Note: Descriptions are shown in the official language in which they were submitted.
1043676
BACKGROUND OF THE INVENTION
It is a matter of utmost criticality that sterility
be assured for each and every unit of parenteral solution
which is administered to a patient. In the absence of such
sterility, the patient, who is typically in a weakened con-
dition anyway, could possibly develop fatal septicemia, or
blood poisoning.
The needs of efficiency and economy dictate that the
sterilizing chambers used for sterilizing loads of parenteral
solution must be very large. Because of this, there is the
need for constant vigilance to be sure that no small corner
of the larger sterilization chamber has for some reason
failed to achieve a sterilizing temperature for an appropriate
length of time; and that no parenteral solution container in
the lot is therefore exposed to less than satisfactory heat
of sterilization. Expensive thermocouples can be distributed
through the load of solution units as they are placed in the
sterilizing chamber, but thermocouples have a tendency to fail
at such temperatures, giving inaccurate results. Also, the
expense and efort of such an operation on a regular basis
could be prohibitive, and the maximum number of thermocouples
which may be installed is limited practically by their lead
wires and the like.
Also, at the present time rigorous and complicated
quality control testing is practiced by every manufacturer,
generally using bacteria incubation testing of sterilized
solutions. This technique has recognized disadvantages.
Accordingly, there is a need for a simple method for
determining whether a load of product in a sterilizing apparatus
.
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1(~4~
has been exposed to a desired total sterilizing heat level,
which heat level is a function of both temperature and time
of exposure. The calculation of such heat level can be quite
complex, because there must be included in the calculation
the temperature levels and times during which the temperature
of the load of materials to be sterilized rises toward the
maximum sterilizing temperature. Thereafter, there must also
be included in the calculation the temperature-time contri-
bution of the period in which the temperature falls back
again to room temperature.
In accordance with U.S. Patent No. 3,344,670, it has
been suggested to determine the total heat applied, as a
function of time and temperature, by the use of dry paper
treated with silver nitrate or the like, which gives a
colorimetric response. This cannot be effectively and
accurately used in an aqueous environment, and cannot in any
event give quantitatively accurate readings. Similarly, it
has been suggested in the Journal of Food Science, Vol. 36
~1971), pp. 692-698, that the heat sterilization of foods and
the like can be estimated in a manner similar to the calcula-
tion of the degradation of thiamine or other materials present
in the food, since bacteria die and thiamine degrades in
àccordance with first order kinetics.
The suggestion has also been made (Thesis by J.
Zahradnik, Controlling Regimes and Survival Rates of Salmonella
During Heating, Massachusetts Institute of Technology, 1965)
that one might determine the total heat applied in a given
sterilization cycle ~from which a resultant bacteria kill can
be calculated by known techniques) by following the thermal
:
.
~ ()43~i~6
struction of aqueous solutions of sucrose. Unfortunately,
however, the activation energy of destruction of this ma- '
terial is less than 30 kilocalories per mole. Due to this
fact, at room temperature, sucrose solutions, as well as '
thiamine solutions, can degrade at an appreciable rate over a
period of weeks or months. Accordingly, thiamine and sucrose
solutions cannot be stored for a significant length of time -'
since a large degradation of thiamine or sucrose can result in
inaccurate analytical results, showing that a sterilizing amount
of heat had been applied, while in fact, such heat had not been
applied, with a substantial number of bacteria remaning via~le. ''~
Similarly, thiamine and sucrose solutions can de~
~ - .
grade rapidly at nonsterilizing temperatures above room temper-
ature but less than less than effective bacteria kill tempera- '
~.; .,
tures, giving a false impression that a sterilizing amount of ' ~'
heat had been applied. '~
,~
The invention of this application provides an improved ' -
,
method'and composition for~quantitative measurement of the heat
sterilization process in which the above disadvantages are
Z0 significantly reduced or eliminated.
