CLEANING AND DECONTAMINATING DIALYZERS BY PER-COMPOUND SOLUTIONS
Field of the Invention
The invention relates to chemical compositions and methods for
using the compositions for cleaning and decontaminating dialyzers.
Background
The medical industry and other industries utilize devices that are
required to be cleaned and decontaminated. Cleaning is the removal of foreign
material, including organic soil such as blood, feces, respiratory secretions,
etc., from objects. It has been reported that failure to remove foreign material
from a medical device such as an endoscope before a disinfection or
sterilization process is likely to render the process ineffective. (Rutala, WA,
APIC Guideline for Selection and Use of Disinfectants, Am J Infect Control,
August 1 996; Vol . 24,4:31 3-342) . The presence of organic material or soil
may contribute to the failure of disinfection by harboring embedded microbes
and preventing the penetration of the germicide. Additionally, some
disinfectants are inactivated by organic material (Martin, MA, Reichelderfer, M,
APIC Guideline for Infection Prevention and Control in Flexible Endoscopy, Am
J Infect Control, 1 994;22: 1 9-38) . Decontaminating is defined as the removal
of hazardous or unwanted materials such as bacteria, mold spores or other
pathogenic life forms and the like, with high-level disinfection and sterilization
representing different levels of decontamination. High-level disinfection is a
process that eliminates many or all pathogenic microorganisms, with the
exception of bacterial spores, from inanimate objects Sterilization is a process
that completely eliminates or destroys all forms of microbial life, including
fungal and bacterial spores.
High-level disinfection can be expected to destroy all
microorganisms, with the exception of high numbers of bacterial spores A
Food and Drug Administration (FDA) regulatory requirement for high-level
disinfectants is that they achieve 1 00% kill of 1 00,000 to 1 ,000,000
organisms of Mycobactenum tuberculosis in the presence of 2% horse serum
in a quantitative tuberculocidal test An additional FDA regulatory requirement
for high-level disinfectants is that they must also achieve sterilization over a
longer exposure time than the disinfection regimen time Sterilization is tested
with a spoπcidal activity test utilizing spores of Bacillus subtilis
Common commercially available high level disinfectants include
glutaraldehyde solutions between 2.4-3 4%, which typically require activation
with an alkaline buffer just prior to use Also available are an acidic
(pH 1 .6-2.0) 7.5 %w/v hydrogen peroxide (H202) solution (Sporox®, Reckitt and
Colman, Inc ) and an acidic (pH 1 .87) mixture of 1 .0% H202 plus 0.08%
peracetic acid (PAA) (Peract™ 20, Minntech Corp. or CidexPA®, Johnson &
Johnson). The minimum effective concentration of PAA for high-level
disinfection at 25 minutes (mm) and 20 ° C is 0 05 % (500 ppm) (Peract™) The
minimum effective concentration of H202 for high-level disinfection at 30 mm
and 20° C is 6.0% (Sporox®)
High-level disinfecting solutions are also typically designed for a
reuse option, depending upon the medical device For example a
glutaraldehyde high-level disinfecting solution for endoscope reprocessing may
be reused for as long as 28-30 days, while kidney dialyzers are disinfected
with single-use solutions.
Kidney dialyzers pose an additional problem in high level
disinfecting in that the materials utilized require particular performance criteria
of the cleaning and disinfection solutions Types of dialyzers include ( 1 ) coil,
which incorporates a membrane in the form of a flattened tube wound around
a central, rigid cylinder core, with a supporting mesh between adjacent
portions of the membranes; (2) parallel plate, which incorporates a membrane
in tubular or sheet form supported by plates in a sandwiched configuration,
and (3) hollow-fiber, which incorporates the semipermeable membrane in the
form of the walls of very small fibers having a microscopic channel running
through them. Most parallel plate and hollow-fiber membranes are made from
cellulose acetate, cellulose triacetate, regenerated cellulose, cuprophan or
polysulfone.
The semipermeable membranes used in dialyzers have large areas
and high porosities, and after use become coated with blood proteins and
other organic and cellular material Dialysis fibers are also often clotted with
blood cells, proteins and other debris. As a result, the membrane of a used
dialyzer has a reduced capacity for dialysis and is highly susceptible to
microbial growth. Effective killing of microorganisms on such a used
membrane for the purpose of reusing the dialyzer is difficult to accomplish
without damaging the membrane.
When initially introduced, dialyzers were one-use devices . Since
1 980, dialyzer reuse has risen dramatically in order to reduce the overall cost
to the patient and the health care delivery system. Hemodialyzers,
reprocessed in conformance with the Association for the Advancement of
Medical Instrumentation (AAMI) specific guidelines and performance tests,
have an average use number, that is, the number of times a particular
hemodialyzer has been used in patient treatment. This number has been
increasing over the years, from a United States average of 1 0 reuses in 1 986
to 1 5 reuses in 1 996. The cost benefits achieved by reprocessing are
significant. For example, a new dialyzer costs about $20-30. With
reprocessing, a dialyzer can be used between 5-20 times without substantial
loss of efficacy. The cost of reprocessing is approximately $6.60-7.72 per
unit, including reprocessing solutions. The cost per reuse for reprocessing
solutions is $0.99-1 .1 4 (average $ 1 .08) . The amortized dialyzer cost per
reuse is $ 1 .35-2.00, based upon an average reuse of 1 5 times. Additionally,
the cost per reuse for dialyzer hazardous medical waste disposal is
$0.50-0.55, reuse technician labor costs are $ 1 4/hr, and the associated labor
cost of manual cleaning/dislodging clots is $0.23. Accordingly, with
reprocessing, the dialyzer cost per treatment is conservatively less than about
$ 1 0, as opposed to $30 if a new dialyzer were used for each treatment. A
typical patient receives approximately 1 56 treatments per year. In 1 998 in the
United States alone there were approximately 280,000 patients on
hemodialysis, and about 86% of hemodialysis centers have a dialyzer reuse
program. Therefore, there are about 35,060,480 reuses in the United States
(280,000 x 0.86 x ( 1 56 - 1 56/1 5)) . The U.S. market for reprocessing
solutions in 1 998 is estimated to be $34.7-40.0 million.
In addition to cost savings with dialyzer reuse, there are health
advantages. Researchers have determined that reused dialyzers significantly
mitigate patients' "new dialyzer" symptoms as well as immune reactions that
often occur. The inherent clinical advantage of reused dialyzers has been
attributed to both the reduction in trace contaminants such as ethylene oxide
sterilant, and to the masking of immune reaction sites located on the
membrane surface by protein deposits.
