Method for purification of albumin

This application is a national phase entry application of Patent Cooperation Treaty Application No. PCT/CA2018/050321, filed Mar. 16, 2018 (which designates the U.S.), which claims the benefit of U.S. Provisional Application No. 62/472,007, filed Mar. 16, 2017, which is are incorporated herein by reference in #s their entirety.

The present disclosure relates to a novel method for the purification of albumin from plasma. The method involves contacting the plasma with sodium caprylate (NaCP) at an amount dependent on total protein concentration and the ratio of NaCP to total protein in the plasma, pasteurizing the plasma at a near neutral pH range, and separating the albumin from non-albumin-phase. The said method provides for a high yield and purity albumin solution.

Human and animal blood is a source of molecules that possess therapeutic properties. Many of these molecules are proteins that may be found in plasma or serum. These proteins have been the target for specific isolation with the aim of purifying and standardising the molecules for use as human therapeutic agents. For examples, albumin, immunoglobulin G, Factor VIII and alpha-1-proteinase inhibitor are all available as isolated therapeutic products.

One of the conventionally used methods for the fractionation of blood plasma or blood serum proteins has been described in U.S. Pat. No. 2,390,074 which discloses a method for the fractionation of plasma or serum proteins in large-scale by utilizing ethanol precipitation and regulating temperature, pH, ionic strength and time to control precipitation of certain proteins from human plasma. The fractionation method involves the stepwise addition of ethanol to the plasma raw material in order to obtain several fractions and corresponding supernatants comprising different enriched protein solutions.

Ethanol precipitation method suffers the drawback that some proteins tend to denature in the presence of ethanol resulting in decreased yield of the protein to be isolated and contamination with aggregates that need to be removed before an acceptable therapeutic product can be obtained. Furthermore, precipitated proteins require resolubilization for further processing which leads to significant levels of insoluble protein and lipid material that hinder the purification of the final product, resulting in further reduction in yield.

Furthermore, human serum albumin (HSA) in particular has been purified from plasma by various fractionation methods. Albumin is the most abundant protein in human blood plasma, constituting up to 60% of serum protein. Therapeutic albumin is formulated by following a pharmaceutical guideline. Under regulatory guideline, the formulation contains N-acetyl tryptophan and caprylate followed by pasteurization at 60±0.5° C. for 10.5±0.5 hour for the purpose of viral inactivation. The amount of both reagents as stabilizer is determined based on the amount of HSA, which is set at 0.08±0.016 mmol/g-HSA protein.

The integrity of HSA is maintained during the pasteurization by the presence of the caprylate/tryptophan regimen. The caprylate ion is believed to decrease the rate of reversible partial unfolding by binding to HSA, thereby inhibiting one step in the pathway which leads to irreversible precipitation. Faroongsarng and Kongprasertkit (2014) observed that the denaturing temperature of HSA increases from 67.23° C. to 72.85° C. in the presence of 0.08 mmol NaCP per gram of HSA.

The basic concept and prior art in the field of albumin isolation have been described, for examples, in U.S. Pat. Nos. 2,705,230, 3,992,367, 4,156,681, 5,118,794, 6,022,954, and 6,504,011. These documents are incorporated herein by reference.

U.S. Pat. No. 6,022,954 relates to a process for preparing purified albumin from a human or animal physiological solution, such as plasma or a plasma fraction. This document teaches a process of delipidation using an anionic detergent and two chromatographic separation stages using ion-exchange resin. The delipidation treatment consists of contact of the albumin solution from the first chromatography stage for approximately 1 hour at 60° C. with a detergent and in the presence of low amount of NaCP as a stabilizer. The pasteurization treatment consists of heating the solution at over 60° C. for at least 10 hours.

U.S. Pat. No. 5,118,794 relates to a process for stabilizing human albumin during heat treatment in a container which involves the addition of a surfactant agent. This document discloses a process that involves pasteurizing the solution with low amount of NaCP, at an amount of 0.08-0.64 mmol NaCP per gram of albumin, at pH 7.0. This document further discloses final pasteurization of the product at 60° C. for 10 hours.

In U.S. Pat. No. 3,992,367, the process of albumin purification involves a heating step where the solution consists of NaCP in weight between 15% and 30% at a pH between 4.8 and 5.25. In U.S. Pat. No. 2,705,230, albumin isolation involves pasteurization heat treatment that was carried out at pH 8 using 50 and 500 mM NaCP.

U.S. Pat. No. 4,156,681 relates to a method for abstracting serum albumin from blood which includes the steps of separating the blood plasma from the solid constituents of the blood, isolating the dissolved non-albumin constituents from plasma, and adding an albumin-stabilizer and treating such fluid with a lower aliphatic alcohol. The method involves adding low amount of NaCP to a large volume of plasma for albumin purification and carrying out the heat treatment at a pH between 4.5 and 7.5.