- ~
' In one particular aspect the present invention pro~
vides~the method of confirming a desired heat effect upon ma- ~
terial which~is subjected to a temperature-time exposure,which '~ -
comprises~including with said material in;said temperature- '
time~exposure an~isolated, aqueous solution of polysaccharide
which hydrolyzes to produce a monosaccharide at a rate which is
tèmperature-dependent,~and the rate constant corresponds to the "~
Arrhenius equation, where the activation energy of said hydrolysis
to form said monosaccharide is greater than 30 kilocalories per -
mole~oE hydrolyzed-sacçharide-saccharide~linkage, and the fre-
quency factor is from 10'4to -Io2~ min. -''; and thereafter analyzing
the aqueous solution for effect of said-exposure to compare said '~
effect with a predetermined standard effect wbich is indicative
;
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iO43t;~6
of receipt of said temperature-time exposure.
In another aspect the present invention provides
the method of confirming the heat sterilization of a meterial
which is subjected to a sterilizing temperature-time exposurel ;
which comprises including with said material, in said tempera-
ture-time exposure, an isolated, aqueous solution of maltose
containing sufficient strong acid to provide a pH of no greater'
than 2; and thereafter analyzing the aqueous solution for an
effect produced by hydrolysis on the maltose under said steri-
lizing temperature-time exposure, and comparing said effect
with predetermined standard effect which is indicative of
exposure to said sterilizing temperature-time range. `~
In a further aspect the present invention provides
an aqueous solution for confirming a desired heat effect upon ;
a material which is subjected to a temperature-time exposure
which comprises from 0.1 to 1 percent (weight/volume~ of a
polysaccharide which hydrolyzes to produce a monosaccharide
at a rate which is temperature dependent, and the rate constant
corresponds to the Arrhenius equation, in which the activation
energy of said hydrolysis to form said monosaccharide is greater
than 30 kilocalories per mole of hydrolyzed saccharide-saccharide
linkage, and the frequency factor is from 10~ 4 to 102 ~ min. ~;
DESCRIPTION OF THE INVENTION
In accordance with this invention, the sterility
of a meterial which is intended to be subjected to a temperature-
time exposure to receive a sterilizing or pasteurizing heat
application can be confirmed by including with the material
in said temperature-time exposure a separate container of an
aqueous solution of a polysaccharide, which hydrolyzes to pro-
duce a monosaccharide at a first order or pseudo first order
ph/ - 4a -
, . . . .. ... , . . . . . ;.
L~ ~6 ~ ~
rate gencrally corresponding to the Arrhenius equation, in
which the activation energy of the hydrolysis reaction to form
~aid monosaccharide is greater than 30 kil~calories per mole
of hydrolyzed saccharide-saccharide linkage, and the fre-
quency factor is from 1014 to 1022 min. 1. Thereafter, the
aqueous solution is analyzed to determine the effect of said
exposure, and this determination is compared with a pre-
determined standard effect which is indicative of exposure to
said sterilizing temperature-time range.
At sterilizing temperatures, an isolated or a separate
container of an aqueous solution such as described above will
form monosaccharide by hydrolysis at a rate which can be
correlated with the actual death rate of bacteria present in
the material to be sterilized. However, at room temperature
lS the solution will degrade so slowly that it can be stored for
a time sufficient to permit its commercial distribution with-
out introducing serious error in subsequent analytical de-
terminations.
Hence, one can insert any desired number of separate,
sealed containers of the solution of this invention in among
the material to be sterilized or pasteurized, and one can
thereafter analyze for monosaccharide, such as glucose, in
accordance with any well known analysis method. Automated
analysis of many small containers of the composition of this
invention can be performed with instruments presently commer-
cially available. These instruments additionally can be
modified so that an alarm is signaled when an anal~is shows
a given container containing less than that given maximum
glucose level which is indicative of assured sterility. An
, . . . . . . .
1(~4~i76
example of such an automated analysis instrument is the
ROTOCHEMTM analyzer, with basic unit and sampler, marketed
by Travenol Laboratories, Inc., of Morton Grove, Illinois.
Optionally, a direct readout unit can also be added to
provide immediate data access.