Dialyzer reprocessing involves three basic steps: ( 1 ) cleaning,
(2) dialysis efficacy confirmation, and (3) high-level disinfecting involving soak
times long enough to achieve sterilization. The cleaning step involves
removing residual blood, organic and cellular material from the blood side and
removing dialysate from the dialysate side of the semipermeable membrane.
A number of cleaning solutions are known, including sodium hypochlorite
bleach, PAA and H202. Purified water has also been used for cleaning . The
cleaning solution must be rinsed from the dialyzer, typically with water.
Sodium hypochlorite bleach at a concentration of 0.5-1 .0%w v for
3 min exposure is utilized for cleaning . However, significant decreases in
patient urea and creatinine clearance have been observed with high-flux
polysulfone (F80B) dialyzers reprocessed with formaldehyde and bleach
(Murthy et. al., Effect of Formaldehyde/Bleach Reprocessing on In Vivo
Performances of High-Efficiency Cellulose and High Flux Polysulfone Dialyzers
J Am Soc Nephrol .464-472, 1 997) Also, Kaplan and colleagues observed
up to 20 g blood protein and specifically 1 5 g albumin loss into the dialysate
per treatment with bleach-reprocessed, high-flux polysulfone (F80) dialyzers
Elimination of bleach from the reprocessing protocol led to a significant
increase in serum albumin levels (Kaplan et al Dialysate Protein Losses With
Bleach Processed Polysulfone Dialyzers Kidney In t 47,573 578 1 995 ) It
is believed that reprocessing certain polysulfone dialyzers with bleach
somehow alters membrane structure. Loss of the usual immune protection
achieved with reused dialyzers has been shown to occur when sodium
hypochlorite, particularly at elevated concentrations, is used for reprocessing,
resulting in complement activation and neutropenia restored to near original
levels. The problems associated with utilizing bleach in the reprocessing
protocol have widespread ramifications; in the United States as of 1 996, 42%
of all patients were utilizing high-flux dialysis and 78% of those were utilizing
polysulfone dialyzer membranes Finally, while not reported within the kidney
dialysis industry, it is known that chlorine bleach solution has a tendency to
form so-called haloforms with organic compounds These compounds are
considered to be carcinogenic and are therefore also hazardous from the health
perspective. In this context, it was recently reported that dialysis patients had
an increase in cancer of 1 5 % as compared to the general population ( The
Lancet, 354. 93-99, 1 999)
Dialyzers reprocessed with prior art acidic H202 solutions have a
significant reduction in ultrafiltration rate, indicating the presence of
hydrolytically resistive protein deposits resistant to removal by H202. In
addition, while prior art H202 solutions are useful in that they react vigorously
with hemoglobin, can be effective in dissolving some clots in dialyzer headers
and blood channels, and can restore dialyzer fiber bundle volume in some
cases, elevated concentrations of H202 can rapidly generate gaseous oxygen
reaction products, as evidenced by the reported bursting of noncompliant
membrane capillary fibers. Acidic PAA reacts similarly with protein deposits,
as PAA contains an equilibrium mixture of H202, PAA and acetic acid . Thus,
acidic PAA will not remove protein deposits but can be effective in dissolving
some clots.
Lastly, water used in the reprocessing cleaning step is generally
ineffective in removing protein deposits or bound clots, as is the case with
formaldehyde and glutaraldehyde.
The use of citric acid in connection with the cleaning of dialysis
machines has been disclosed in a number of patents. Tell et al., U .S . Patent
No. 4,690,772, discloses a sterilizing and cleaning solution comprising sodium
chlorite, citric acid and a sodium bicarbonate buffer. U.S. Patent
No. 5,480,565 to Levin discloses a method for reprocessing dialyzer cartridges
used with kidney dialysis machines. The method involves filling the blood and
dialysate compartments of the dialyzer with an aqueous solution containing
citric acid at a concentration of about 1 .0-5.0%w v and then subjecting the
dialyzer to an elevated temperature above 90°C and below 1 00°C for a period
of at least 1 5 h. It is known, however, that citric acid is incapable of
removing bound protein deposits from polymer surfaces at these temperatures.
Moreover, the sodium chlorite solutions in the '772 patent have the capacity
to crosslink proteins in surface deposits, making them even more resistant to
removal . Also, the heat utilized in the '565 patent will further denature
proteins and possibly create more deposits, as well as deposits which are more
resistant to removal.
The efficacy confirmation step for dialyzer reprocessing involves
confirming that membrane integrity and performance is substantially equivalent
to that of a new dialyzer. Specifically, with respect to membrane
performance, when the measured fiber bundle volume (FBV) of the membrane
drops by 20%, the dialyzer is no longer reused .
The disinfection step involves subjecting the dialyzer to high level
disinfection with a process or chemical disinfecting agent. Chemical
disinfecting agents such as formaldehyde, glutaraldehyde or an acidic
equilibrium mixture of PAA, H202 and acetic acid are typically employed . In
the United States in 1 996, 36% of dialysis centers used formaldehyde, 54%
used PAA, 7% used glutaraldehyde and 3% used heat to disinfect and
sterilize. A commonly used glutaraldehyde solution is Diacide® (Gulfstream
Corp.), a 26%w/v concentrate of acidic glutaraldehyde which is activated with
alkali just prior to use and then diluted with water to a final concentration of
0.8%w/v. In 1 998, the most commonly used PAA-based product was Renalin "
Dialyzer Reprocessing Concentrate (Renal Systems Division, Minntech Corp.) .
Renalin® is a concentrated solution of acidic 4%w/v PAA and 24%w v H202,
designed to be diluted to a 3.5 %v v concentration in water, yielding a final
concentration of about 0.1 4%w/v ( 1 400 ppm) PAA and about 0.84%w/v (8400
ppm) H202. The minimum effective concentration of PAA to achieve either
high level disinfection in a short time interval or sterilization over a longer soak
period is 500 ppm.
Another PAA solution is described in Japanese patent JP
1 1 076380A. An English translation of this patent discloses a cleaning and
disinfecting composition consisting of an aqueous solution containing 3.5-6%
hydrogen peroxide, 5-30% of an organic acid and 0.4-3.4% of an organic
peracid in a weight ratio so that the sum of the acid and peracid to the
peroxide is at least 1 . The preferred organic acid is acetic acid. The method
of cleaning and disinfecting comprises diluting the composition 20 to 1 00 fold
with water. The composition may be used to clean the dialysis line of an
artificial dialyzer through single step cleaning, allows easy removal of mass
precipitation of calcium salts, and has high storage stability and high safety in
handling. The '380 patent does not explicitly reveal the solution pH; however,
its reference to a solution having "high storage stability" (which Test 1
indicates is either four or eight weeks storage in darkness at 36°C) would
require a pH of substantially less than 5. It is thus essentially equivalent to
other highly acidic PAA solutions such as Renalin®.
