WO2016137471A1

WO2016137471A1 - Method for enhanced heparin quality

Info

Publication number: WO2016137471A1
Application number: PCT/US2015/017768
Authority: WO
Inventors: Ralph Yamamoto; Enrico Giuseppe BELLOMO; Yumi Kim; Rachana VYAS
Original assignee: Nantpharma, Llc
Priority date: 2015-02-26
Filing date: 2015-02-26
Publication date: 2016-09-01

Abstract

The invention provides methods for preparing heparin that result in a reduction in global color and heparin-associated color. The present invention further provides methods for preparing heparin that result in a reduction in global color and heparin-associated color that are scalable. The invention also provides methods for distinguishing global color and heparin-associated color.

Description

METHOD FOR ENHANCED HEPARIN QUALITY

TECHNICAL FIELD

[0001] The invention is directed to the technical field of heparin production.

BACKGROUND OF THE INVENTION

[0002] Heparin was first discovered to be a clinically useful natural anticoagulant in 1916. Heparin is currently used to treat and prevent thromboembolic disorders, such as acute venous thrombosis and pulmonary embolism. During cardiopulmonary bypass surgery, heparin is used to prevent blood clotting within the bypass circuit. Additionally, heparin is fractionated to make low molecular weight heparins, or LMWHs, which are salts of sulfated GAGs having a molecular weight of less than 8,000 Daltons. In contrast to heparin, which acts at many points in the coagulation cascade, the shorter-chain LMWHs act more specifically, resulting in a reduction of the hemorrhagic side effects associated with heparin. See id. These many uses of heparin have resulted in a global demand of about 100 tons of heparin annually. See Pavao et al., "Challenges for Heparin Production: Artificial Synthesis or Alternative Natural Sources?" Glycobiology Insights, Vol. 3, pages 1-6 (2012).

[0003] Heparin is composed of acidic glycosaminoglycans (GAGs), which are long unbranched polysaccharide chains composed of repeating disaccharide units. Specifically, heparin is composed of a major trisulfated disaccharide repeating unit, as well as a number of additional disaccharide structures, which comprise a pentasaccharide that is the binding site for antithrombin III, or AT, a serine protease inhibitor that is integral to heparin's

anticoagulant activity.

[0004] Heparin works by accelerating the inhibition of coagulation factors such as thrombin and factor Xa, in a so-called coagulation cascade, wherein one coagulation factor activates the next coagulation factor, until prothrombin, or factor II, is converted to thrombin, or factor Ila, by factor Xa. Thrombin then acts on fibrinogen to form a fibrin clot. Heparin accelerates the formation of an inhibitory complex with AT, a serine protease inhibitor that inhibits factor Xa, thereby inhibiting the conversion of prothrombin to thrombin in the formation of a fibrin clot. (See Linhardt et al, "Production and Chemical Processing of Low Molecular Weight Heparins," Seminars in Thrombosis and Hemostasis, Vol. 25, Suppl. 3 (1999)).

[0005] Heparin is most commonly extracted from the mucosa of slaughterhouse animals, e.g., from porcine intestine or bovine lung. The typical manufacturing process employs five general steps: first, the tissue is collected from an animal at a slaughterhouse; second, heparin is extracted from the tissue via elevated temperatures and pressures with proteases that solubilize GAGs, and/or hydrolysis at an alkaline pH with proteolytic enzymes; third, raw heparin is bound to and recovered from an anion- exchange resin, where heparin-like GAGs selectively adsorb onto the resin, and crude heparin is filtered, precipitated, and vacuum- dried; fourth, crude heparin is extracted either via dissolution in purified water, followed by filtration at low pH, and oxidation to alkaline pH, or via cation exchange chromatography; and fifth, extracted heparin is recovered either by precipitating and vacuum drying or re- dissolving it in purified water, or by various filtration and freeze-drying steps.

[0006] The second step of this process is often referred to as "digestion." Historically, mucosa digestion takes place at elevated temperatures, at alkaline pH, and over a time period of several hours, wherein the digestion includes alkaline proteases. Additionally, the third step of this process historically involves using an anion exchange resin for recovering the raw heparin.