This invention can also be used to map isotherm
patterns of heat intensity and changes in isotherm patterns
between different heating cycles. Other data relating to
detailed heating behavior of chambers and reaction containers,
at any number of desired points within the chambers or
containers~can also be determined. Thus this invention can
be used to study sterilizer chamber performance and to guide
engineering re-design, by the use of hundreds of solution
containers in a sterilizer chamber, to obtain a detailed
heat map of the chamber.
Typically, the polysaccharide used in the compo-
sition of this invention is the dissacharide maltose, which
has an activation energy of hydrolysis of bètween 31 and
33 kilocalories per mole. The maltose is generally present
in a concentration of 0.1 to 1 gram per 100 ml. of solution
and the aqueous solution of this invention typically has
an acidic pH of no higher than 2, to provide a frequency
factor of over 1014. The acidic conditions are typically
imparted by the presence of 0.1 to 1 N. sulfuric acid. By
adjusting the pH of the maltose-containing aqueous solution
the frequency factor can be varied to obtain the most accurate
correlation with the appropriate bacterial death curve. The
hydrolysis rate increases with decreasing pH.
Other polysaccharides having the proper activation
-- 6 --
~A. . . , : .- , . - . .
.
~3~i~6
energy, such as trehalose, can also be used in accordance
with this inve~tion in a solution having a pH which brings
the hydrolysis frequency factor within the range stated
above. For example, in O.S0 N. sulfuric acid solution, 0.4
grams of trehalose per 100 ml. of solution exhibits an
activation energy of about 32-33 kilocalories per mole and
a frequency factor of about 5 x 1016 min. 1.
The Arrhenius equation is well-known in physical
chemistry, and is of the following form: k=Aé E/RT, in which
k is the reaction rate constant, A is-the frequency factor, e
is the well-known natural logarithm base having a rounded off
value of 2.718, E is the activation energy, R is a constant
having the value of 1.987 calories per degree Kelvin per mole,
and T is the temperature in degrees Kelvin of the reaction.
The formula is typically solved mathematically from empirical
chemical reaction data to provide the values of any desired
unknown quantity in the ~quation.
The method of this application not only exhibits
increased aacuracy in the determination of the bacteria
killed by the sterilization process over that which is known
in the prior art, but it is a notably inexpensive technique and
lS susceptible to automated assay, which makes it the current
method of choice for use in determining the heat sterilization
of commercial lots of parenteral solution. The method and
composition of this invention can also be used for determining
the similar sterilization or pasteurization produced by an
appropriate temperature-time exposure of foods during canning
or bottling, or thereafter, and other similar items. For
example, a vial of the solution of this invention can be held
by a support in the center of a closed food-containing can or
1~367~;
bott~e, and inserted in a pasteurizing or sterilizing apparatus
with other food-containing cans or bottles. The amount of poly-
saccharide degradation in the vial indicates the heat level
received at the center of the cans or bottles, which is of course
the least-heated area thereof. From this, the adequacy of the
pasteurization or sterilization process can be determined.
During a given temperature-time exposure of the
composition of this invention, the frequency factor is
relatively unchanging due to the fact that the net hydrogen
ion concentration, and the resultant pH, are relatively unchanging
during the hydrolysis. However, by the initial adjustment of
the amount of hydrogen ions present, the frequency factor can
be varied, providing differing reaction curves, as desired, of
the degradation of the polysaccharide used herein. An increase
in hydrogen ion concentration generally increases the frequency
factor and accelerates the reaction.
The currently preferred composition of this inven-
tion for heat sterilization determination is a solution of 0.4
gram of maltose per 100 ml. of solution in 0.15 N. sulfuric acid
solution to provide a frequency factor of about 1016 to 1018
min.~l. If desired, other acid sources can be used, such as
hydrochloric acid, nitric acid, or any other strong acid which
does not provide serious side reactions.
It has been contemplated to insert the solution of
this invention in a sealed vial and distribute such vials
throughout the load of parenteral solution or other containers
to~be sterilized. Preferably, a parenteral solution container
itself can be filled with the solution of this invention, or one
of the separate vials can be dropped into, or suspended at the
'
'`" 1~436q6
center of, the filled parenteral solution container. In
either of these latter three conditions, the solution of this
invention is exposed to conditions which are more closely
identical to the actual conditions experienced by the actual
parenteral solutioh or other material to be sterilized, since
the solution of this invention is, during the sterilization
process, within the actual container in which the parenteral
solution is sold.