The chemical disinfecting agent must be able to be rinsed out of
the dialyzer to below toxic levels, with a rinse-out period established for the
particular agent. Typically, for glutaraldehyde disinfectants, 1 liter of isotonic
sterile saline is perfused through the dialyzer fibers prior to dialyzer use, with
sterile purified water additionally used to rinse the dialysate chamber
Moreover, since the dialyzer is connected to the vascular system during use
any residual chemical entity which may be reversibly bound to the
semipermeable membrane and which may desorb from the dialyzer following
the rinse should be non-immunogenic, i.e., it should not provoke an immune
response.
While some of the above solutions enjoy some commercial
success, all have inherent problems which limit their use. The alkaline
glutaraldehyde solutions have an appreciable noxious odor and a low vapor
threshold for toxicity, and thus require the concomitant use of a ventilation
system. Glutaraldehyde also cross-links proteins and thus likely further clogs
previously uncleaned dialyzers and further limits solute transport through the
dialyzer fibers. The current commercial acidic H202 and PAA solutions do not
efficiently clean dialyzers and result in limited dialyzer re-use life.
One potential solution to the aforementioned problems with highly
acidic H202 and PAA solutions is the invention disclosed in U.S. Patent
No. 5,827,542. The '542 patent discloses a low odor, aqueous, quick acting,
room temperature disinfecting and/or sterilizing solution that is non-corrosive
to metals and elastomers used in medical instruments which are in need of
sterilization and disinfection, having a pH within the range of from about 2.0
to about 6.0. The solution consists essentially of from about 1 % to about
30% by weight of a peroxide and from about 1 % to about 30% by weight of
malonic acid, or salt form thereof, the solution being effective at room
temperature to disinfect medical instruments within 30 min without corroding
surfaces of the medical instruments The '542 patent also discloses that the
amount of peroxide component and the amount of malonic acid or carboxylic
acid component are balanced such that the pH will be within the range of
about pH 2 0-6.0, preferably about pH 3.0-5.0 However, all six patent
examples at or above pH 5.0 do not achieve sterilization. Also disclosed is the
composition packaging, wherein the composition may be prepackaged in
solution form, ideally in two packages, one the peroxide and one the organic
acid component, to be mixed at the point of use. This packaging is described
as enhancing freshness. However, due to the slow approach to equilibrium
between peroxide, organic acid and peracid, the production of the disinfecting
peracid will not take place with the thirty mm disinfection time specified by the
patent. Thus, premixing of a peroxide and an organic acid component in the
'542 patent is inoperative Diluents such as alcohols can also be employed
While this invention discloses solutions with higher pH, up to pH 6 0 which
are inherently much less corrosive to metals, it employs a high concentration
of peracid generated from the combination of the peroxide and the malonic or
other carboxylic acid High peracid concentrations are known to be
incompatible with medical device adhesives used to bond metal and plastic
parts together Solution compatibility tests with medical device adhesives
were not disclosed, and claims for adhesive compatibility were also not made
An additional disadvantage of the '542 invention is the potential toxicity of the
high concentrations of disclosed peracids, despite the unsupported statement
to the contrary The '542 invention did not disclose cleaning of medical
devices such as kidney dialyzers
Thus, it can be seen that there remains a need for a safe,
practical, and efficient cleaning and high-level disinfecting and sterilizing
composition and method for reprocessing kidney dialyzers.
Summary of the Invention
The invention is directed to a composition for cleaning and
decontaminating a dialyzer. The composition comprises a source of at least
one per-compound oxidant and a buffer in amounts to provide the per-
compound oxidant at a concentration and pH effective for cleaning and
decontaminating the dialyzer. The composition has a pH between about 5-1 1 .
The per-compound oxidant may be at least one peracid or hydrogen peroxide
(H2O2) and at least one peracid. In different embodiments, the H202
concentration may be in the range of about 1 -50%w/v, about 3-20%w v, or
about 6.5-8%w/v. The peracid may be at a concentration in the range of about
0.0050-1 0.0%w/v. The per-compound oxidant may be a mixture of peracetic
acid at about 0.0050-0.5%w/v and H202 at about 0.5-50.0%w/v or peracetic
acid at about 0.005-0.2%w v and H202 at about 0.5-8.0%w/v . The buffer may
be acetic acid, propanoic acid, glycine, monobasic dihydrogen phosphate,
dibasic hydrogen phosphate, bicarbonate, and/or carbonate, either with or
without non-immunogenic counter ions.
The invention is also directed to a method of cleaning and
decontaminating a dialyzer. A solution is produced by combining at least one
per-compound oxidant and a buffer, mixed prior to use, in amounts to provide
the per-compound oxidant at a concentration and pH effective for cleaning and
decontaminating the dialyzer. The dialyzer is contacted with the solution for
a period of time effective for cleaning and decontaminating The method may
included a further step of removing the solution from the dialyzer, for example,
by rinsing with sterile water or sterile saline. The dialyzer may be stored to
prevent recontamination. A soiled dialyzer may be treated to remove soil
before it is contacted with the solution, for example, by contact with an
enzyme solution.
The invention is also directed to a method of cleaning and
decontaminating a dialyzer having a blood chamber and a dialysate chamber.
At least one per-compound oxidant and a buffer are combined in amounts and
at a pH effective to form a cleaning and decontaminating solution, then the
blood chamber is contacted with the solution for a time effective for cleaning
and decontaminating. The solution may also contact the dialysate chamber
The dialyzer may be vented to allow escape of a gas formed from combining
the per-compound and buffer.
These and other objects and advantages of the present invention
shall be made apparent from the accompanying description and examples.
Brief Description of the Drawing
The figure is a graph showing used dialyzer fiber bundle volume
recovery after treatment with a solution of the invention
Detailed Description
It has been discovered that a simple one-step mixing of a per-
compound oxidant or per-compound oxidant mixture in a particular
concentration range with a buffer to adjust the pH of the per-compound
oxidant solution just prior to use results in a sufficiently stable solution which
achieves simultaneous cleaning and high-level disinfection and sterilization of
kidney dialyzers. Cleaning is defined herein as any significant restoration of
dialyzer function such that its reuse life would be extended by at least one or
more dialysis treatments. Cleaning efficacy can be measured by evaluating
dialyzer membrane performance. The fiber bundle volume (FBV) of a hollow-
fiber dialyzer is one measure of membrane performance as is membrane
ultrafiltration rate. Mixing prior to use is achieved conventionally, for example,
through hand or assisted mixing of two solutions in a suitable container. It is
desirable to use readily mixed and soluble components so that the entire
mixing operation lasts only a few seconds to not more than a few minutes in
the case wherein one or more solid components is utilized . The present
invention is particularly suited for the simultaneous cleaning and high-level
disinfecting and sterilizing of artificial kidney dialyzers such as Polysulfone*
dialyzers (Fresenius USA Corporation, Lexington, MA), but can also be used
to clean other types of dialyzers including peritoneal dialyzers. In disclosing
compounds and methods used to clean and decontaminate dialyzers, it is
contemplated that the tubing or lines connecting to and from the dialyzer, for
example, patient lines, are also encompassed by the invention.
