WO2011126957A1

WO2011126957A1 - Process for trypsin and chymotrypsin purification utilizing hydrophobic interaction chromatography

Info

Publication number: WO2011126957A1
Application number: PCT/US2011/031036
Authority: WO
Inventors: Bhaktavachalam Thiyagarajan; Steve Magee; Nandu Deorkar
Original assignee: Avantor Performance Materials LLC
Current assignee: Avantor Performance Materials LLC
Priority date: 2010-04-05
Filing date: 2011-04-04
Publication date: 2011-10-13
Anticipated expiration: 2012-10-05
Also published as: TW201138974A

Abstract

A method for the separation and purification of trypsin and chymotrypsin in which a crude mixture of trypsin and chymotrypsin is passed through a hydrophobic interaction chromatography media in a chromatographic column. The hydrophobic interaction chromatography media is preferably one having weak anion exchange functionality and alkyl, alkanoyl, phenyl, benzoyl, phenylalkyl or phenylalkanoyl functional groups, such that chymotrypsin is bound to the chromatographic hydrophobic interaction chromatography media and trypsin passes through the media without binding to said media to produce purified trypsin, and thereafter bound chymotrypsin is eluted from the media to produce purified chymotrypsin.

Description

PROCESS FOR TRYPSIN AND CHYMOTRYPSIN PURIFICATION UTILIZING HYDROPHOBIC INTERACTION CHROMATOGRAPHY

FIELD OF THE INVENTION

[0001] The invention relates to the use of chromatographic media for the separation and purification of proteins. More particularly, the current invention discloses use of hydrophobic chromatographic media for the separation and purification of closely related proteolytic enzymes trypsin and chymotrypsin. The manufacturing process provided in this invention is intended for separation of mixture of serine proteases such as trypsin and chymotrypsin from their contaminants using hydrophobic chromatography commonly referred to as HIC (Hydrophobic Interaction Chromatography). Further more, the process also provides viral clearance (removal) for trypsin and chymotrypsin.

BACKGROUND TO THE INVENTION

[0002] There are three principal digestive proteinases found in the pancreas, namely trypsin, chymotrypsin and pepsin. Trypsin and chymotrypsin are members of the serine proteases family. In the digestive process trypsin acts with the other proteinases to break down dietary proteins molecules to their component peptides and amino acids. Trypsin continues the process of digestion, begun in the stomach, in the small intestines where a slightly alkaline environment, about pH 8, promotes its maximal enzymatic activity. Trypsin, produced in an inactive form by the pancreas, is remarkably similar in chemical composition and in structure to the other chief pancreatic proteinase, chymotrypsin. Both enzymes appear to have similar mechanisms of action; residues of histidine and serine are found in the active sites of both. The chief difference between trypsin and chymotrypsin molecules appears to be their specificity, that is, each is active only against the C- terminal ends of peptide bonds in protein molecules that have carboxyl groups donated by certain amino acids. For trypsin those amino acids are arginine and lysine, except when either is followed by proline; for chymotrypsin, which recognizes aromatic residues, those amino acids are tyrosine, phenylalanine, tryptophan, methionine and leucine., while trypsin recognizes lysine and arginine. The recognition of a particular side chain is fully determined by the structure and properties of the binding pocket. [0003] Trypsin is the most discriminating of all the proteolytic enzymes in terms of the restricted number of chemical bonds that it will attack. Good use of this fact has been made by chemists interested in the determination of the amino acid sequence of proteins. Trypsin is widely employed as a reagent for the orderly and unambiguous cleavage of such molecules. The process is commonly referred to as protein proteolysis or trypsination and protein that have been digested/treated with trypsin is said to have been trypsinized.

[0004] Both trypsin and chymotrypsin are extracted from pancreas and pancreatic juices of various animals including bovine and porcine pancreas and pancreatic juices. They are generally isolated using multiple precipitation, fractionation, and filtration steps. Because of the similarity between trypsin and chymotrypsin structures and similar physicochemical properties, commercial sources of trypsin almost always contains significant amount of chymotrypsin and vice versa. The purification of trypsin can be performed by a number of methods including but not limited to ammonium sulfate precipitation, ion-exchange chromatography and affinity chromatography. Depending on the purification method, purified trypsin contains varying amount of chymotrypsin. There is therefore a need for a process for producing trypsin with an insignificant, minimal or non-detectable amount of chymotrypsin.

[0005] Also, mammalian-derived trypsin may contain adventitious agents, such as viruses that are detrimental for the final use of the trypsin or chymotrypsin. There is therefore a need for a process for producing purified trypsin and chymotrypsin with no or essentially no viruses contaminating the trypsin or chymotrypsin.

BRIEF SUMMARY OF THE INVENTION

[0006]. This invention relates to a method of separating the biologically active compounds trypsin and chymotrypsin upon selective adsorbents using hydrophobic interaction chromatographic media. According to the invention a method for the separation and purification of trypsin and chymotrypsin comprising contacting a crude mixture of trypsin and chymotrypsin with a hydrophobic interaction chromatography media wherein the method comprises passing the crude mixture through a hydrophobic chromatographic media whereby chymotrypsin is bound to the hydrophobic chromatographic media and trypsin passes through the media without binding to said media to produce purified trypsin, and thereafter bound chymotrypsin is eluted from the media to produce purified chymotrypsin. More particularly preferred is the use of a hydrophobic mixed mode media containing both hydrophobic groups and weak anion exchange groups. Hydrophobic interaction chromatography (HIC) differs from reversed-phase HPLC in that the latter requires strong organic solvent to elute the molecules, whereas HIC usually operates using aqueous buffers and binding and elution of the solute molecules tuned by salt concentration. Hydrophobic interaction chromatography utilizes the interaction between the hydrophobic surface of the chromatographic media and the hydrophobic groups on the proteins molecules. This interaction is strongly influenced by the presence of certain salts. A high salt concentration of certain salts from about 0.8 M to about 1.5 M generally increases the interaction between the proteins and the hydrophobic surface and a low salt concentration decreases the interaction. In the present invention there is described a novel process for the separation and purification of trypsin and chymotrypsin using hydrophobic interaction chromatographic media. More particularly, in a preferred embodiment, the process is conducted employing a hydrophobic interaction chromatographic media having both hydrophobic groups and weak anion exchange functionality.

[0007] Any suitable hydrophobic interaction may be employed in the process of this invention. Especially preferred for use in the process of this invention are hydrophobic interaction chromatographic media having alkyl, alkanoyl, phenyl, benzoyl, phenylalkyl or phenylalkanoyl functional groups. More particularly, hydrophobic interaction chromatographic mixed mode media having hydrophobic groups such as alkyl, alkanoyl, phenyl, benzoyl, phenylalkyl or phenylalkanoyl functional groups along with weak anion exchange functionality are preferred. In this process, chymotrypsin is bound to the hydrophobic interaction chromatographic media and trypsin passes through the media without binding to said media under high salt conditions of from about 0.8M to about 1.5M. The hydrophobic media could be one of several commercially available media such as butyl Sepharose, octyl Sepharose, phenyl Sepharose from GE Healthcare or Toyopearl butyl, Toyopearl phenyl, Toyopearl hexyl from Tosoh Bioscience LLC. More particularly and preferably, the media is a hydrophobic interaction chromatographic mixed mode media having alkyl, alkanoyl, phenyl, benzoyl, phenalkyl or phenylalkanoyl functional groups attached to some of the polyethyleneimine ligands on a polymeric surface of a polymeric or inorganic chromatographic media. The non-functionalized polyethyleneimine groups covalently attached on the polymeric surface or inorganic materials provide weak anion exchange sites. The polymeric media may be based on any suitable natural or synthetic polymers, such as for example, agarose, polymethacrylate, and polystyrene. Inorganic media may be based on titanium dioxide, zirconium dioxide, or silica. The alkyl or alkanoyl groups of the alkyl, alkanoyl, phenylalkyl or phenylalkanoyl functional groups can have from 1 to 8 carbon atoms, preferably 1 to 4, more preferably 1 to 3, carbon atoms in the alkyl or alkanoyl groups. Such hydrophobic chromatographic media may be purchased from the aforementioned suppliers and the mixed mode hydrophobic chromatographic media based on polyethyleneimine covalently attached on the surface of polymers, particularly polymethacrylate polymers, or inorganic media, particularly silica gel, may be prepared according to the disclosures in US Patent Application Publication No. US20080203029 and US Patent 4, 551, 245, which documents are incorporated herein by reference thereto. The mixed mode hydrophobic media are commercially available from JT Baker under the product number 7588 for polymethacrylate based material and 7182 for silica based material. The use of the hydrophobic interaction chromatographic media in the invention process enables running the purification process at lower pH (~ 3) and this lower pH is known to inactivate viruses.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The invention is illustrated by, but not limited to, the illustration shown in the drawing figures.

[0009] Fig. 1 is a graph of USP units/ml versus fraction number for the purification process exemplified in Example 1.

[0010] Fig. 2 is a graph of USP units/ml versus fraction number for the purification process exemplified in Example 2.

[0011] Fig. 3 is a graph of USP units/ml versus fraction number for the purification process exemplified in Example 3.

[0012] Fig. 4 is a graph of USP units/ml versus fraction number for the purification process exemplified in Example 4.

[0013] Fig. 5 is a graph of USP units/ml versus fraction number for the purification process exemplified in Example 5.

[0014] Fig. 6 is a graph of USP units/ml versus fraction number for the purification process exemplified in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

[0015] This invention relates to a method of separating biologically active compounds trypsin and chymotrypsin upon selective adsorbents using mixed mode having weak anion and hydrophobic (reverse phase) functional groups.

[0016] One aspect of the invention comprises a method for the separation and purification of trypsin and chymotrypsin comprising contacting a crude mixture of trypsin and chymotrypsin with a hydrophobic interaction chromatography media, more preferably a mixed mode hydrophobic interaction chromatographic media, in a chromatographic column, wherein the hydrophobic interaction chromatographic media is preferably a hydrophobic interaction chromatographic mixed mode media having weak anion exchange functionality and alkyl, alkanoyl, phenyl, benzoyl, phenylalkyl or phenylalkanoyl hydrophobic functional groups, whereby chymotrypsin is bound to the chromatographic media and trypsin passes through the media without binding to said media to produce purified trypsin, and thereafter bound chymotrypsin is eluted from the media to produce purified chymotrypsin.

[0017] Another aspect of the invention comprises the method wherein the bound chymotrypsin is eluted from the chromatographic media under low salt conditions, i.e., containing less than about 50 to lOOmM commonly used salts, such as for examples acetates or sulfates and the like. In a preferred embodiment of the invention the crude mixture is passed through the hydrophobic interaction chromatographic media under high salt conditions of from about 0.8 M to about 1.5 M salt and the bound chymotrypsin is eluted from the hydrophobic interaction chromatographic media under low salt conditions of from about 50 to about 100 mM salt.

[0018] Another aspect of the invention comprises the method wherein mixed mode hydrophobic interaction chromatographic media is one having alkyl, alkanoyl, phenyl, benzoyl, phenylalkyl or phenylalkanoyl groups attached to polyethyleneimine ligands on a polymeric surface of a polymeric polymethacrylate media or silica gel media.

[0019] Another aspect of the invention comprises the method wherein the polymeric surface comprises a polymethacrylate polymeric surface and the alkyl, alkanoyl, phenylalkyl and phenylalkanoyl groups have from 1 to 8 carbon atoms in the alkyl or alkanoyl groups.

[0020] Another aspect of the invention comprises the method wherein the alkanoyl groups and phenylalkanoyl groups have from 1 to 4, more preferably 1 to 3, carbon atoms in the alkanoyl groups.

[0021] Another aspect of the invention comprises the method wherein the hydrophobic functional groups are propanoyl groups.

[0022] Another aspect of the invention comprises the method wherein the hydrophobic functional groups are butanoyl groups.

[0023] Another aspect of the invention comprises the method wherein the hydrophobic functional groups are phenylpropanoyl groups.

[0024] Another aspect of the invention comprises the method wherein the hydrophobic functional groups are phenylbutanoyl groups

[0025] Another aspect of the invention comprises the method wherein the bound chymotrypsin is eluted with acetic acid.

[0026] Another aspect of the invention comprises the method wherein the crude mixture is contacted with the hydrophobic interaction chromatographic media as a pH 3 solution of the crude mixture with ammonium sulfate.

[0027] Another aspect of the invention comprises the method wherein the flow throughpurified trypsin is freeze-dried to a semi-crystalline powder.

[0028] Another aspect of the invention comprises the method wherein the purified trypsin has a trypsin activity of 3500 USP units/mg or greater and a trypsin/chymotrypsin activity ratio of more than 50.

EXAMPLES

[0029] Example 1 171.8 g of ammonium sulfate was dissolved in 800 mL 50 mM acetic acid and the pH was adjusted to 3.0 with sulfuric acid. The solution was diluted to 1 L with 50 mM acetic acid to give 1.3 M ammonium sulfate at pH 3.0. 20 g crude trypsin containing 1775 USP units/mg with the trypsin/Chymotrypsin activity ratio being 8.2 was dissolved in 2L of 1.3 M ammonium sulfate at pH 3.0 to give 10 mg/mL solution. The solution was filtered and loaded, at the flow rate of 50 mL/min (150 cm/hr) on to a column of dimension (5.0 cm ID and 200 mL volume) loaded with a chromatographic media of a polymethacrylate polymer having polyethyleneimine ligand bound to the surface thereof and functionalized with hydrophobic propanoyl groups produced according to Example 19 of US Patent Application Publication No. US20080203029. The column was then washed with 600 ml (3 column volumes (CV)) of 1.3 M ammonium sulfate at pH 3.0 and then eluted with step gradient (4 CV) to 35 % elution buffer of 50 mM acetic acid followed by linear gradient to 100 % 50 mM acetic acid (8 CV). The fractions were analyzed for Trypsin and Chymotrypsin activity using US Pharmacpoeia method (USP Monographs, 3823- 3824, USP 32, Volume-Ill, 2009) and shown in the Figure 1 and the fractions with higher trypsin content were combined and desalted and freeze-dried to get a pure form of trypsin. The lyophilized pool contains 3500 USP units of trypsin/mg with the trypsin/chymotrypsin activity ratio being 86. A280 is the total absorbance at 280 namometers.

[0030] Example 2 28.1 g of crude trypsin containing 2795 USP units/mg with the trypsin/Chymotrypsin activity ratio being 8 was dissolved in 562 mL of 0.25 % (w/w) sulfuric acid and the pH was adjusted to 3 with concentrated ammonium hydroxide. 562 mL of 2.5 M ammonium sulfate in 100 mM acetic acid was added to the solution to bring the trypsin concentration of 25 mg/ml at pH 3 with 1.25 M ammonium sulfate. About 10 grams of celite was added to the solution and the solution was filtered through a 0.45 micron filter. The resulting clear solution was then loaded, at the flow rate of 50 mL/min (150 cm/hr), onto a column (5cm diameter, 200mL) containing the mixed mode media of the type employed in Example 1. After the loading the column was washed with 3 CV of 1.25 M ammonium sulfate, pH 3 and eluted with 50 mM acetic acid with 4 CV. The fractions were analyzed for trypsin and chymotrypsin activity using US Pharmacopoeia method (USP Monographs, 3823-3824, USP 32, Volume-Ill, 2009) and are shown in the Figure 2. A280 is t he total absorbance at 280 namometers. The fractions with higher trypsin content were combined and desalted and freeze-dried to provide a purified form of trypsin having a trypsin/chymotrypsin activity ratio of 48.

[0031] Example 3 22.4 g of crude trypsin containing 3022 USP units/mg trypsin was dissolved in 560 mL of 0.25 % (w/w) sulfuric acid and the pH was adjusted to 3 with concentrated ammonium hydroxide. 560 mL of 2.6 M ammonium sulfate in 100 mM acetic acid was added to the solution to bring the trypsin concentration of 20 mg/ml at pH 3 with 1.3 M ammonium sulfate. About 10 grams of celite was added to the solution and the solution was filtered through a 0.45 micron filter. The resulting clear solution containing trypsin to chymotrypsin activity ratio of approximately 6 was then loaded, at the flow rate of 50 mL/min (150 cm/hr), onto a column (5cm diameter, 200mL) packed with the media as employed in Example 1 After the loading the column was washed with 3 CV of 1.25 M ammonium sulfate, pH 3 and eluted with 50 mM acetic acid with 4 CV. The fractions were analyzed for trypsin and chymotrypsin activity using the US Pharmacopedia method (USP Monographs, 3823-3824, USP 32, Volume-Ill, 2009) and are shown in Fig. 3. A280 is the total absorbance at 280 namometers. The fractions with higher trypsin content were combined and desalted and freeze-dried to get a purified form of trypsin with an activity of higher than 3500 USP units/mg. The trypsin/chymotrypsin activity increased from 11 to 69 for the collected pool that contains purified trypsin activity of 3500 UPS units/ml.

[0032] Example 4 1.68 g of crude trypsin containing 3022 USP units of trypsin/mg was dissolved in 42 mL of 0.25 % (w/w) sulfuric acid and the pH was adjusted to 3 with concentrated ammonium hydroxide. 42 mL of 2.6 M ammonium sulfate in 100 mM acetic acid was added to the solution to bring the trypsin concentration of 20 mg/ml at pH 3 with 1.3 M ammonium sulfate. About 2 grams of celite was added to the solution and the solution was filtered through 0.45 micron filter. The resulting clear solution was then loaded onto a column (1cm diameter, 7.8 mL) at the flow rate of 1.8 mL/min (140 cm/hr). The column contained a mixed mode media comprising polymethacrylate polymer having polyethyleneimine ligand bound to the surface thereof and functionalized with hydrophobic butanoyl groups, prepared according to process described in Example 19 of US Patent Application Publication No. US20080203029 with substitution of valeric anhydride for butyric anhydride reactant. After the loading, the column was washed with 3 CV of 1.25 M ammonium sulfate, pH 3 and eluted with 50 mM acetic acid with 4 CV. The fractions were analyzed for trypsin and chymotrypsin activity using the US Pharmacopedia method (USP Monographs, 3823-3824, USP 32, Volume-Ill, 2009) and are shown in Fig. 4. A280 is the total absorbance at 280 namometers. The fractions with higher trypsin content were combined and desalted and freeze-dried to get a purified form of trypsin. The trypsin/chymotrypsin activity ratio increased from 2.4 to 82 for the fraction that contains the purified form of trypsin having more than 3500 USP trypsin units /mL.

[0033] Example 5 1.68 g of crude trypsin containing 3022 USP units of trypsin/mg was dissolved in 42 mL of 0.25 % (w/w) sulfuric acid and the pH was adjusted to 3 with concentrated ammonium hydroxide. 42 mL of 2.6 M ammonium sulfate in 100 mM acetic acid which was adjusted to pH 3 with sulfuric acid was added to the solution to bring the trypsin concentration of 20 mg/ml at pH 3 with 1.3 M ammonium sulfate. About 2 grams of celite was added to the solution and the solution was filtered through 0.45 micron filter. The resulting clear solution was then loaded, at the flow rate of 1.8 mL/min (140 cm/hr), onto a column (1cm diameter, 7.8 mL). The column contained a mixed mode media comprising polymethacrylate polymer having polyethyleneimine ligand bound to the surface thereof and functionalized with hydrophobic phenoyl groups, prepared according to process described in Example 19 of US Patent Application Publication No. US20080203029 with substitution of benzoic anhydride for butyric anhydride reactant. After the loading the column was washed with 3 CV of 1.25 M ammonium sulfate, pH 3 and eluted with 50 mM acetic acid with 4 CV. The fractions were analyzed for trypsin and chymotrypsin activity using the US Pharmacopedia method (USP Monographs, 3823-3824, USP 32, Volume-Ill, 2009) and are shown in Fig. 5. A280 is the total absorbance at 280 namometers. The fractions with higher trypsin content were combined and desalted and freeze- dried to get a purified form of trypsin. The trypsin/chymotrypsin activity ratio was increased from 6 to more than 50 for the pool of purified trypsin that contains more than 3500USP trypsin units/mg.

[0034] Example 6 1.60 g of crude trypsin containing 3022 USP units trypsin/mg was dissolved in 40 mL of 0.25 % (w/w) sulfuric acid and the pH was adjusted to 3 with concentrated ammonium hydroxide. 40 mL of 2.6 M ammonium sulfate in 100 mM acetic acid was added to the solution to bring the trypsin concentration of 20 mg/ml at pH 3 with 1.3 M ammonium sulfate. About 2 grams of celite was added to the solution and the solution was filtered through a 0.45 micron filter. The resulting clear solution was then loaded, at the flow rate of 1.8 mL/min (140 cm/hr), onto a column (1cm diameter, 7.8 mL). The column contained a mixed mode media comprising polymethacrylate polymer having polyethyleneimine ligand bound to the surface thereof and functionalized with hydrophobic phenylpropanoyl groups, prepared according to process described in Example 19 of US Patent Application Publication No. US20080203029 with substitution of to the benzoic anhydride for butyric anhydride reactant. After the loading the column was washed with 3 CV of 1.25 M ammonium sulfate, pH 3 and eluted with 50 mM acetic acid with 4 CV. The fractions were analyzed for trypsin and chymotrypsin activity using the US Pharmacopedia method (USP Monographs, 3823-3824, USP 32, Volume-Ill, 2009) and are shown in Fig. 6. A280 is the total absorbance at 280 nanometers. The fractions with higher trypsin content were combined and desalted and freeze-dried to get a purified form of trypsin. The trypsin/chymotrypsin activity ratio was increased from 5 to more than 50 for the pool that contains the purified trypsin having more than 3500 USP trypsin units/mg.