At the end of the sterilization process, the vials of
the solution of this invention, or the containers which in-
corporate the solution of this invention, are separated from
the commercial items and analyzed to determine the glucose
content of each solution. As stated above, the glucose con-
centration is a reliable indicator of the heat applied as a
function of temperature and time to which each particular
sample of the solution of this invention has been exposed.
The glucose may be analyzed colorimetrically, using,
for example, glucose oxidase. Other suitable analysis methods,
either manual or automated, can be used, such as analysis for
reducing sugar concentration, optical activity, or other
properties of the solution which are affected by the hydrolysis
of the chosen polysaccharide to a monosaccharide.
The most accurate analytical results are generally
obtained on the composition of this invention, for any desired
temperature-time exposure to which the composition is to be
subjected, when about one-half of the available maltose is
hydrolyzed to glucose during the exposure. A typical tempera-
ture-time exposure for parenteral solutions is to ma`intain a
sterilizer chamber temperature of at least 113C. for a period
of 35 minutes, and the preferred composition of this invention
.. ; . . .
. . -
1(~436~6
provides approximately a 50% to 75% maltose hydrolysis when
subjected to those conditions. Many other temperatures and
times can also be used.
The following example is offered for illustrative
purposes only, and is not in~ended to limit the scope of
the invention of this application, which is defined in the
claims below.
Example 1
A large sample of aqueous solution of 0.4 percent
(weight/volume) of maltose in 0.15 N. sulfuric acid was pre-
pared and placed in standard one liter Travenol parenteral
solution bottles. This solution is sufficiently stable for
several months at room temperature ti.e., below 85F.) to
remain useful and effective for that time period. A solution
is generally deemed "useful and effective" as long as the
solution contains less than 25% hydrolysis of the initial
maltose concentration prior to sterilization. In this appli-
cation, the term "weight/volume" implies grams per 100 ml.;
that is, the above maltose solution contains 0.4 gram of
maltose per 100 ml. of solution, which is 0.15 N sulfuric acid.
The initial glucose content immediately after
preparation of the solution was about 1 mg. per 100 ml.
Into each of the bottles of the maltose or test
solution so prepared was placed, through a conventional Trave-
nol closed hole solution bottle stopper, a thermocouple in a
stainless steel probe with a wire lead, so that the thermo-
couple probe was inserted in the solution.
Up to eleven of these bottles containing the thermo-
couples were distributed throughout a load of about 1500 similar
bottles containing aqueous solution in a large, water-cooled
sterilizer ~sed commercially by Travenol Laboratories, Inc.
The wire leads from the thermocouples were connected to a
-- 10 --
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1(3~3~76
read-out device, and the sterilizer was closed and heated to
a temperature, as recorded by the sterilizer thermometer, which
remained above 235F. for at least 35 minutes. The sterilizer
was then rapidly cooled by running water through its interior,
and the maltose, or test solution bottles, removed for analysis.
Samples of the test solution from each of the bottles
were analyzed for the concentration of glucose present with a
Technicon Autoanalyzer, using a modified glucose oxidase pro-
cedure with Glucostat, a glucose oxidase product of the Worthing-
ton Biochemical Corporation, Freehold, New Jersey. Duringanalysis, the test solution was buffered with a buffer com-
prising 6.805 grams of KH2PO4 and 6.0 ml. of 5 N. sodium hy-
droxide solution, the above being diluted with water to 1 liter.
The Technicon instrument takes up about 0.07 ml. of the test
solution per assay, and an appropriate amount of the buffer
solution and other reagents are automatically mixed in. The
concentration of glucose in each sample is automatically read
out as a relative concentration compared with a series of glu-
cose solutions of known standard concentration in an acid con-
centration similar to the test solution.