The proper combination of per-compound, per-compound
concentration and pH must be employed to achieve protein degradation and
removal from a kidney dialyzer (e.g., cleaning) simultaneously with disinfection
and sterilization. Generally, lower molecular weight per-compounds at higher
concentrations and higher pH values are more efficacious. The term per-
compound oxidant, as used herein, is any compound which delivers active
oxygen in solution and which can achieve high-level disinfection and
sterilization of kidney dialyzers. Thus, for example, peracetic acid (PAA) or
other peracids, alone or in combination, which achieve high-level disinfection
and sterilization are included in this definition .
In one embodiment, peracids produced from C C4 mono or
dicarboxylic acids, such as performic acid, peracetic acid and perpropionic
acids are used. Peracids produced from carboxylic acids with linear, saturated
or unsaturated hydrocarbon chains having 5-20 carbon atoms such as linoleic
acid (C18H3202) may also be used. Combinations of hydrogen peroxide (H202)
or other peroxides with one or more peracids are also included. Solid sources
of H202 such as sodium percarbonate and sodium perborate can be used in
combination with one or more peracids or one or more solid peracid precursors,
such as tetracetylethylenediamine (TAED) or acetylsalicylic acid. Generally, the
concentration of peracid or peracid mixtures in the present invention ranges
from about 0.0050%""" to about 1 0%w ". In one embodiment combinations of
PAA and H202, wherein the concentration of PAA is between about
0.0050%""" and 0.5%""" and the concentration of H202 is between about
0.5 %""" and 50.0%""", are used. In another embodiment, the concentration
of PAA is between about 0.08%w/v and 0.2%""" and the concentration of H202
is between about 0.5 %w/" and 8.0%w v. Since peracids are generally unstable
at higher pH and must be typically kept at a pH less than 3-4 to achieve shelf
stability, they are kept separate from the buffer until just prior to use.
Peracid solutions are typically pH stabilized to between about
pH 1 -3 prior to mixing with the buffers of the invention. The peracid solutions
are even more stable between about pH 1 .6-2.0. This is achieved with an
acidic compound such as an inorganic or organic acid. Organic acids with the
greatest stabilizing effects are those which are precursors for the particular
peracιd(s) employed, e.g., acetic acid most efficiently stabilizes PAA This is
due to the well known equilibrium between H202, acetic acid and PAA. The
amounts of each in a stable solution will be determined by the known
equilibrium constant for the formation of PAA from H202 and acetic acid
Similar considerations apply to other peracids. Additionally, transition metals
such as iron, manganese and copper also destabilize peroxides and peracids
Such metal-induced destabihzation can be prevented with sequestrants such
as hydroxy-ethylidene diphosphonic acid (Dequest 201 0™ Monsanto Co.), or
with the use of conventional chelating agents such as ethylenediamme
tetraacetic acid (EDTA) . Concentrations of Dequest 201 0™ between about
0.1 0 and 1 .0%w/v are used in one embodiment Other conventional means of
stabilizing peroxides and peracids can also be employed, such as the use of
nitrilotπsmethyl-, methyl- and other phosphonic acids
A liquid or solid per-compound(s) may be utilized in the
compositions and methods of the present invention. The solid per-
compound(s) such as sodium peroxide and a peracid precursor such as TAED
would be mixed just prior to use with water and the buffer, or alternatively
directly with a liquid buffer.
Generally, a pH between about 5-1 1 is useful in the present
invention. The particular pH selected for a particular composition is dependent
upon several testable factors, such as type amount and stability of per
compound(s) and the particular kidney dialyzer application, e.g , type and
amount of protein-containing deposit to be removed, rate of disinfection and/or
sterilization required and device compatibility. The present invention generally
destabilizes the per-compound due to the pH range employed. This
destabilization is controlled for the particular application Control of the
destabihzation of a particular per-compound is achieved through conventional
studies of the particular equilibrium or equilibria involved in the formation and
stabilization of the peracid as well as studies of decomposition rates versus pH
and other solution parameters For example it is known that peracetic acid
decomposition follows a second-order dependence on peracetic acid
concentration with a maximum rate at pH 8 2 (Z Yuan et al , The Canadian
Journal of Chemical Engineering, Vol. 75, February, 1 997, pp 37-41 ) Buffers
useful in the present invention will generally have pKa's of between about 4.5
and 1 1 .5. The buffers must be compatible with the per-compound(s) with
which they are paired in terms of buffer and per-compound short-term stability
after mixing, e.g., about 48 hours or less for an application for reprocessing
(cleaning and disinfecting or sterilizing) a used kidney dialyzer The buffer also
must maintain the desired pH during the use period Buffer concentrations
between about 2 mM and about 2 M can be used in compositions of the
present invention Buffers such as acetic acid (pKa = 4 76), propanoic acid
(pKa = 4.86), glycine (pKa2 = 9.78), monobasic dihydrogen phosphate, dibasic
hydrogen phosphate, bicarbonate or carbonate, each with a suitable counter
ion such as sodium or potassium or another counter ion, can be used. Acetic
acid can be used to buffer the solution to between pH 5 0-5 8 However, in
a peracetic acid system, the amount of peracetic acid which can be stably
maintained with an acetic acid buffer at pH 5 0 5 8 is less than 700 ppm This
is due to the shift in the equilibrium relationship between hydrogen peroxide,
peracetic acid and acetic acid and the high rate of peracetic acid loss at higher
pH . Non-immunogenic buffers and counter ions are generally used with kidney
dialyzers. Potentially immunogenic buffers and counter ions can also be used
with kidney dialyzers, as long as they can be completely rinsed from the
dialyzer prior to use. A hydroxide buffer such as sodium or potassium
hydroxide can also be used, alone or in combination with other buffers in
some circumstances. The buffer may be a liquid or solid
The purity of the raw materials used in the compositions of the
invention for kidney dialyzer use must be considered All per-compounds,
peroxides and buffers should be of the highest possible purity, taking into
account raw material costs, in light of the need to avoid provoking the immune
system through exposure of trace amounts of undesirable chemicals should any
contaminants in the raw materials desorb in trace amounts into the blood from
a kidney dialyzer during dialysis Moreover, compositions of the present
invention need to be kept as simple as possible in terms of number of
components for dialyzer reprocessing applications, to minimize and avoid
immune response complications Ideally, all components of the compositions
of the present invention should be structurally similar if not identical to
naturally occurring chemical entities found in the body so as to avoid immune
responses from trace amounts which may inadvertently come into contact with
the vascular system during dialysis
In addition to the compositions of the invention not provoking an
immune response in a patient, the compositions also should not be harmful to
the patient in small amounts that may not be completely removed from the
dialyzer For example, a composition that contained anti corrosive agents such
as benzotπazole, chromates, dichromates, molybdates, vanadates, tungsdates
etc. would be harmful to patients. The compositions of the invention achieve
cleaning and decontaminating of dialyzers without harm to patients.