[0035] Example 7 1.60 g of crude trypsin containing 3022 USP units trypsin/mg is dissolved in 40 mL of 0.25 % (w/w) sulfuric acid and the pH is adjusted to 3 with concentrated ammonium hydroxide. 40 mL of 2.6 M ammonium sulfate in 100 mM acetic acid is added to the solution to bring the trypsin concentration to 20 mg/ml at pH 3 with 1.3 M ammonium sulfate. About 2 grams of celite is added to the solution and the solution is filtered through a 0.45 micron filter to produce a crude solution containing 20 mg/mL crude trypsin/chymotrypsin. This crude trypsin solution is purified by processing through a 1 cm diameter column with a column volume of 7.8 mL packed with the hydrophobic media Toyopearl Butyl 650S from Tosoh Bioscience LLC, Montgomeryville, PA. The crude trypsin is processed through the column at a flow rate of about 1.8 mL/min. After the column loading is complete the elution is completed using 59 mM acetic acid after the column is washed with 1.25M ammonium sulfate at pH 3. The resulting flow through fraction will contain more than 3500 USP trypsin units/mg and the trypsin/chymotrypsin activity ratio will increase from 5 to about 50.

[0036] While the invention has been described herein with reference to the specific embodiments thereof, it will be appreciated that changes, modification and variations can be made without departing from the spirit and scope of the inventive concept disclosed herein. Accordingly, it is intended to embrace all such changes, modification and variations that fall with the spirit and scope of the appended claims.

Claims

1. A method for the separation and purification of trypsin and chymotrypsin comprising contacting a crude mixture of trypsin and chymotrypsin with a hydrophobic interaction chromatography media wherein the method comprises passing the crude mixture through a hydrophobic interaction chromatographic media whereby chymotrypsin is bound to the hydrophobic chromatographic media and trypsin passes through the media without binding to said media to produce purified trypsin, and thereafter bound chymotrypsin is eluted from the media to produce purified chymotrypsin.

2. The method according to claim 1 wherein the hydrophobic interaction chromatographic media contains hydrophobic functional groups selected from the group consisting of alkyl, alkanoyl, phenyl, benzoyl, phenylalkyl, and phenylalkanoyl functional groups.

3. The method according to claim 2 where in the hydrophobic functional groups are selected from alkanoyl, benzoyl, and phenylalkanoyl functional groups and these functional groups are attached to polyethyleneimine ligands on a polymeric surface of a hydrophobic polymeric or inorganic chromatographic media .

4. The method according to claim 1 wherein the crude mixture is passed through the hydrophobic interaction chromatographic media under high salt conditions of from about 0.8 M to about 1.5 M salt and the bound chymotrypsin is eluted from the hydrophobic interaction chromatographic media under low salt conditions of from about 50 to about 100 mM salt.

5. The method according to claim 2 wherein hydrophobic interaction chromatographic media is one having alkanoyl, benzoyl or phenylalkanoyl groups attached to polyethyleneimine ligands on a polymeric surface of a polymeric polymethacrylate media.

6. A method according to claim 5 wherein the polymeric surface comprises a polymethacrylate polymeric surface and the alkyl, alkanoyl, phenylalkyl and phenylalkanoyl groups have from 1 to 8 carbon atoms in the alkyl or alkanoyl groups.

7. A method according to claim 6 wherein the alkyl, alkanoyl, phenylalkyl and phenylalkanoyl groups have from 1 to 3 carbon atoms in the alkyl or alkanoyl groups.

8. A method according to claim 7 wherein the functional groups are propanoyl groups.

9. A method according to claim 7 wherein the functional groups are butanoyl groups.

10. A method according to claim 7 wherein the functional groups are phenylpropanoyl groups.

1 1. A method according to claim 1 wherein the bound chymotrypsin is eluted with acetic acid.

12. A method according to claim 1 wherein the crude mixture is contacted with the hydrophobic interaction chromatographic media as a pH 3 solution of the crude mixture with ammonium sulfate.

13. A method according to claim 1 wherein the flow through purified trypsin is freeze-dried to a semi-crystalline powder.

14. A method according to any one of claims 1 to 13 wherein the purified trypsin has a trypsin activity of 3500 USP units/mg or greater and a trypsin/chymotrypsin activity ratio of more than 50.