For each of the above samples of heated maltose solutionr
a "Maltose Ratio" was calculated, which is an expression re- -
lating to the amount of maltose degraded by the heat treat-
ment, and is defined by the expression mo-ga , where mO is the
mO-grt
initial concentration of maltose in the solution as formulated,
ga is the concentration of glucose present after the heating
treatment, and grt is the concentration of glucose immediately
prior to the heat treatment.
-- 11 --
. . , . ~ . . .
.
. . ..
~: . . . .
1~143~
Additionally, the course of heating was monitored
at one minute intervals by each thermocouple prohe mounted in
each test solution container over the heating cycle. From
these thermocouple data, a corresponding theoretical equiva-
lent bacterial kill effect (defined as Fs) at 250F., expressedin minutes for a bacterial system having a z value of 18, was
calculated for each bottle in accordance with the procedures
described in C. R. Stumbo, Thermobacteriology in Food Process-
ng, second edition (1973), Academic Press, New York, with
emphasis on Chapters 9 and 10, and especially pages 146 to 151.
The variables Fs and z are fully explained in the Stumbo
reference.
Briefly, Fs as used herein is the integrated lethal
capacity of heat received by all points in a container during
a heating cycle. It is a measure of the capacity of a specific
heat process to reduce the population of a living organism that
is destroyed exponentially, expressed in terms of minutes of
heating at lethal temperatures necessary to achieve the
bacteria killing effect of the specific heat process.
For purposes of this example, an Fs minimum value of
2.73 equivalent minutes at 250F. was selected as indicative
of a sufficient temperature and time to provide adequate steri-
lization. In this example, this corresponds to a maltose
ratio of about 0.38. If a greater assurance of sterilization
is desired, a higher minimum Fs value, giving a correspondingly
lower maltose ratio, should be selected.
The following Table A is a summary of the thermocouple
probe heat history data, and the maltose ratios of each sample
of test solution associated with each thermocouple probe.
- 12 -
1043~7S
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The above sterilization run is believed to be accept-
able for the reliable sterilization of commercial parenteral
solution bottles under the conditions specified.
Example 2
The experiment of Example 1 was repeated, except that
the sterilizer was heated to a sterilizer temperature which
remained above 235F. for only about 23 minutes, as read on
the sterilizer instruments. This cycle is inadequate to pro-
vide reliable, adequate sterilization, as used in this Example.
The thermocouple heat history data, and the correspond-
ing maltose ratios, are as in the followin~ Table B.
7~
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It should be noted that all of the maltose ratios aresignificantly above 0.38, indicating that the total heat re-
ceived was insufficient for safe sterilization, as confirmed
by the thermocouple data in the same run.
Example 3
The experiment of Example 1 was repeated, except that
the sterilizer was heated to a sterilizer temperature which
remained above 250F. for 20 minutes, as read on the sterilizer
instruments.
The following FS values from the thermocouple heat
history, and the maltose ratios, were obtained as previously
described.
(Table C).
- 16 -
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From the above data, it can be seen that all maltose
raiios are significantly below 0.38, indicating that the
total heat received was well above that necessary for safe
sterilization.
By comparison of the maltose ratios calculated for
the solution bottles prepared in this example with the corres-
ponding Fs values derived from the thermocouple data, the Fs
values can be correlated with corresponding maltose ratios.
Accordingly subsequent and similar commercial sterilization
cycles under similar conditions can be monitored without
thermocouples by a simple analysis of the test solutions in-
cluded in the load, followed by calculation of maltose ratios
for each sample after sterilization. Proper and reliable
sterilization is indicated when the maltose ratios of all
lS samples of maltose Rolution are below a specified maximum
maltose ratio that indicates reliably, for the particular heat
cycle used, with a substantial margin for error, adequate heat
sterilization, e.g., a maximum maltose ratio of 0.38. This is
calculated by graphing each Fs on one axis of a graph against
its corresponding maltose ratio on the other axis to record
a point on the graph for each Fs-maltose ratio pair. From
the resulting curve of points, an Fs of 2.73, mentioned above
as a desired value, is seen to correspond to a maltose ratio
of 0.38 in this example.
- 18 -
. .