Renalin®, the solution which upon dilution yields a final acidic
(pH < 2.5) concentration of 0.1 4%w/v PAA and 0.84%w/" H202, can also be
utilized in combination with a suitable buffer to raise the pH to a level which
effects cleaning of a kidney dialyzer without compromising disinfection and
sterilization. Other peracid concentrates which are diluted with an aqueous
buffer just prior to use can also be used. Peract™20 (Mmntech Corp.) or Cidex
PA® (Johnson and Johnson), which is the acidic (pH 1 87) mixture of 1 0%w v
H202 and 0.08%w/v PAA, can also be utilized in combination with a suitable
buffer to raise the pH to a level which affects cleaning of a kidney dialyzer
without compromising disinfection and sterilization
The method of the invention for cleaning and decontaminating a
kidney dialyzer generally involves the following steps- ( 1 ) combining a per-
compound oxidant and a buffer in amounts effective for cleaning and
decontaminating a kidney dialyzer, and (2) contacting the kidney dialyzer with
the per-compound and buffer for a time sufficient to simultaneously clean and
decontaminate the kidney dialyzer The contacting time can be very short for
example 1 0 min or 48 hours or even longer, as long as the standards herein for
cleaning and decontamination are achieved.
One embodiment of the present invention used for cleaning and
decontaminating kidney dialyzers employs a gas-venting filter cap placed over
at least one of the dialyzer fiber bundle (blood chamber or dialysate chamber)
access ports to vent any oxygen gas produced from the per-compound and
buffer solution. Per-compound solutions are generally stable at values below
about pH 3-4. Higher pH values are known to destabilize per-compound
solutions, with greater destabilization occurring with higher pH . This
destabilization results in the production of oxygen gas. The amount of gas
formation can be very high in some solutions, depending upon the type,
concentration and pH of the per-compound solution. An example of this is the
known production of 1 00 ml of oxygen from 1 0 ml of 3%w " H202 when the
latter is completely catalytically neutralized. The fiber bundle volume of a new
dialyzer is approximately 1 00 ml and that of a used dialyzer is no less than
80% of the new volume. The result is that a considerable amount of oxygen
gas can be potentially produced from a per-compound and buffer solution of
the present invention. Thus, the method of the present invention for cleaning
and decontaminating a kidney dialyzer generally involves the following steps:
( 1 ) combining a per-compound oxidant and a buffer in amounts effective for
cleaning and decontaminating the dialyzer; (2) venting the dialyzer to allow
increased gas to escape; and (3) contacting the dialyzer with the per-compound
and buffer for a time sufficient to simultaneously clean and decontaminate the
dialyzer. Alternatively, step (2) may be performed prior to step ( 1 ) or after step
(3) . More specifically, the inside of the empty dialyzer fiber bundle and
optionally the inside of the dialysate chamber, are filled with one of the
solutions of the invention. One end of the dialyzer fiber bundle blood chamber
is capped with a gas- and solution-impermeable cap, and the other end opening
is capped with a gas-only venting cap, typically comprised of a hydrophobic
barrier filter placed inside a regular cap with a physical hole or other gas
channel or transport construction. Alternatively, both openings of the dialyzer
fiber bundle blood chamber are capped with a gas-venting cap . Alternatively,
any or all openings of both the blood and dialysate chamber are capped with
a gas-venting cap. The hydrophobic barrier filter prevents water passage but
allows gas passage through the gas channel on the opposite side of the barrier
filter from the blood or dialysate chamber access port. The hydrophobic barrier
filters and caps useful in the present invention are of conventional types and
are commercially available from a variety of sources. An example of a suitable
filter and cap is a Gore-Tex® membrane (W.L. Gore Associates, Inc., Elkton,
MD) and a polypropylene cap (Rexam Closures, Evansville, IN) . The entire
blood contact surface of the dialyzer is contacted with the solutions of the
invention. Both ends of the fiber bundle should, in most cases, be covered
with the solution, as a considerable amount of clotted blood and bound protein
is typically found on used dialyzers at the ends of the fiber bundles.
The first step in the general method of the invention wherein the
per-compound and buffer are combined can be preceded or followed by a step
to remove soil from the dialyzer. This soil removal step can be comprised of
contacting the dialyzer with an enzyme. Another embodiment of the method
of the invention includes a step wherein the per-compound and buffer solution
is removed from the dialyzer following the contacting step. This removal can
be accomplished with a sterile water or saline rinse. Other sterile solutions
such as dextrose or glucose solutions can also be used. The solution removal
step can be followed by storage of the device in a way which prevents
recontamination either by microorganisms or soils.
Other energy input may be employed to potentiate the solution's
cleaning and decontaminating effects. For example, heat and ultrasonic energy
are known to potentiate the speed at which chemical cleaning and disinfecting
agents work. The practice of this invention is not to be limited by temperature
except by those temperature extremes which would substantially inactivate the
capability of the chemistry employed and/or damage the kidney dialyzer, as can
be determined through routine performance and stability testing.
EXAMPLE 1
Cleaning and Disinfecting Efficacy at Various pH Values
Two used kidney dialyzers obtained from a dialysis clinic were
used for testing the cleaning efficacy of H202 cleaning and disinfecting
solutions of the present invention at different pH values. The solutions in this
example consisted of 7.5 %""" H202 at pH 4.0 and 8.5, the latter which was
adjusted with NaOH . The fiber bundle volumes of the used dialyzers were
measured as follows: after both blood chamber and dialysate chamber of a
dialyzer were filled with tap water, the dialyzer was tapped repeatedly to rid
the chambers of air bubbles and all four outlets/inlets of the dialyzer were
plugged to prevent water loss. The dialyzer was weighed before and after the
water in the blood chamber was evacuated. Evacuation was accomplished by
blowing compressed air through the chamber. The blood chamber (fiber
bundle) volume was determined as the difference between the two weight
values divided by the density of water. The used dialyzer initial (before
treatment with the cleaning/disinfecting solution) volumes (Vused), measured
with the above method are consistent with the labeled minimum volumes
(measured by the dialysis clinic) which are about 80% of the new dialyzer full
volumes before the fiber bundles were clotted with patient blood.
