Ultrapure hypoallergenic solutions of sacrosidase

Abstract

The invention provides a method for treating a subject who lacks endogenous sucrase activity by orally administering a solution of sacrosidase in about 1:1 glycerol/water having an enzymatic activity of at least about 7500 IU/mL and a residual papain concentration of less than about 10 ng/ml.

Claims

1. A method for treating a subject who lacks endogenous sucrase activity, comprising orally administering an effective amount of a protein composition consisting essentially sacrosidase having a band volume ratio of sacrosidase to other proteins comprising papain of at least about 35:1 by SDS-PAGE of about 20 g of said composition, wherein the primary structure of the sacrosidase is a 513 amino acid polypeptide that is gycosylated.

2. The method of claim 1 wherein the band volume ratio is about 35-55:1.

3. A method of treating a subject who lacks endogenous sucrase activity comprising orally administering an effective amount of a protein composition consisting essentially of sacrosidase derived from Saccharomyces having residual papain in a concentration of less than about 10 ng/mL, papain, and having a band volume ratio of sacrosidase to other proteins comprising papain of at least about 35:1 by SDS-PAGE of about 20 g of said composition, wherein the primary structure of the sacrosidase is a 513 amino acid polypeptide that is glycosylated.

4. The method of claim 3 wherein the protein composition is administered in a solution.

5. The method of claim 4 wherein the sacrosidase has an enzyme activity of at least about 7500 IU/ml.

6. The method of claim 4 wherein a daily dose of the solution of about 2-10 mL per day does not induce an allergic reaction in a human patient afflicted with congenital sucrase-isomaltase deficiency.

7. The method of claim 3 wherein the sacrosidase contains less than about 3.0 ng/mL papain.

8. The method of claim 3 wherein the sacrosidase contains no detectable papain by an enzyme-linked immunosorbant assay having a lower limit of quantification of 3 ng/mL.

9. The method of claim 4 wherein the solution of sacrosidase contains no detectable papain by SDS-PAGE of up to about 15 g of said solution.

10. The method of claim 5 wherein the solution of sacrosidase has enzymatic activity of about 7500-10,000 IU/ml.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a flow diagram of the current sacrosidase drug substance manufacturing process.

(2) FIG. 2. Optimal Loaded SDS-PAGE gel Stained with Coomassie Blue. Sacrosidase bulk drug substance (FIG. 1) from two manufacturers (5 g protein). Lane 1: Low range MW marker 17, 28, 34, 40, 73 and 102 kD; Lane 2: Reference Standard C4546; Lane 3: STS-199NS; Lane 4: STS-220NS; Lane 5: STS-241NS; Lane 6: 125275; Lane 7: 125434; Lane 8: 125722; Lane 9: Reference Standard C3955; Lane 10: Broad range MW marker (21, 34, 45, 66, 97 kD and 200 kD).

(3) FIG. 3. Overloaded SDS-PAGE gel Stained with Coomassie. Sacrosidase bulk drug substance from two manufacturers (20 g protein). Lane 1: Low range MW marker; Lane 2: Reference Standard C4546; Lane 3: STS-199NS; Lane 4: STS-220NS; Lane 5: STS-241NS; Lane 6: 125275; Lane 7: 125434; Lane 8: 125722; Lane 9: Reference Standard 03955; Lane 10: Broad range MW marker.

(4) FIG. 4. Overloaded Native gel stained with Coomassie. Sacrosidase bulk drug substance from two manufacturers (15 g protein). Lane 1: Reference Standard C4546; Lane 2: STS-199NS; Lane 3: STS-220NS; Lane 4: STS-241NS; Lane 5: 125275; Lane 6: 125434; Lane 7: 125722; Lane 8: Reference Standard C3955.

(5) FIG. 5. The primary amino acid structure of Sacrosidase consists of 513 amino acids. Sacrosidase has the amino acid sequence shown below (SEQ ID NO:1).