U.S. Pat. No. 6,504,011 relates to a process for purifying recombinantly produced serum albumin by incubation with an anion exchange adsorbent, followed by affinity chromatography employing a hydrophobic solid phase and using a water soluble lipid anion as desorbens in the aqueous phase. This document teaches albumin purification by chromatography without a pasteurization stage and the use of NaCP in an elution buffer for the recovery of albumin.

Previous reports did not lend themselves to understand the complex relationship between total protein concentration and the ratio of NaCP to total protein in the plasma during pasteurization for the purification of albumin. The high yield and purity achieved by the presently disclosed method provides an advantage in maximizing the quality and quantity of recovered albumin.

The present disclosure describes an improved method for the purification of albumin from plasma.

Accordingly, the present disclosure provides a method for purifying albumin from plasma comprising:

(a) contacting the plasma with sodium caprylate (NaCP),

wherein the amount of NaCP is between 0.1 and 5 mmol NaCP per gram of total protein in the plasma;

(b) heating the plasma at a temperature between 60 and 70° C.; and

(c) separating the albumin from the non-albumin phase.

In one embodiment, the plasma is concentrated prior to step (a) to a total protein concentration between 20 and 150 g/L, preferably between 45 and 65 g/L.

In another embodiment, the amount of NaCP is between 0.2 and 3, optionally between 0.5 and 2, optionally between 0.8 and 1.3 mmol NaCP per gram of total protein.

In another embodiment, the NaCP added is in a concentration between 20 and 140 mM, optionally between 40 and 100 mM, optionally between 50 and 90 mM, optionally between 60 and 85 mM.

In another embodiment, the heating of the plasma in step (b) is carried out at a pH between 6.0 and 8.0, optionally between 6.5 and 7.5, optionally between 6.7 and 7.3, optionally between 6.7 and 6.9.

In another embodiment, the conductivity of the plasma is less than 9 mS/cm, optionally less than 5 mS/cm.

In another embodiment, the heating of the plasma is carried out at a temperature between 60 and 70° C., optionally between 62 and 65° C., for a period between 0.5 and 24 hours, optionally between 2 and 12 hours, optionally between 3 and 12 hours, optionally between 4 and 6.5 hours.

In another embodiment, the separation of the albumin from the non-albumin phase in step (c) comprises precipitation of non-albumin protein and lipid impurities.

In another embodiment, the separation of the albumin from the non-albumin phase comprises cooling the plasma to below 30° C.

In another embodiment, the plasma is filtered in step (c), optionally through a Pall Seitz K700P depth filter in the presence of Harborlite filter aid (2% w/v H900, 2% w/v H1900), tangential flow filtration (TFF) with a cassette, or hollow fibre TFF, preferably hollow fibre TFF.

In another embodiment, the separation of the albumin from the non-albumin phase comprises adjusting the pH of the plasma to between 4.8 and 5.4, optionally between 5.1 and 5.3.

In another embodiment, the adjustment of the pH of the plasma is carried out by the addition of acid.

In another embodiment, the method for purification of albumin can be linearly scalable.

In addition, the present disclosure includes a recovered albumin solution with an albumin content of at least 90% (w/w) of protein, optionally at least 92% (w/w) of protein, optionally at least 94% (w/w) of protein, optionally at least 96% (w/w) of protein, optionally at least 98% (w/w) of protein, optionally at least 99% (w/w) of protein.

Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific Examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The disclosure will now be described in relation to the drawings in which:

FIG. 1 shows an illustration of purities and recoveries of HSA after each purification step.

FIG. 2 shows size exclusion chromatography chromatograms of the heated samples.

FIG. 3 shows a non-reduced SDS-PAGE gel.

FIG. 4 shows HSA purity as a function of pasteurization time.

FIG. 5 shows size exclusion chromatography chromatograms of the PAS2 samples at each pasteurization time.

FIG. 6 shows a non-reduced SDS-PAGE gel.

FIG. 7 shows an illustration of purities and recoveries of HSA after each purification step.

FIG. 8 shows size exclusion chromatography chromatograms of the PAS1 samples after each treatment step.

FIG. 9 shows non-reduced and reduced SDS-PAGE gels.

FIG. 10 shows purities of HSA after each treatment.

FIG. 11 shows size exclusion chromatography chromatograms overlay view.

FIG. 12 shows an illustration of purities and recoveries of HSA after each purification step.

FIG. 13 shows size exclusion chromatography chromatograms of the samples after each purification step.

FIG. 14 shows a SDS-PAGE gel, 8% Invitrogen Gel (200 V×22 minutes).

FIG. 15 shows a Western blotting for HSA.

FIG. 16 shows size exclusion chromatography chromatograms of the samples after each purification step.

FIG. 17 shows size exclusion chromatography chromatograms of the samples.

FIG. 18 shows a Western blot, 7.5% Invitrogen Gel (200 V×22 minutes) under a non-reduced condition.

FIG. 19 shows a SDS-PAGE gel, 7.5% Invitrogen Gel (200 V×22 minutes).

FIG. 20 shows size exclusion chromatography chromatograms of the samples from different conductivities.

FIG. 21 shows size exclusion chromatography chromatograms of the samples pH adjusted with different acids.

FIG. 22 shows purity and recovery profile of the samples from different conductivities.

FIG. 23 shows purity and dimer formation profile of the samples from different acids used in pH adjustment.

FIG. 24 shows a SGS-PAGE gel, reduced.

FIG. 25 shows purity, dimer and aggregate formation profile of the samples from different samples including PAS3 samples.

FIG. 26 shows a SDS-PAGE, 7.5% gel and under reduced condition.

FIG. 27 shows purity, dimer and aggregate formation profile of the samples.

FIG. 28 shows non-reduced and reduced gel of the samples.

FIG. 29 shows SEC HPLC overlay views.

FIG. 30 shows SEC HPLC overlay views.

FIG. 31 shows purity of recovered HSA on a SDS-PAGE gel.

FIG. 32 shows a contour plot illustrating the effect of total protein concentration ([TP]) and the ratio of NaCP to total protein during pasteurization on HSA recovery.

FIG. 33 shows a contour plot illustrating the effect of total protein concentration ([TP]) and the ratio of NaCP to total protein during pasteurization on HSA purity.

FIG. 34 shows a Pareto chart of standardized effects for HSA recovery as relating to (A) total protein concentration ([TP]) and (B) the ratio of NaCP to total protein in the material during pasteurization.

FIG. 35 shows a Pareto chart of standardized effects for HSA purity as relating to (A) total protein concentration ([TP]) and (B) the ratio of NaCP to total protein in the material during pasteurization.

FIG. 36 shows an overlay contour plot of HSA recovery and purity indicating the optimal operating region for the parameters of focus; non-feasible region indicated in grey.

HSA was purified from plasma and partially fractionated plasma by using NaCP at a concentration dependent on total protein concentration and the amount of NaCP per gram of total protein in the plasma, at near neutral pH, followed by separation of albumin and non-albumin phase by cooling and decreasing the pH of the pasteurized plasma. The purification process was carried out without the use of ethanol or extensive processing steps. The final recovered HSA is of high yield at exceptional purity.

Accordingly, the present disclosure provides a method for purifying albumin from plasma comprising:

(a) contacting the plasma with sodium caprylate (NaCP),

wherein the amount of NaCP is between 0.1 and 5 mmol NaCP per gram of total protein in the plasma;

(b) heating the plasma at a temperature between 60 and 70° C.; and

(c) separating the albumin from the non-albumin phase.

Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.

As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

In the absence of any indication to the contrary, reference made to a “%” content throughout this specification is to be taken as meaning % w/v (weight/volume).

As used herein, the term “purified” and its derivatives is meant purified from other common components present in plasma, fractionated plasma, cryo poor plasma, plasma intermediate, solution of plasma proteins, or mixtures thereof. For example, purified albumin is purified away from the other proteins, nucleic acids, lipids and small metabolites present in plasma fractionated plasma, cryo poor plasma, plasma intermediate, solution of plasma proteins, or mixtures thereof. A purified albumin is at least 60% pure by weight, optionally at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or even 100% pure, from other proteins, nucleic acids, lipids, or small metabolites present in plasma, partially fractionated plasma, cryo poor plasma, plasma intermediate, solution of plasma proteins, or mixtures thereof.

As used herein, the term “plasma” and its derivatives includes partially fractionated plasma, cryo poor plasma, plasma intermediate, solution of plasma proteins, or mixtures thereof. It is readily apparent to the person skilled in the art how to obtain partially fractionated plasma, cryo poor plasma, plasma intermediate, solution of plasma proteins, or mixtures thereof. For example, partially fractionated plasma may be prepared from filtration through Seitz® EK1 filter (0.45-0.65 μm).

The method of the disclosure may in principle be used for purifying any known albumin such as albumin derived from human, non-human primate, sheep, goat, bovine, donkey, canine, feline, rabbit, rodent, hamster, guinea pig and fowl.

The total protein in the plasma can be concentrated prior to the addition of the NaCP. The concentration can be done using techniques known in the art including the addition of water or any suitable buffer known to a person skilled in the art. The NaCP is preferably added to the plasma at a pH range of 6.6 to 7.2, most preferably at a pH range of 6.7 to 6.9.

It is important to regulate the total protein in the plasma. In particular, the total protein in the plasma should be in a concentration between 20 and 150 g/L, such as at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 g/L, and at most 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 g/L. In a specific embodiment, the total protein is in a concentration between 35 and 80 g/L, or 45 and 65 g/L.

Regulating the amount of NaCP used is an important aspect of the method. In particular, the amount of NaCP is between 0.1 and 5 mmol NaCP per gram of total protein in the plasma, such as at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.05, 1.15, 1.2, 1.3 or 1.4 mmol NaCP per gram of total protein in the plasma, and at most 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2, 2.5, 3, 3.5, 4 or 5 mmol NaCP per gram of total protein in the plasma. In a specific embodiment, the amount of NaCP is between 0.2 and 3, 0.5 and 2, or 0.8 and 1.3 mmol NaCP per gram of total protein in the plasma. It is readily apparent to the person skilled in the art that the values may be converted and expressed in terms of weight, molar amount, concentration including molarity, or a combination thereof.

In another embodiment, the NaCP is added in a concentration between 20 and 140 mM, such as at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, or 85 mM, and at most 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 mM. In a specific embodiment, the NaCP is in a concentration between 40 and 100 mM, 50 and 90 mM, or 60 and 85 mM.

After the NaCP is added the plasma is heat treated or pasteurized. The pasteurization or heating process of the plasma is carried out at a temperature of between 60 and 70° C., such as at least 60, 61, 62, 63, 64, 65, 66, or 67° C., and at most 64, 65, 66, 67, 68, 69, or 70° C. In a specific embodiment, the pasteurization or heating process of the plasma is carried out at a temperature between 60 and 65° C., or 62 and 65° C. It is readily apparent to the person skilled in the art how to regulate the temperature, for example, by using a pasteurizer.

The pasteurization or heating process of the plasma is preferably carried out at a pH of between 6.0 and 8.0, such as at least 6.0, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0 and at most 6.7, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.5 or 8.0. In a specific embodiment, the pasteurization or heating process of the plasma is carried out at a pH between 6.5 and 7.5, 6.7 and 7.3, or 6.7 and 6.9.

The conductivity of the plasma may also be an important factor in the process. In one embodiment, the conductivity is less than 9, 8, 7, 6, 5, 4, 3, 2 or 1 mS/cm. The conductivity of the plasma may be measured by an electrical conductivity (EC) meter.

The pasteurization or heating process of the plasma is preferably carried out for between 0.5 and 24 hours, such as at least 0.5, 1, 2, 3, 4, 5 or 6 hours, and at most 5, 6, 6.5, 7, 8, 9, 10, 11, 12, 16, 20 or 24 hours. In a specific embodiment, the pasteurization or heating process of the plasma is carried out for a period between 0.5 and 12 hours, 2 and 12 hours, 3 and 12 hours, or 4 and 6.5 hours.

In a specific embodiment, the pasteurization or heating process of the plasma is carried out at a temperature between 62 and 65° C., for a period between 4 and 6.5 hours.

In a specific embodiment, the pasteurization or heating process of the plasma is carried out at a pH between 6.7 and 6.9 at a temperature between 62 and 65° C. for a period of at least 4 hours, wherein amount of NaCP is between 0.8 and 1.3 mmol NaCP per gram of total protein, wherein the total protein is in a concentration between 45 and 65 g/L.

In step (c), the albumin is separated from the non-albumin proteins in the plasma. The separation of the albumin from the non-albumin phase involves precipitation of non-albumin protein and lipid impurities. In a specific embodiment, the separation of the albumin from the non-albumin phase involves cooling the pasteurized plasma to below 30° C.

In another embodiment, the separation of the albumin from the non-albumin phase involves adjusting the pH of the plasma to between 4.8 and 5.4, such as 4.8, 4.9, 5.0, 5.1, 5.2, 5.3 or 5.4. In a specific embodiment, the separation of the albumin from the non-albumin phase involves adjusting the pH to between 4.9 and 5.3, optionally between 5.1 and 5.3. It would be readily apparent to the person skilled in the art to employ filter aid for the separation step, including the addition of filter aid such as Celpure® P1000, Celpure® P300, or a mixture thereof to the pasteurized plasma prior to filtration (i.e. separation of albumin from non-albumin through filtration).

In an aspect, the separation of albumin from non-albumin can be carried out over a depth filter, tangential flow filtration (TFF) with a cassette, or hollow fibre TFF. Hollow fibre cross flow filters are open channel devices that are ideal for use with liquids that are turbid and can handle solids concentrations up to 40% (v/v). Hollow fibre filters are less sensitive to variability in levels of particles/colloids/cells, or liquids that are viscous or become viscous upon concentration. These filters are often chosen when product recovery from the filter is important (for example vaccine processes). Since hollow fibre filters have a smooth and unobstructed internal flow path, they are ideal for recovery of product from the filter. They are available in different flow path geometries (nominal fibre diameters from 0.5 to 1.75 mm inner diameter (ID) with nominal fibre length options of 30, 60 or 110 cm length). A person skilled in the art would appreciate that variances of hollow fibre TFF can alternatively be used for separating albumin from non-albumin.

Cassettes are most often used for concentration and diafiltration of “clear” low viscosity fluids. The use of screen type turbulence promoters, enhances de-polarization of solute at the membrane surface, increasing sample flux. Under the same process conditions, pore size and filter area, cassette may have up to 2× the flux of a hollow fibre filter. However, cassettes are not suitable for solutions containing particles, colloidal suspensions or have high viscosity, due to the “plugging”/pressure drop of the screen spacers.

In an embodiment, the separation of albumin from non-albumin comprises filtration or clarification over a depth filter, tangential flow filtration (TFF) with a cassette, or hollow fibre TFF, preferably hollow fibre TFF. In an embodiment, the separation of the albumin from the non-albumin phase by depth filter, TFF with a cassette, or hollow fibre TFF is carried out at pH between 4.8 and 5.4, optionally between 5.1 and 5.3. In another embodiment, the separation of albumin from non-albumin through filtration or clarification is carried out at feed flow between 1, 2, 3, 4, or 5, and 6, 7, 8, 9, or 10 L/min per m 2 , optionally between 1 and 10 L/min per m 2 , optionally between 2 and 10 L/min per m 2 , optionally between 3 and 10 L/min per m 2 , optionally between 8 and 9 L/min per m 2 . In another embodiment, the separation of albumin from non-albumin through filtration or clarification is carried out with between 1× and 1.2× initial volume reduction concentration, preferably 1.2×, and between 3 and 4 diavolumes, preferably 4 diavolumes. In an embodiment, the separation of albumin from non-albumin comprises filtration or clarification is carried out with hollow fibre TFF at pH between 4.8 and 5.4, optionally between 5.1 and 5.3, at feed flow between 3 and 10 L/min per m 2 , optionally between 8 and 9 L/min per m 2 . In a specific embodiment, the separation of albumin from non-albumin comprises filtration or clarification is carried out with hollow fibre TFF at pH between 4.8 and 5.4, optionally between 5.1 and 5.3, at feed flow between 3 and 10 L/min per m 2 , optionally between 8 and 9 L/min per m 2 , with initial volume reduction concentration between 1× and 1.2×, optionally 1.2×, and between 3 and 4 diavolumes, optionally 4 diavolumes.

The adjustment of the pH of the plasma may be carried out by the addition of acid, such as an organic and/or inorganic acid. In a specific embodiment, the organic acid is selected from citric acid, acetic acid and trifluoroacetic acid. In another specific embodiment, the organic acid is acetic acid. In a further embodiment, the inorganic acid is selected from hydrochloric acid, sulfuric acid and phosphoric acid.

The plasma can be filtered through a Pall Seitz K700P depth filter in the presence of Harborlite filter aid (2% w/v H900, 2% w/v H1900). It is readily apparent to the person skilled in the art how to assess the purity of the resulting albumin solution. For instance, size-exclusion chromatography-HPLC (SEC-HPLC) or capillary electrophoresis-SDS (CE-SDS) analysis may be carried out to assess the purity of albumin.

The method for purification of albumin recovers at least 40, 50, 60, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of albumin from the plasma.

In another embodiment, the method for purification of albumin can be linearly scalable.

In addition, the present disclosure includes an albumin solution with an albumin content of at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% (w/w) of protein obtained by the method disclosed herein.

The following non-limiting Examples are illustrative of the present disclosure:

Faroongsarng D, Kongprasertkit J. 2014. The role of caprylate ligand ion on the stabilization of human serum albumin. AAPS PharmSciTech. 14(2): 465-471.

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U.S. Pat. No. 4,156,681 A, May/1979, Schneider et al.

U.S. Pat. No. 5,118,794 A, June/1992, Grangeorge et al.

U.S. Pat. No. 6,022,954 A, February/2000, Dernis et al.

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