Both blood and dialysate chambers of the dialyzers were soaked
with the above cleaning/disinfecting solutions, one dialyzer per solution, after
the water in the dialysate chambers was removed. Figure 1 shows the
relationship between the percentage fiber bundle (blood chamber) volume
recovery and the cleaning/disinfecting solution soak time. The percentage fiber
bundle volume recovery (P) was determined with the equation :
P = 400 x Fiber Volume lncrease/Vused
Percentage fiber bundle volume recovery is also = (V
lncrease/(V(new)-V(used))) x 1 00, wherein V(newl is assumed to be 1 .25 x V(used).
This example shows that the alkaline H202 solution can effectively remove the
blood proteins clotted in the dialyzer fiber bundles (about 50% recovery of
clotted fiber bundle volumes within 4 hours) and that the acidic H202 solution
has no significant effect on removing the clotted blood protein in the dialyzers.
The negative results obtained with the acidic H202 solution are consistent with
the fact that these used dialyzers had been processed repeatedly from their
new state to the end of their useful life with the diluted Renalin® Dialyzer
Reprocessmg Concentrate from the Renal Systems Division of the Minntech
Corporation. Renalin® is an acidic (pH 2.5) solution of about 0.8%w/" hydrogen
peroxide (H202) and about 0 1 4%w " peracetic acid (PAA) when diluted
The compositions and the methods using the compositions of the
present invention in many cases do not need to be neutralized prior to disposal,
as do the currently marketed solutions such as Renalin® The sanitary disposal
of used chemical solutions including cleaners, germicides and liquid chemical
germicides are regulated by federal, state and local agencies Sanitary sewer
system disposal regulations are developed by the governing Publicly Owned
Treatment Works (POTW) in compliance with the Federal Clean Water Act
Disposal requirements can vary with each POTW and cover a wide range of
physical and chemical properties including flammabihty, pH, pollutant control,
health hazards, viscosity, etc. Solutions with acid pH values, such as pH 2.5
which occur with Renalin®, meet the classification of a hazardous waste under
the Resource, Conservation and Recovery Act (RCRA, 40 C.F.R. § 261 .22) and
hence must be neutralized for routine disposal into a sanitary sewer system
EXAMPLE 2
Sporicidal activity of hydrogen peroxide (H2O2) solutions at various pH values.
The Association of Official Analytical Chemists (AOAC) test for
Sporicidal Activity of Disinfectants (AOAC Official Methods of Analysis, 1 5th
edition, 1 995) was employed to evaluate the sporicidal activity of H2O2
solutions. No organic soil load was used. Clean porcelain penicylinders
(outside diameter (O.D.) 8 mm ± 1 , inside diameter (I .D ) 6 mm ± 1 , length
1 0 mm ± 1 ) were sterilized for 2 h at 1 80 ° C Porcelain penicylmder carriers
were immersed for 1 5 min in a 72 ± 4 h old broth culture containing spores
of Bacillus subtilis (ATCC #1 9659) in soil extract-nutrient medium at a ratio of
1 carrier per ml broth culture and placed into a glass petπ dish matted with two
layers of filter paper. The contaminated carriers were transferred in a vacuum
desiccator containing CaCI2 and the vacuum was drawn to 69 cm (27 inches)
Hg for 20 min. The contaminated carriers were dried in a desiccator for 42 h.
Solutions of 7.5 %w/v H202 at various pH values were utilized for
this test. The solutions were prepared from a 35.1 %w/w solution of H20
(Aldrich Chemical Company) that was diluted with the required amount of
deionized water. The solution at pH 8.5 additionally contained a small
concentration (0.04 mg/ml) of protein load for the purpose of simulating the
protein deposit, and which did not interfere with sporicidal activity. Deionized
water was tested as a negative control.
Test solutions of H202 were placed into duplicate test tubes and
equilibrated to 20 ° C in a water bath for 1 0 min. Five of the contaminated
porcelain penicylinder carriers were placed into each of two tubes containing
the individual test solutions. After a defined period of contact, representing
the sterilization time, the carriers were individually removed by hook needle to
a subculture medium of Fluid Thioglycollate Medium USP. After completion of
subcultures, each carrier was re-transferred to a fresh tube of the same
subculture medium. Subculture tubes were incubated for 21 days at 37 ° C ±
2 °C. After 21 days the tubes were examined for growth as determined by
turbidity. Tubes demonstrating growth were subcultured onto agar medium for
confirmation of the test organism. Tubes demonstrating no growth of the test
organisms were heat shocked for 20 mm at 80°C ± 1 °C and remcubated for
72 h ± 4 h at 35 °C-37°C Tubes without growth following heat shock were
considered negative The results are shown in Table 1
Table 1 . Sporicidal activity of 7.5%w/v H2O2 solutions of various pH values at exposure time of 4 h with 10 carriers.
As shown in Table 1 , there was no change in sporicidal activity
with a change in pH of the H202 solution at an exposure time of 4 h This
indicated that H202 solutions having a higher pH maintain their sporicidal
activity without significant loss
In contrast to these results, it has been reported (J Applied
Bacteriology, 1 983, 54, 41 7-423) that H202 at 0 88 m/l (3 0%w ) lost some
sporicidal rate of kill activity at pH 8.0 versus at pH 5 0, while remaining
sporicidal at both pH 5 and pH 8, killing all Bacillus subtilis spores in 6 h at
pH 8.0, and in 3 h at pH 5.0 Similarly, the report found that PAA at
0.01 3 m/l (0.1 0%w/v) lost some sporicidal rate of kill activity at pH 8 0 versus
pH 5.0, while remaining sporicidal at both pH 5 and pH 8, killing all Bacillus
subtilis spores in > 6 h at pH 8 0 and in 1 h at pH 5 0 PAA at pH 2 6
exhibited a similar rate of kill at pH 5.0. However PAA at between 0 1 3-
0.38 m/l (0.99-2.89%w ") did not show a reduction in sporicidal rate of kill
activity between pH 8.0 and pH 5.0 Together, these sporicidal activity tests
indicate that the compositions of the invention using peracids alone or in
combination with H202 can achieve adequate sporicidal activity, well within the
20 h sporicidal kill FDA standard for high-level disinfectants and sterilants.
EXAMPLE 3
Tuberculocidal effectiveness of hydrogen peroxide (H2O2) without peracetic acid.
Several hydrogen peroxide solutions were formulated without any
additional preservatives or other antimicrobial agents and without surfactants,
chelators or organic buffers, as shown in Table 2.
