unchanged for the thiotic acid treated interferon
while the interferon titer for untreated interferon decreases
from 103-5 to 102 after 14 weeks at 4C. Thus thiotic acid
present in concentrations of 10 5 to 10 3 molar preferably at
least 10 4 molar prolong shelflife of interferon.
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