[0007] Recently, production batches of heparin have exhibited higher global color or color of solution, as well as higher heparin-associated color. This higher color is an undesirable characteristic because it is associated with a diminution in heparin's anticoagulant activity and may require, during extraction of heparin, additional or drastic chemical oxidation steps to reduce/reverse both global and GAG-associated color, which could also lead to a diminution of anticoagulant activity. Absorbance at a wavelength of 420 nm is a measure of yellow-to-brown color and as an example, is a common method used in the wine industry to assess accelerated browning in wine See Kallithraka et al., "Changes in Phenolic composition and antioxidant activity of white wine during bottle storage:

Accelerated browning test versus bottle storage", Food Chemistry, Vol. 1 13, Pages 500-505 (2008); Li et al., "Mechanisms of Oxidative Browning in Wine", Food Chemistry, Vol. 108, Pages 1 -13 (2008).

[0008] Accordingly, there exists a need for a process of preparing heparin resulting in a reduction in global color or color of solution and/or heparin-associated color. Additionally, there exists a need for a process of preparing heparin that is scalable also resulting in a reduction in global color or color of solution and/or heparin-associated color. Further, there is a need for methods of measuring heparin color so that the extraction processes can be monitored.

[0009] To address these long felt needs, it is an object of the present invention to provide a process for preparing heparin that results in a reduction in global color or color of solution and/or heparin-associated color. It is a further object of the present invention to provide a process for preparing heparin that results in a reduction in global color or color of solution and/or heparin-associated color that is scalable.

SUMMARY OF THE INVENTION

[0010] This invention provides methods for extracting heparin from the tissue of mammals that contains heparin and DNA. These methods comprise obtaining suitable tissue from a mammal, and digesting the tissue with proteases for a certain length of time, at a particular temperature and pH. In certain embodiments of the invention, the tissue is digested with proteases for about 5-8 hours, at a temperature of between about 50 °C and 60 °C, and at a pH of between 7.7 and 8.8. These methods further comprise digesting the tissue in the presence of sodium chloride and sodium bisulfite, and in certain embodiments, the sodium chloride is present in an amount of about 0.150-0.200 pounds per "runner" (animal carcass) and the sodium bisulfite is present in an amount of about 0.5-2% (w/w). The contribution from one runner is typically about 3 pounds. After digestion of the tissue, these methods further comprise capturing the extracted heparin and DNA on an anion exchange resin, followed by differentially eluting the DNA and heparin off of the anion exchange resin. In certain embodiments, the DNA is eluted off of the anion exchange resin with 1 M sodium chloride solution, and the heparin is eluted off the anion exchange resin with 3 M sodium chloride.

BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1 : Depicts the inverse Relationship of Heparin Activity and P420 Values.

[0012] FIG. 2: Depicts Time Course of Mucosa Digest under different salt (NaCl)) concentrations.

[0013] FIG. 3: Depicts the effect of Sodium Chloride Content during Mucosa Digestion with respect to Normalized A400 color.

[0014] FIG. 4: Depicts the effect of Sodium Chloride content during Mucosa Digestion with respect to Normalized P420 color.

[0015] FIG. 5: Depicts the reproducibility of the P420 assay.

[0016] FIG. 6: Depicts the linearity of the P420 assay over a range of dilutions.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is directed to methods for extracting heparin from the tissue of mammals, wherein this mammalian tissue contains heparin and DNA, wherein these methods minimize unwanted chemical reactions that colorize heparin molecules while simultaneously maximizing heparin yield, activity, and which are scalable. These methods comprise obtaining suitable tissue from a mammal, digesting the tissue, capturing the extracted heparin and DNA on an anion exchange resin, and the differential elution of the DNA and heparin off of the anion exchange resin.

[0018] To better monitor the color change in heparin, a biochemical assay, referred to as "the P420 assay," was developed to distinguish between global color and GAG-associated color (including heparin color.) The P420 assay comprises the following steps: mixing liquid heparin with 65-75% (v/v) ethanol, preferably 70% ethanol; isolating a solid precipitate; dissolving the solid precipitate in any suitable form of water; reading the absorbance as measured at 420 nm; and normalizing the absorbance by dividing it by the heparin concentration.

[0019] Methods in accordance with the invention can use any suitable method for determining the heparin concentration, including but not limited to the USP potency assay.

[0020] While not desiring to be committed to any particular theory, the coloration of heparin may occur as a result of a Maillard reaction during protease digestion, especially detectable during late stages of digestion (i.e., times greater than 10 hours). Accordingly, known methods for suppressing the Maillard reaction where considered. The current invention achieves vast improvements in heparin color which are surprisingly better than would be expected from conventional protocols for suppressing the Maillard reaction.

[0021] As used herein, "Scalable" means that the method can be used with markedly increase amounts of each component provided that the components maintain their proportions relative to one another. As such, the claimed method can be done both on a lab scale and at a production scale without changing the ratios of each component to the other components and the pH, times of incubation, and temperature are the same at any sacle of the method.

[0022] As used herein, "Runner" means any single suitable animal carcass, including, but not limited to, those of cows, pigs, horses, and poultry.

[0023] As used herein, "Heparin-on-resin" means heparin bound to the anion exchange resin.

[0024] As used herein, "Normalized P420 assay" refers to the assay performed by mixing heparin with 70% (v/v) ethanol, resolubilizing the resultant solid precipitate with water, and measuring its absorbance at 420 nm, P420. The absorbance value P420 is then normalized to a heparin activity correction factor, such that the P420 absorbance is divided by the heparin activity correction factor, anti-FXa as expressed in Units/mL. [0025] As used herein, "Normalized A400 assay" refers to the assay performed by appropriately diluting heparin and measuring its absorbance at 400 nm, A400. The absorbance value A400 is then normalized to a heparin activity correction factor, such that the A400 absorbance is divided by the heparin activity correction factor, anti-FXa as expressed in Units/mL.

[0026] Tissue for the extraction of heparin can be obtained from any suitable animal, including, but not limited to, cows, pigs, horses, and poultry. Desirably, the tissue is obtained from cows or pigs, and most desirably, the tissue is obtained from pigs. The tissue that is obtained can be any tissue that contains both heparin and DNA. Preferably, the tissue is intestinal tissue or lung tissue. Most preferably, the tissue is porcine intestinal tissue or bovine lung tissue.

[0027] The tissue can be digested by any suitable means. For example, the tissue can be digested with alkaline proteases, including Savinase proteases, Alcalase proteases, or their equivalents or combinations thereof.

[0028] The tissue is digested in the presence of sodium chloride. In particular, preferred embodiments, the tissue is digested in the presence of sodium chloride in an amount of about 2-10% (w/v), more preferably in an amount of about 3.5-5% (w/v), and most preferably in an amount of 5% (w/v). Alternatively, sodium chloride can be added to the digestion on a per- runner basis. Specifically, the inventors have surprisingly found that the tissue can be digested in the presence of sodium chloride in an amount of about 0.150-0.200 pounds per runner, wherein the contribution of each runner is about 3 pounds.

[0029] The tissue is digested preferably also in the presence of sodium bisulfite. In particularly preferred embodiments, the tissue is digested in the presence of SBS in an amount of 0.4-2.2% (w/v) 0.4-2.1% (w/v) 0.5-2% (w/v). In a particularly preferred embodiment, the tissue is digested in the presence of both sodium chloride and sodium bisulfite, wherein sodium chloride is present in an amount of 3-5% (w/v), and SBS is present in an amount of 0.5% (w/v).

[0030] The digestion can occur at any suitable temperature, preferably at a temperature of between 50 °C and 70 °C, more preferably at a temperature of between 50 °C and 60 °C, and even more preferably at a temperature of about 55 °C. Likewise, the digestion can occur at any suitable pH, preferably at a pH of between 7.7 and 8.8, more preferably at a pH of about 7.8. The digestion should occur for a time less than 8-12 hours, more preferably for 5-8 hours, and most preferably for about 5-6 hours. [0031] It has been determined that detectable color formation occurs during late digestion times, around the same time that heparin activity plateaus. While not meaning to be bound by any particular theory, it is thought that global color reduction can be mediated by controlling Maillard reactions. This is because heparin is one of 15 GAGs attached to one of the 24 ser-gly rich repeats of Serglycin, tethered to the protein core through a xylosyl linker. During the protease-driven digestion of the tissue, proteins, including Serglycin, are digested and peptides and amino acids are formed and are likely to participate in Maillard reactions, especially during late stages of digestion (i.e., times greater than 10 hours).

[0032] After digestion, the heparin and DNA are captured on an anion exchange resin. Any suitable anion exchange resin can be used. With scale up in mind, it has been determined that effective separation of heparin from other GAGs is difficult, as anion exchange resin is typically eluted in "batch mode," meaning that eluates obtained from washing the heparin-on-resin are collected and pooled together. Typically, heparin-on-resin is washed with 1 M sodium chloride and then eluted twice with 3 M sodium chloride. The two 3 M sodium chloride eluates are pooled together. Thus, any reactive impurities that are collected with the eluates may cause an increase in heparin color. It has been determined that the addition of sodium bisulfite in the second eluate results in a 3 -fold reduction of P420 color. In addition, it has been discovered that post-digestion Maillard reactions may occur during elution, with Maillard-reactive intermediates remaining bound to the anion exchange resin. These reactions can be controlled by soaking the heparin-on-resin in sodium bisulfite. In a preferred embodiment of the invention, the heparin-on-resin is soaked in about 2-5% (w/v) SBS, and in a more preferred embodiment, the heparin-on-resin is soaked in about 2- 5% (w/v) SBS for up to one week at a temperature of 4 °C. This soaking of heparin-on-resin in sodium bisulfite occurs prior to treatment with 1 M sodium chloride and elution with 3 M sodium chloride.

[0033] Thus, the inventors have determined that the number of uses of regenerated resin may be a critical factor with respect to global and heparin-associated color. In a preferred embodiment of the invention, virgin anion exchange resin is used. This prevents any intermediates from participating in a Maillard reaction during elution from the heparin-on- resin. Recycling of resin can be used in subsequent digestion processes provided adequate cleaning and regeneration procedures are performed.

[0034] The heparin produced by the methods of this invention has an average color value, as determined by the P420 assay, which is below an acceptable value determined to be 150 x 10^"6. To conduct the P420 assay, heparin is mixed in an alcoholic solvent, such as 50-70% (v/v) ethanol. invention also provides methods for evaluating heparin color comprising the following steps:

a) mixing liquid heparin with 65-75% (v/v) ethanol;

b) isolating a solid precipitate;

c) dissolving the solid precipitate in water;

d) reading the absorbance is measured at 420 nm; and normalizing the absorbance by dividing it by the heparin concentration. The method can be used with a solid precipitate by dissolving it in water using dilution of 1 :5 to 1 :50, preferably 1 :10 to 1 :30, more preferably 1 :20.

[0035] The following examples further illustrate the invention but should not be construed as in any way limiting its scope.

EXAMPLE 1

[0036] The inventors have confirmed that there is an inverse relationship between heparin activity and heparin color (FIG. 1).

[0037] The next study was conducted to determine the effect that the use of sodium chloride during digestion and the length of digestion time has on heparin color. The digestion was conducted on laboratory scale (0.200 L) at a pH of 8.8 and a temperature of 60 °C, and sodium bisulfite was included in the digestion at 0.5% (w/v). The study was conducted at 0, 2, 4, 6, 8, and 16 hours, both in the presence and absence of sodium chloride (i.e., 0% or 2% NaCl (w/v)). The results are compiled in Table 1 and are further shown in FIG. 2.

TABLE 1

[0038] As demonstrated by Table 1 , the maximal heparin activity occurred prior to the 8 hour mark. Further, the addition of 2% (w/v) sodium chloride improved color, as indicated by the lower values obtained when the global color was measured at a wavelength of A400 nm. This study showed that color forms during late processing times, around the same time that heparin activity plateaus. Thus, these results indicate that digestion should not last longer than 8 hours, and sodium chloride should be included in the digestion.

EXAMPLE 2

[0039] This study was conducted to determine the effect of digestion temperature on heparin color. The digestion was conducted at a pH of 8.8 with 0.5% (w/v) sodium bisulfite for 5 hours. The temperature of digestion was 50 °C, 60 °C, and 70 °C. The results of this study are compiled in Table 2.

TABLE 2

[0040] As can be seen in Table 2 above, color as measured by normalized P420 increases as temperature increases. Thus, these results indicate that lower temperatures should be used for the digestion.

EXAMPLE 3

[0041] This study was conducted to determine the effect of the amount of sodium chloride and sodium bisulfite included in the digestion on heparin color. The digestion was conducted at a pH of 7.8 and a temperature of 55 °C for 5 hours, with 0.5% (w/v) sodium bisulfite added to the digestion. The amount of sodium chloride was varied at either 2% or 5% NaCl (w/v), and the amount of sodium bisulfite was varied at either 0.5% or 2% SBS (w/v). The results are complied in Table 3.

TABLE 3

[0042] As can be seen in Table 3, as the amount of sodium chloride increased, color was reduced. Surprisingly, the best combination of the two salts was determined to be 0.5 % SBS (w/v) and 5% NaCl (w/v).

EXAMPLE 4

[0043] This study was conducted to determine the effect of sodium chloride used during digestion on the efficiency of heparin binding to anion exchange resin. Solutions of purified heparin were added to various concentrations of sodium chloride in water, 0%, 2%, 3.5%, 5%, and 10% (w/v). Two grams of virgin resin were added to each solution and incubated for one hour. Each sample was then washed with 1 M NaCl and eluted with 3 M NaCl. The results are complied in Table 4.

TABLE 4

[0044] As can be seen in Table 4, it was determined that there is no significant difference in heparin binding to the anion exchange resin in the presence of up to 3.5% (w/v) sodium chloride. While a slight decrease in binding efficiency was determined in the presence of 5% (w/v) sodium chloride, a greater binding inhibition was observed in the presence of 10% (w/v) sodium chloride.

EXAMPLE 5

[0045] Heparin can be isolated from a variety of animals. There are differences in the anti-coagulant properties of heparins from different animals. In addition, because heparin preparations made from ruminants, but not pigs, can harbor prion diseases (e.g., "mad cow disease") heparin preparations are tested to confirm the species source of the heparin. In particular, quantitative PCR protocols have been developed to confirm the species of origin for heparin extracts. However, PCR can be inhibited by heparin or high salt conditions. In view of these concerns, the verification that the high salt conditions employed in the current invention do not preclude the use of PCR is surprising. [0046] A study was conducted to determine the effect of sodium chloride used during digestion on the qPCR speciation assay post-digestion. Mucosal digests were prepared with two concentrations of sodium chloride, 3.5% and 5% (w/v). Following digestion, each resin was purified by washing with 1 M NaCl and then analyzed via qPCR speciation assay. As referenced in PCT/US2012/059918 the results indicate that including sodium chloride in the digest at an amount up to 5% did not negatively impact the ability of the DNA to bind to the anion exchange resin. Thus, a qPCR speciation assay can still be performed when sodium chloride is present in the digest in an amount of 5% (w/v) or less.

EXAMPLE 6

[0047] A study was conducted to determine the effect that storage has on the stability of heparin-on-resin. Heparin on resin was held at 4 °C and 38 °C for up to one week, with and without prior soaking in 2% or 5% (w/v) sodium bisulfite. Following storage, the heparin on resin was treated with 1M NaCl and 3 M NaCl, and then concentrated by tangential flow filtration.

TABLE 5

[0048] As can be seen in Table 5, as the concentration of sodium bisulfite increases, the normalized P420 color decreases. Also, P420 color is lower when stored at 4 °C as compared to 38 °C. EXAMPLE 7

[0049] Three pilot scale-up batches were prepared - one using no salt conditions, and the other two utilizing inventive conditions, with the concentration of sodium chloride varying from 3.5% and 5% (w/w). The reaction parameters set forth in Table 6 below were used:

TABLE 6

[0050] As can be seen from Table 7 and FIGS. 3 and 4, the inventive parameters with the incorporation of salt resulted in a surprisingly large decrease in normalized P420.

TABLE 7

[0051] Additionally, heparin from pilot-scale test batches #1 and #2 were extracted under laboratory conditions, and the results are set forth in Table 8: TABLE 8

White standard tile = 76

²USP Heparin Sodium for assays (2144 U for 9.5 mg)

EXAMPLE 8

[0052] In addition to overall salt concentration in the digest, the effect of salt/runner ratio on resulting color was investigated. Experiments were performed on the lab scale (0.200L) by varying salt/runner ratio over the range 0.104 - 0.208 lbs/runner and a concentration range of 2% - 5% (w/w) salt. Results were evaluated by comparing normalized A400 as indicated in Table 9 below.

TABLE 9

100531 As salt/runner ratio increases, normalized A400 decreases. Interestingly, when salt/runner ratio is low (0.104 lb/runner) coupled with a low overall salt concentration (2%), the color is above a visual threshold of 1 x 10^"3 for normalized A400 as compared to a similar salt/runner ratio (0.104 lb/runner) and 4% salt. Therefore, the combination of both factors is critical to controlling color during digestion.

[0054] Fig. 3 includes the normalized A400 results for laboratory batches and comparative pilot scale and full scale batches. Two Full-scale batches were prepared - one using a salt/runner ratio of 0.105 and the other one using a salt/runner ratio of 0.150. Table 10 summarizes the surprising superiority of the invention by indicating a comparison of the full scale batches with the pilot scale batches described previously. As the salt/runner ratio increases, the normalized P420 and normalized A400 results decrease.

TABLE 10

EXAMPLE 9

[0055] Reproducibility testing (n=6) using samples of heparin CE at the upper and lower range of normalized P420 color indicate that the assay is reproducible over a broad range encompassing both color corrected (Full Scale Batch #2) and non color corrected (CH-1455) samples. The results are provided in Table 1 1 and FIG. 5 and indicate a 1 14-fold difference in mean normalized P420 values.

TABLE 1 1

EXAMPLE 10

[0056] Linearity was evaluated over a broad dilution range, which covers the standard dilution (1 :20) as well as noixaalized dilutions of 0.25X (1 :5) and 2.5X (1 :50). Samples of heparin CE with high (CH-1455), medium (CE-0207) and low (Full Scale Batch #2) color were evaluated over the broad range of dilutions. For each case, linearity was exhibited, as indicated in Table 12 and FIG. 6.

TABLE 12

[0057] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0058] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly

contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0059] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A method for extracting heparin from mammalian tissue containing heparin and DNA comprising:

(a) Obtaining suitable mammalian tissue;

(b) digesting said tissue with proteases for 5-8 hours, at a temperature of 50 °C- 60 °C and at a pH of 7.7-8.8;

(c) wherein NaCl is added the at the start of the digest in an amount of 0.150- 0.200 lbs/runner and maintained at a concentration of 3% - 5% w/v, wherein the contribution of each runner is about 3 pounds, and SBS is present in the digestion amount of about 0.5-2% (w/v),

(d) capturing heparin on an anion exchange resin, and

(e) eluting the heparin off the anion exchange resin with a 3M NaCl solution.

2. The method of claim 1 , wherein the mammalian tissue is from a pig.

3. The method of claim 1, wherein the mammalian tissue is porcine intestine.

4. The method of claim 1, wherein the protease is Savinase or Alcalase or combinations thereof that show proteolytic activity at alkaline pH are used during step (b).

5. The method of claim 1, wherein said tissue is digested for about 6 hours.

6. The method of claim 1 , wherein said tissue is digested at a temperature of about 55 °C.

7. The method of claim 1, wherein the pH is about 7.8.

8. The method of claim 1 , wherein the resin bound heparin is soaked in about 2% (w/v) SBS.

9. The method of claim 1 , wherein the resin bound heparin is soaked in 2% (w/v) SBS and stored at a temperature of about 4 °C.

10. The method of claim 1 , wherein the resin bound heparin comprises virgin resin or regenerated resin from prior digestion processes that incorporated salt and under claims 1-9.

11. The method of claim 1 , wherein the resin bound heparin is further twice soaked in about 2% (w/v) SBS before collection and elution.

12. The method of claim 1 , wherein DNA is also generated from the digestion, wherein and said DNA is also bound to said anion exchange resin,

the DNA is eluted from the anion exchange resin with 1 M NaCl solution, and this DNA is used for speciation.

13. The method of claim 1, wherein said method is scalable.

14. The method of claim 1, wherein said resin can be regenerated and recycled for use in subsequent digestions.

15. Heparin prepared by the method of any one of claims 1-14 and having a normalized P420 average color value less than 150 x 10^~6

16. A method for evaluating heparin color comprising the following steps:

a) mixing liquid heparin with 65-75% (v/v) ethanol;

b) isolating a solid precipitate;

c) dissolving the solid precipitate in water;

d) reading the absorbance is measured at 420 nm; and

e) normalizing the absorbance by dividing it by the heparin concentration.

17. The method of claim 16, wherein the solid precipitate is dissolved in water using dilution of 1 :5 to 1 :50.