Table 2
* FMC Corporation
Their tuberculocidal effectiveness was evaluated with a quantitative
tuberculocidal test (suspension test) designed to determine the tuberculocidal
effectiveness of a disinfectant/sterilant following the EPA Guidelines for the
Quantitative Tuberculocidal Procedure.
The specific test conditions were as follows. Two sterile glass
test tubes each containing 1 8 ml of a particular test formula at 25 ° C were
each inoculated with 2 ml of a standardized suspension of Mycobacterium
bovis (ATCC 35743) containing 1 .0 x 1 06 Colony Forming Units (CFU)/ml. At
contact times of 10 min (1 56B only) and 30 mm (1 56A, B, and C), ahquots of
the test suspension were removed and diluted in a neutralizer solution
containing catalase to inactivate the H202. Serial tenfold dilutions of the
neutralized suspension were then made in sterile saline. Each dilution was
filtered through a 47 mm membrane filter with 0.45 micron porosity with the
aid of a vacuum. Each filter was removed and aseptically placed on the
surface of a Middlebrook 7H 1 1 agar plate. The plates were incubated at 37 °C
± 2 °C for 1 5-25 days. Colonies were counted and the average CFU/ml was
calculated for each contact time. The log10 reduction was determined by
subtracting the survivors at each contact time from the log10 CFU/ml at time
zero (t0) . The results are presented in the following table.
Table 3. Tuberculocidal activity test results
The results of these tests indicate that H202 solutions alone up to
6-8%w/" or more, without any additional preservative or other antimicrobial
agents and without surfactants, chelators or organic buffers, cannot achieve
in a short time interval one of the fundamental antimicrobial efficacy
requirements for a high-level disinfectant, namely tuberculocidal activity.
Example 4 demonstrates tuberculocidal activity in a short time interval .
Example 4. Tuberculocidal tests of hydrogen peroxide (H2O2) plus peracetic acid (PAA) solutions.
Several H202 plus PAA solutions were formulated and their
tuberculocidal effectiveness was evaluated with a quantitative tuberculocidal
test (suspension test), as in the previous example.
Table 4. Formulations (all concentrations expressed as %w/v
Table 5. Tuberculocidal activity test results.
The results in Table 5 show that all of the solutions representing
compositions of the present invention tested at all time intervals exhibited
maximum tuberculocidal activity, with a 6 log10 reduction, or total kill, of the
entire initial mnoculum challenge Benzotπazole does not contribute to the
tuberculocidal activity of the solution The solution pH 3 is also not considered
to be significant, as the work of Baldry and others, together with the above
example, indicate that solutions comprising peracetic acid at a higher pH can
achieve adequate tuberculocidal activity. Specifically, even the lowest
concentration of peracetic acid tested at pH 3 in Table 5, 0.1 0%w/", achieved a
6 log10 reduction in 1 0 mm The work of Baldry indicated a factor of 6 times
increase in sporicidal kill time for peracetic acid at pH 8 0 versus 5.0, and a
possible factor of 2 times increase in kill time at pH 2 6 versus pH 5 0 Overall
the sporicidal activity of peracetic acid as measured by Baldry did not decrease
by more than a factor of 3- 1 0 times between pH 2 6 and pH 8 0 In this
context, the antimicrobial activity of undissociated peracetic acid is known to
exceed that of the anion formed upon dissociation, and the pKa of peracetic acid
is 8.2. Thus, the observed changes in antimicrobial activity of peracetic acid are
consistent with its pKa. Tuberculocidal activity is expected to exhibit the same
pH relationship, given the indiscriminate mode of action of peracetic acid on
cells. Therefore, even a 1 0 times increase in the 1 0 min tuberculocidal kill time
in the example above results in a 1 00 min required contact time at a higher pH .
The total allowed time for high-level disinfection and sterilization, established by
FDA guidelines, is a maximum of 20 h. Kidney dialyzers are disinfected and
sterilized between dialysis treatments, which are typically every other day and
provide a 48 h period between dialysis treatments with which to disinfect and
sterilize the dialyzer. Thus the foregoing results and conclusions are reasonable.
They are also supported by the known neutral pH sterilization capacity of the
0.2%""" peracetic acid solution used in the Steris® System l ™ Sterile Processing
System for endoscope reprocessing (Steris Corp., Mentor, OH) . This system
achieves sterilization at a neutral pH in 1 2 min at 43-48 ° C.
Example 5. Proteolysis tests of hydrogen peroxide solutions.
Tests were performed to evaluate the proteolytic effects of
hydrogen peroxide solutions at various pH values. Azocasein, comprised of the
milk protein casein with covalently-bound azo dye molecules linked to amino acid
side chains, was exposed to various hydrogen peroxide solutions to determine
if the solutions could lyse the casein molecule into small peptide and amino acid
fragments, which remain soluble upon exposing the entire solution to
trichloracetic acid (TCA) . The soluble fragments absorb light at 390 nm due to
the presence of the azo dye. Intact azocasein is insoluble in TCA. Therefore,
the method can detect proteolysis and provides a quantitative measure of protein
removal (e.g ., cleaning) capacity. This is also a standard method to detect
enzymatic proteolysis.
Azocasein powder was added to 7.5 %w/" H202 and 1 0 mM
potassium phosphate buffer (K2HP04) or 1 0 mM potassium phosphate buffer
solutions at various pH values (adjusted with 1 N NaOH) to yield an azocasein
concentration of 0.4%""" After 30 m at 23 °C, 5 ml of the azocasein solution
was mixed with 5 ml of 1 0%w " TCA solution The precipitates of the solutions
were removed after 1 5 mm by filtration, and the absorbance of the supernatant
was measured at 390 nm (Abs390) using a UV-visible spectrophotometer The
absorbance of the azocasein solution (0 4% ") containing phosphate buffer
alone, without hydrogen peroxide, was measured as a control While it can be
dissolved in all of the above solutions at pH 8 and pH 5 within a few minutes
with stirring, azocasein virtually does not dissolve at pH 2 The results are
presented in Table 6.
Table 6. Absorbance at 30 min contact time of hydrolyzed azocasein solution at 390 nm containing 7.5%w/ H2O2 and/or potassium phosphate buffer at various pH values at 23°C.