DETAILED DESCRIPTION OF THE INVENTION

(6) This invention provides an ultrapure hypoallergic sacrosidase that meets the pharmaceutical standards for human prescription use, with reduced allergenic properties. The sacrosidase is also organoleptically improved, since aqueous solutions thereof are odorless. Solutions of sacrosidase that are less pure can exhibit unpleasant organoleptic properties, such as a yeasty smell, unappetizing color and other odors.

(7) Common Names: Invertase, Sacrosidase

(8) Trade Name: Sucraid (sacrosidase) oral solution (Drug Product)

(9) USAN: Sacrosidase

(10) Chemical: -D-fructofuranoside fructohydrolase

(11) Synonyms: -D-fructofuranosidase

(12) -D-fructofuranosidase -fructofuranosidase -fructofuranoside fructohydrolase -fructopyranosidase -fructosidase -invertase Fructosylinvertase Invertase Invertin Glucosucrase Saccharase Sucrase Sucrose hydrolase Exo--(2,6)-Fructofuraosidase Maxivert L 1000 Yeast Sucrase (YS)
CAS Number: 85897-35-4 9001-57-4
Summary of the Purification Process

(13) This invention provides a chromatography free process to produce ultrapure sacrosidase protein that meets pharmaceutical standards for human prescription use. Never before has a non-chromatographic method been developed to produce such ultra-high purity protein, particularly sacrosidase, overcoming prior shortcomings due to inadequate purity. A non-chromatographic purification process engenders significant cost and time savings during manufacturing. Provided herein is study drug characterization, purity evidence, and finally clinical proof of the improved unique hypoallergenic formulation.

(14) The steps of a preferred embodiment of the present sacrosidase isolation and purification process are summarized in FIG. 1. 1. Digest yeast saccharomyces cerevisiae with papain in an aqueous buffered solution at a pH of about 7.0 and at a temperature of about 30-34 C. 2. The papain digestion cleaves all proteins into small molecular weight fragments that can later be effectively diafiltered away from the very large, papain resistant, sacrosidase enzyme, which in solution has an apparent native solution molecular weight of 500,000+ grains per mole (Daltons). Sacrosidase having the extensive glycosylation produced in yeast is resistant to papain digestion. 3. Use of diatomaceous earth with two different pore and/or particle sizes is preferred to remove most of the insoluble components, and likely other soluble and insoluble impurities. At the chosen pH, ionic strength, and concentrations, a large increase in purity occurs using these two steps in series that also prepares the resulting particulate free solution for diafiltration. Removal of insoluble components also prevents the dialysis membrane filters in the ultrafiltration unit from clogging. This increases the processing speed. 4. Diafiltration using an experimentally determined optimal pore size filter to balance purity with yield. The present process employs a minimum 40 kiloDalton cutoff filter to achieve an ultrapure hypoallergenic odorless solution of sacrosidase. The solution has a sacrosidase activity of at least about 22,000 IU/mL, preferably at least about 17,000 IU/mL. Larger pore filters give equivalent purity, but at the cost of yield. Smaller pore size filters do not provide sufficient final purity. Also experimentally determined was the number of batch volumes against which to diafilter: only 4+ diafiltration volumes allows for purity adequate to meet the requirements for a regulated bulk drug substance for human pharmaceutical use. 5. Final formulation of the drug substance with glycerol, preferably employs sterile filtration to ensure lot bioburden that meets FDA standards. The drug substance is stored in drums at about 18 C. to 25 C. for long term storage, until processed/concentration adjusted/packaged to yield the Sucraid drug product. At this point, the sacrosidase activity is at least about 10,000 IU/mL.

(15) The invention will be further described by reference to the following detailed examples, wherein the terms SDS-PAGE and RASA as used herein are defined with reference to the following techniques.

(16) Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis

(17) Samples of the sacrosidase bulk drug substance containing predetermined amounts of total protein, were analyzed by Charles Rivers Laboratory, Malvern, Pa. and the assays were validated by Tekagen, Inc., Malvern, Pa.

(18) Reducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is used to separate mixtures of polypeptides (in an electric field) into distinct bands. Mobility under these conditions is primarily determined by molecular size. Staining with Colloidal Coomassie Blue allows for visual and/or densitometric analysis of the resolved bands. This method is linear for loads from 0.25 g to 15 g with a lower limit of detection of 0.1 g per band, e.g., the limits of detection and quantification of sacrosidase were determined to be 0.1 g. The measured sacrosidase band area correlated linearly with the protein load.

(19) In the assays herein below, the sacrosidase-containing samples described were reduced with 2-mercaptoethanol in SDS sample buffer. Samples were adjusted to about 1.0 mg/mL with water and treated with one volume of the mercaptoethanol in SDS buffer at 100 C. for 3 min. to denature/reduce the protein. Samples were run in an XCell SureLock Mini-Cell (Invitrogen) using Novex 4.Math.20% Tris-Glycine Gel (Invitrogen) using an SDS running buffer for 90 min. at 120 volts. Gels were stained with Coomassie Blue and washed in water. Gels were scanned using a Personal Densitometer SI (Molecular Dynamics).

(20) Papain Quantification by Enzyme-Linked Immunosorbent Assay (ELISA)

(21) Samples of the sacrosidase bulk drug substance were dialyzed against PBS and any papain was inhibited with E-64. The sample was assayed for papain content by Warnex Laboratories, Quebec, Canada, using a quantitative sandwich-type ELISA. A polyclonal capture antibody (rabbit) specific to papain is coated in each well of a microplate. When present in the samples, papain binds to the capture antibody. A second anti-papain antibody (goat) conjugated with horse-radish peroxidase (HRP) is added which binds to any captured papain. After washing, the O-phenlenediamine substrate is added with 0.03% H.sub.2O.sub.2 to produce an enzymatic color reaction with HRP. The reaction is stopped with a solution of sulfuric acid (stopping solution). The color intensity formed by the enzymatic reaction is measured by spectrophotometry.

(22) The quantity of papain in the samples was calculated based on a standard curve, where the average OD values of each of the standards are on the Y-axis and the corresponding concentration of papain is on the X-axis. To determine the final concentration of papain in the samples the concentration obtained from the standard curve is multiplied by the dilution factor. The quantity of papain is calculated automatically by the Gen5 software. The lower limit of quantification of papain is 3.13 ng/mL. The lower limit detection of papain is 1.56 ng/mL.

Example 1: Manufacturing Process for Ultrapure Sacrosidase

(23) Deionized water (1700 kg) is charged to a 1000 gallon reactor and heated to 322 C. at which time Saccharomyces cerevisiae yeast (360 kg) is added and the slurry is mixed for 20-30 min. The pH is checked and if it is found to be below 6.9, the pH is adjusted using a 25% sodium hydroxide solution until the pH is 7.00.1.

(24) Papain (3.4 kg) 100 TU/mg is added followed by hydrogen peroxide (35%, 2.8 kg). The slurry is stirred at 322 C. for the next 17.5 to 20 h; after every 3.5-4 h the pH is adjusted to 7.00.1 and hydrogen peroxide (34%, 2.8 kg each portion, 11.2 kg total) is added.

(25) Once the 17.5-20 hours of mixing is complete the mixture is cooled to between 22 C. and 26 C. at which point 25 kg of 85% phosphoric acid is added over a 20-30 min period. The pH of the slurry is checked and the pH is further adjusted to 4.00.1 by addition of 85% phosphoric acid.

(26) Diatomaceous earth (Celpure S1000, 210 kg) is added to the mixture followed by an additional 700 kg of deionized water and the slurry is mixed for 30-40 min. The slurry is filtered in four equal portions through a sparkler filter to remove the cell debris and any other solids from the product in solution. For each portion of the filtration, once the filtrate collection rate stops the filter bundle is washed with fresh deionized water (400 kg). These washes are combined with the initial filtrates.

(27) After the fourth filtration is complete the combined filtrates and washes are returned to the 1000-gallon reactor and treated with hydrogen peroxide (34%, 2.0 kg). The solution is decolorized by treatment with activated carbon (2040 mesh, 60 kg) and aluminum sulfate (9.4 kg); the resulting slurry is mixed for 1.5-2 h.

(28) Diatomaceous earth (Celpure S100, 32 kg) is added and mixed for an additional 30 min. The solids are removed by filtration through the sparkler filter. The reactor is cleaned to remove any residual solids that may be present and the filtrate from decolorization is returned to the reactor through a 0.2-micron in-line filter. The solution is then concentrated to about 250 kg using an ultrafiltration unit that has been equipped with 40 kilo-Dalton cutoff filters.

(29) The concentrated sacrosidase solution is then purified by diafiltration against at least four volumes (1000 L) of citric acid buffer solution. This buffer solution is prepared from deionized water (1000 kg), citric acid (9.2 kg), sodium citrate (11.2 kg) and the pH of the solution is adjusted to 4.60.1 by addition of 25% sodium hydroxide. Additional volumes of buffer can be used to increase the overall protein purity while continuing to reduce residual levels of impurities in the final formulated solution. Typically four volumes (1000 L) of citric acid buffer has been sufficient during diafiltration to purify the sacrosidase protein retained in the solution to >99% purity and to achieve papain levels <50 ppb.

(30) Once the diafiltration is complete the solution is further concentrated until the material has an assay value 26,000 IU/mL in the sacrosidase assay test. The pH of the solution is then checked and adjusted to 4.30.1 by either use of 25% sodium hydroxide solution if the pH is <4.15 or by use of 85% phosphoric acid if the pH of the solution is >4.45.

(31) Once the pH adjustment is complete the solution is transferred to a mixing vessel and an about equal weight of glycerin is added to produce the sacrosidase drug substance. The resulting water-glycerin solution is mixed for 10 min. The target drug substance has >10,000 IU/mL of sacrosidase and about 45-54 wt-% glycerol. Once the mixing is complete the solution is transferred to drums through two 0.2 micron nylon in-line filters that are set up in series. The drummed solution or drug substance is stored at room temperature for 8-36 h prior to being moved into long-term storage at about 20 C. Portions can be removed as needed and diluted and packaged to yield the Sucraid drug product.

Example 2: Coomassie Blue Stained Reduced SDS-PAGE Gel with DensitometryOptimal Load

(32) The gel shown in FIG. 2 is a Coomassie stained, reduced SDS-PAGE comparing 3 lots of a prior sacrosidase manufacturer (Lanes 3, 4, 5) that was manufactured with an older manufacturing process, with 3 lots of sacrosidase (Lanes 6, 7, 8) manufactured using the new ultra-pure hypoallergenic manufacturing process. Reference standard C4546 is the reference standard currently in use and was produced from the older process. Reference standard C3955 is an older-yet reference standard from the original Sacrosidase manufacturer.

(33) The densitometry scan of this optimally loaded SDS-PAGE gel is shown below (Table 1). These results clearly show that the sacrosidase manufactured using the new process is ultrapure with no detectable protein impurities. The cGMP validation of this method established a limit of detection of less than 0.1 micrograms.

(34) TABLE-US-00001 TABLE 1 Lot Lane Band Band Sample Type Number No. No. Volume % Density Reference C4546 2. 1. 1823 94.31 Standard 2. 2. 110 5.69 Old Process STS- 3. 1. 2376 94.93 199NS 3. 2. 127 5.07 Old Process STS- 4. 1. 2822 94.83 220NS 4. 2. 154 5.17 Old Process STS- 5. 1. 2590 94.46 241NS 5. 2. 152 5.54 New Process 125275 6. 1. 2356 >99.9 New Process 125434 7. 1. 2331 >99.9 New Process 125722 8. 1. 2159 >99.9 Reference C3955 9. 1. 2256 >99.9 Standard* *Purified by chromatography. Experimental sample only.

Example 3: Coomassie Stained Reduced SDS-PAGE Gel with DensitometryOverloaded Gel

(35) In a second protocol reduced SDS-PAGE gels were overloaded with 20 g of protein (FIG. 3). In the Coomassie stained gels, a small impurity band is visible in the present sacrosidase drug substance, but the absolute purity of the sacrosidase of the invention (97%, Lanes 6, 7, 8) is still superior to the older sacrosidase (90%, Lanes 3, 4, 5). Table 2 summarizes the information from this gel.

(36) TABLE-US-00002 TABLE 2 Protein Sample Sample Qty Lane Band Band Band Band % Type Lot (g) No. No. rf Volume Sum Density Reference C4546 20 2. 1. 0.151 4419 4867 90.80 Standard 2. 2. 0.561 448 9.20 Old STS- 20 3. 1. 0.145 5063 5588 90.60 Process 199NS 3. 2. 0.568 525 9.40 Old STS- 20 4. 1. 0.132 5460 5969 91.47 Process 220NS 4. 2. 0.559 509 8.53 Old STS- 15 5. 1. 0.132 4577 5003 91.49 Process 241NS 5. 2. 0.550 426 8.51 New 125275 15 6. 1. 0.126 5157 5292 97.45 Process 6. 2. 0.542 135 2.55 New 125434 15 7. 1. 0.134 4820 4936 97.65 Process 7. 2. 0.546 116 2.35 New 125722 15 8. 1. 0.128 4832 4931 97.99 Process 8. 2. 0.550 99 2.01 Reference C3955 15 9. 1. 0.257 5640 6326 89.16 Standard 9. 2. 0.450 69 1.09 9. 3. 0.490 135 2.13 9. 4. 0.554 108 1.71 9. 5. 0.609 96 1.52 9. 6. 0.669 143 2.26 9. 7. 0.732 135 2.13

Example 4: Optimal and Overloaded Native Gels

(37) In a third protocol non-reduced samples were run on native PAGE gels. Both optimal (5 g protein) and overloaded (20, 30 & 60 g protein) protein were applied to polyacrylamide gels then stained with Coomassie Blue.

(38) FIG. 4 depicts an overloaded (15 g) Coomassie stained native gel. Densitometry scanning of the gel show purity values of 97.59, 99.02, and 99.31 for sacrosidase made by the older sacrosidase process and 100%, 100% and 100% for the sacrosidase manufactured using the present ultrapure process. This gel clearly demonstrates the ultrapure quality of the sacrosidase manufactured using the new process. Table 3 summarizes the information from this gel.

(39) TABLE-US-00003 TABLE 3 Protein Sample Sample Qty Lane Band Band Band Band % Type Lot (g) No. No. rf Volume Sum Density Reference C4546 15 1. 1. 0.170 10034 10167 98.69 1. 2. 0.492 62 0.61 1. 3. 0.711 71 0.70 Old STS- 15 2. 1. 0.115 8926 9146 97.59 Process 199NS 2. 2. 0.495 51 0.56 2. 3. 0.689 86 0.94 2. 4. 0.896 83 0.91 Old STS- 15 3. 1. 0.120 8473 8557 99.02 Process 220NS 3. 2. 0.679 84 0.98 Old STS- 15 4. 1. 0.150 8670 8730 99.31 Process 241NS 4. 2. 0.662 60 0.69 New 125275 15 5. 1. 0.166 9100 9100 100 Process New 125434 15 6. 1. 0.129 7967 7967 100 Process New 125722 15 7. 1. 0.141 8605 8605 100 Process Reference C3955 15 8. 1. 0.190 13693 13744 99.63 8. 2. 0.573 51 0.37

Example 5: Three Order of Magnitude (1,000 Fold) Reduction in a Protein Impurity

(40) The FDA recalled all topical papain products in 2008 due to risk of hypersensitivity reactions (www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2008/ucm116956.htm) and a REMS was required for sacrosidase that same year (www.fda.gov/downloads/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/UCM144251.pdf). The present process demonstrates an impressive three order of magnitude reduction in the papain levels of the drug substance to nearly undetectable levels with a very sensitive ELISA (LOQ of 3 ng/mL) using this new chromatography-free process. LOQ is the limit of quantitation. These lots below were the end result of three process validation lots from the present process (Lots 125275, 125897, 125907) versus the old manufacturer lots (STS-199NS, STS-220NS, STS-241NS) which carried an FDA imposed papain limit of 10,000 ng/mL.

(41) TABLE-US-00004 TABLE 4 Sacrosidase lot number with papain concentration underneath in ng/mL STS- STS- STS- 199NS 220NS 241NS 125275 125897 125907 7300 8050 5470 <3 <3 <3

(42) This example shows the substantial level of reduction of papain to undetectable levels. Papain at 23 kilo-Daltons is a key marker protein that is likely representative of any other proteins below the 40 KD cutoff limit of the ultrafiltration filter used, along with the proteins cleaved by papain that fall above the 40 KD molecular weight cut-off, with the exception of sacrosidase which is resistant to papain cleavage in its three dimensional native form in solution.

Example 6: Organoleptic Properties Improved

(43) While diafiltering the solution after concentration, an operator was tasked with observing the color and smell of the concentrated sacrosidase solution. As the number of wash volumes increased from no (zero) diafiltration wash volumes up to four (4) diafiltration wash volumes, the operator observed that all yeast smell was removed from the sacrosidase solution and the yellow color was dramatically reduced to almost no yellow color. These organoleptic properties of this new ultrapure formulation are important for young pediatric patient compliance as the yeasty smell is not tolerated well by some young patients leading to poor compliance with their prescribed Sucraid dosing.

Example 7: Clinical Evidence of Improved Safety and Continued Efficacy

(44) A 5 year old female CSID patient was first dosed with the old formulation of Sucraid in August 2012, and as instructed by the FDA approved labeling (package insert) due to the possibility of allergic reactions, she was dosed the first time within her physicians' offices. This patient manifested a whole body case of hives immediately after dosing with the old formulation of Sucraid. Further dosing with Sucraid was therefore precluded. The family was eager to find a solution to their daughters gastrointestinal problems, so acceded to a second challenge with different lot of the old formulation of Sucraid about 2 months later. Again, hives manifested, and so this patient was permanently precluded from using the commercially marketed FDA approved Sucraid to treat her genetically determined sucrase deficiency.

(45) After this new ultrapure hypoallergenic formulation of sacrosidase was developed, an IND was filed and authorized by FDA, along with Institutional Review Board (IRB) approval at the Mayo Clinic in Rochester Minn. for a test dosing protocol in this same patient in November 2013. After treatment with the new ultrapure hypoallergenic formulation of sacrosidase, this 5 year old patient no longer manifested allergic reactions or symptoms, including hives. Further, after taking the new ultrapure hypoallergenic formulation of Sucraid her CSID symptoms were completely ameliorated, including normalizing to 1-2 formed stools per day versus >6+ watery diarrhea type bowel movements per day, elimination of her abdominal pain, and elimination of her abdominal distension, so that this pediatric patient is now able to utilize this life changing medication to treat her disease. The new ultrapure hypoallergenic formulation of sacrosidase is being provided to this patient for chronic use pursuant to an FDA approved IND along with Mayo IRB approval with excellent results for control of her CSID disease.