Although azocasein can be dissolved in the solutions at pH 8 and
5, the absorbance of the solutions as shown in Table 5 remained nearly as low
as that of pH 2 solutions. At all pH values, the azocasein hydrolysis rate is
higher in 7.5 %w/" H202 solutions than in the corresponding phosphate buffer
solutions, although not significantly so, indicating the lack of substantial protein
hydrolysis/cleaning efficiency
Example 6. Proteolytic activity in H2O2 solutions over time
The initial 7.5 %w v H202/potassιum phosphate buffer solutions were
prepared with standard procedures as in Example 5. One half hour prior to the
time points listed in Table 7, azocasein powder was added to aliquots of the
solutions in an amount of 0.4% of the total solution weight The solution
absorbance was measured following the same procedures as in Example 5
except the supernatant of the azocasein/TCA solutions was diluted 6-7 fold to
avoid saturation of the UV spectrophotometer. The results are presented in
Table 7.
Table 7. (Dilution Factor) x (Abs390nm) of hydrolyzed azocasein solution containing 7.5%w/v H2O2 and 10 mM potassium phosphate buffer at pH 8.0 and pH 5.0 at 23°C.
These results show that the cumulative azocasein hydrolysis
increased over 8 h to a significant level at both pH conditions, well beyond that
of phosphate buffer alone (which produces an absorbance about one half that
of the H202-containing solution at pH 8.0, not shown) and that hydrolysis was
greater at pH 8.0 than pH 5.0.
Example 7. Proteolytic activity in H2O2 and PAA solutions at various pH values.
Solutions of 1 .08%""" H2O2/0.2%w/" PAA were prepared by diluting
a stock of 26.5%w/w H202 and 4.9%w/w PAA (Degussa, density 1 .1 2 g/ml) . The
pH values were adjusted with 1 N NaOH in the presence of 1 0 mM K2HP04
solution. Azocasein powder was added to the solutions to yield an azocasein
concentration of 0.4%""". After dissolved azocasein stood for 30 minutes at
23 °C, 5 ml of the azocasein solution was mixed with 5 ml of 1 0% TCA
solution. The precipitates of the solutions were removed after 1 5 min by
filtration and Abs390 of the supernatant was measured using a UV-visible
spectrophotometer. The absorbance of an azocasein solution (0.4%w w)
containing phosphate buffer alone, without H202 and PAA, was measured as a
control. Additional solutions were prepared containing Alcalase- 2.5 L, a seπne-
protease enzyme (Novo Nordisk, Copenhagen, Denmark) to serve as positive
controls, as it has been discovered that such enzymes exhibit equal or greater
activity in these H202/PAA solutions as in phosphate buffer solutions as
disclosed in copending United States Patent Application Serial No. 09/1 83, 1 86,
Simultaneous Cleaning and Disinfecting Compositions and Methods invented by
Huth et al . The H202/PAA solutions were mixed with Alcalase® 2.5 L in a
volume ratio of 1 280: 1 , yielding a solution with a nominal enzyme activity of
0.0021 Anson units/ml.
Table 8. Abs390 of hydrolyzed azocasein solution containing 1 .08%w/ H2O2, 0.2%w/v PAA and potassium phosphate versus potassium phosphate alone, with and without Alcalase® at various pH values at 23°C.
Comparing absorbance data in Tables 6 and 8, one can see that the
mixed solutions of 1 .08% H2O2/0.2% PAA without enzyme can hydrolyze 4-5
times more azocasein than 7.5 %w/" H202 solution without enzyme in the same
time period at pH 5 and pH 8. Furthermore, although 0.4% azocasein was
poorly dissolved in the H202/PAA solutions at pH 2, the partially dissolved
azocasein molecules are hydrolyzed about 2 times faster than in the 7.5 % H202
solutions, without enzyme. Table 8 shows that the absorbance of hydrolyzed
azocasein solutions in the mixed solutions of H202/PAA and enzyme is higher
than that in the corresponding phosphate buffer solutions containing enzyme at
pH 5 and pH 8 or the corresponding solutions that do not contain enzyme. It is
surprising, however, that the H202/ PAA solutions that do not contain enzyme
produce between 1 2-1 4% of the hydrolysis activity of the corresponding
H2O2/PAA enzyme solutions at pH 8.0, and between 28-31 % of the hydrolysis
activity of the corresponding H202/PAA enzyme solutions at pH 5.0. The pH
difference is largely due to the pH activity range for Alcalase® 2.5L, which has
an optimal activity range between about pH 6-1 2, with a peak cleaning activity
between about pH 8-8.5.
Example 8. Proteolytic activity in H2O2/PAA solutions at pH 5.6 over time
A 1 08%""" H2O2/0.2%w " PAA solution containing 1 0 mM
potassium phosphate buffer was adjusted to pH 5 6 with 1 N NaOH The
solution was then mixed with azocasein powder to result in 2%w/w azocasein at
23 °C. Three ml of the above solution was removed at each time point and was
mixed with 3 ml of 1 0% TCA solution The precipitates of the solutions were
removed after 1 5 mm by filtration, and Abs3go of the supernatant was measured
using a UV-visible spectrophotometer Azocasem/TCA solutions were diluted 6
7 fold to avoid saturation of the UV spectrophotometer
Table 9. Abs390 of hydrolyzed azocasein solution containing 1 .08%w H2O2/2.0% PAA and 10 mM K2HPO4 at pH 5.6 at 23°C.
The results show that substantial hydrolysis of azocasein occurs in
this system at both time intervals. Since the azocasein concentration was five
times that in the preceding Example 7 (i.e., 2% versus 0.4%), this difference in
protein substrate (azocasein) concentration possibly accounts for the large
difference in results seen at 30 m (e.g ., Abs390 0 937 in this example versus
Abs390 0.31 81 in Example 7 at 30 mm)
Example 1 shows that 7.5 %""" H202 at pH 8.5 can restore about
50% of clotted dialyzer fiber bundle volume in 4 h However, Example 3 shows
that 6.0%w/v H202 at pH 5.0-8.5 is not tuberculocidal between 1 0-30 mm
Moreover, the data indicate that a 6.0 log10 reduction, the standard for
tuberculocidal activity for a high-level disinfectant within the regimen soak time
would not be achieved by a 6.0%w v or even 7.5 %w/" H202 solution in under
about 5 h, since only a 0.63 log10 reduction was achieved in 30 min by the
6.0%""" H2O2 solution. Examples 4-8 show that mixtures of H202 and PAA can
achieve high-level disinfection standards and 4-5 times greater hydrolysis of
protein than 7.5%w/" H202 alone. This indicates that such solutions can achieve
greater restoration of clotted kidney dialyzer fiber bundle volume than that
observed with the 7.5%w/v H202 solution and also adequately disinfect and
sterilize dialyzers in a reasonable time frame.
It should be understood that the embodiments of the present
invention shown and described in the specification are only preferred
embodiments of the inventors who are skilled in the art and are not limiting in
any way. Therefore, various changes, modifications or alterations to these
embodiments may be made or resorted to without departing from the spirit of
the invention and the scope of the following claims.
What is claimed is: