Title of Invention

"A TWO DIMENSIONAL GEL ELECTROPHORESIS METHOD"

Abstract

A two dimensional gel electrophoresis method for analysing and determining the identity, protein, peptide patterns and stability of a digestive enzyme mixture with lipolytic, proteolytic and amylolytic activity, characterized in that said method comprises the steps of: (a) preparing the protein sample by dissolving the sample in a solvent composition for gel electrophoresis comprising a solvent suitable to dissolve protein materials, an internal standard for quantifying proteins, and a protease inhibiting agent; (b) defining the first dimension of the gel electrophoresis by isoelectric focussing, and applying a gradient to separate protein fractions; (c) subsequently re-buffering the protein fractions; (d) transferring the protein fractions from step (c) to the second dimension of the gel electrophoresis and separating components of the fractions by SDS-PAGE; (e) fixing and staining SDS-PAGE gels resulting from step (d); and (f) evaluating the gels densitometrically by fluorescence scanning.

Full Text

The present invention relates to a process for analyzing a protein. The present invention concerns a novel method to analyze identity, protein and/or peptide pattern and as well the stability of samples containing physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic acfivity, but especially of mixtures of digestive enzymes such as pancreatin, in particular in the context of the manufacture of medicinal products comprising said enzyme mixtures, e.g. precipitated pancreatin or pancreatin mini-microspheres. It is the objective of the invention to provide new analytical method suitable for pharmaceutical preparations comprising mixtures of digestive enzymes such as pancreatin, in particular also in the context of the manufacture of medicinal products comprising said enzyme mixtures, e.g. pancreatin or pancreatin mini-microspheres. In particular it is the object to provide an analytical method suitable and reliable to be validated for pharmaceutical manufacturing for analyzing and determining the identity, protein and/or peptide pattern and as well the stability of said digestive enzyme samples. A further object is to provide said analytical method in conditions which are optimized for the analysis of pancreatin, in particular precipitated pancreatin or pancreatin mini-microspheres samples. According to the invention, physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity, such as suitable enzyme mixtures of microbial origin and/or especially mixtures of digestive enzymes of animal origin such as preferably pancreatin or pancreatin-like mixtures of digestive enzymes, are analyzed according to the analytical methods essentially described in this patent specification. For the present invention, physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity can be analyzed that are of any animal or microbiological origin. The enzyme mixtures with lipolytic, proteolytic and amylolytic activity analyzed by the method of the invention can be both of purely microbial origin, purely animal origin or may also be a mixture of enzymes of animal and microbial origin. CONFIRMATION COPY In one variant of the invention, therefore, the enzyme mixture used is of purely microbial origin. Especially enzymes produced by bacteria, i. e. by the Bacillus or Pseudomonas strains, or by fungal cultures such as moulds, for example of the Rhizopus and Aspergillus strains, are especially suitable as microbial enzymes. Examples of such physiologically acceptable bacterial and/or mould fungi enzymes are already described in the state of the art, e. g. in connection with their synthesis and use for the treatment of maldigestion. Upases may be derived from, for example, Bacillus or Pseudomonas strains, amylases and fipases from mould fungi, for example of the Rhizopus strain, and proteases, for example, also from Aspergillus. One preferred variant of the invention, however, will involve the use of mixtures of digestive enzymes with lipolytic, proteolytic and amylolytic activity that in their properties closely resemble pancreatin. For the present invention, mixtures of digestive enzymes containing pancreatin and especially pancreatin itself are preferably used, and one or more microbial enzymes, i.e. enzymes synthesized by microorganisms, of the group of lipases, proteases and amylases may if desired be added to the pancreatin or the mixtures of digestive enzymes containing pancreatin. Most preferred the method according to the invention is suitable for analysis of precipitated pancreatin or pan-- • • • creatin mini-microspheres samples. Pancreatin is a known enzyme mixture with lipolytic, proteolytic and amylolytic activity which is available for example, under the trade name Creon®, in the form of granules, pellets or capsules containing enteric coated microspheres and is used medically for enzyme replacement, for example in pancreatic insufficiency, digestive insufficiency after stomach operations, liver and biliary diseases, cystic fibrosis and chronic pancreatitis. Pancreatin is generally obtained as a mixture of natural enzymes by extraction from porcine pancreas, for example according to the process described in German patent applications DE 25 12 746 and DE 42 03 315, and is then converted into the desired galenical form in a manner known to the art. The pancreatic enzymes are usually administered orally in the form of solid preparations. In one van'ant of the Invention, the pharmaceutical preparations to be analyzed in accordance with the invention contain preferably pancreatin or mixtures of digestive.enzymes containing pancreatin. These pharmaceutical preparations analyzed according to the invention can contain pancreatin or mixtures of digestive enzymes containing pancreatin and possibly in addition to pancreatin one or more physiologically acceptable enzymes from the group of lipases, proteases and amylases, of the kind that can be obtained from microorganisms. Microbial enzymes used in this supplement include especially the bacterially synthesized enzymes already mentioned above, for example by the Bacillus or Pseudomonas strains, or by fungal cultures such as mould fungi, for example of the Rhizopus or Aspergillus strains. The lipases contained in addition to the pancreatin or the mixtures of enzymes containing pancreatin may originate, for example, from Bacillus or Pseudomonas strains, added amylases and lipases from mould fungi, for example of the Rhizopus strain, and added proteases, for example, also from Aspergillus. It has now been found that the physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity such like pancreatin or non-animal sourced enzyme mixtures which can be obtained from microbial and/or animal sources and described with reference to this invention can be analyzed very efficiently according to the methods of the present invention. The invention provides a powerful and reliable (reproducible) method for e.g. analyzing and determining the identity, protein and/or peptide pattern and as well the stability of said digestive enzyme compositions or samples containing physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity. It te evident to the skilled artisan that he may Vary given parameters to a certain extent without loosing the overall functionality of the method according to the present inven^- tion; e.g. it may be wished to adapt the parameters indicated for performing the method in the following description, the examples, Tables and Figures by +/-10 %, in particular by +/- 5 %. Thus, the invention pertains to an analytical method for characterization and/or specification of protein samples containing physiologically acceptable digestive enzyme mixtures with lipolytic, proteolytic and amylolytic activity, which are used in the manufacture of pharmaceutical preparations for the treatment of disorders and/or disorders, by two dimensional gel electrophoresis (2D GE), said method is comprising (a) sample preparation by solving of an enzyme mixture sample in a solvent composition for gel electrophoresis comprising a specified solvent suitable to solve protein materials, an internal standard for quantification of proteins, and a protease Inhibiting agent; (b) an isoelectrical focussing step for defining the first dimension of the gel electrophoresis and applying a gradient for separation of the protein fractions; (c) a subsequent pre-treatment step comprising re-buffering; (d) transfer to the second dimension and separation by SDS-PAGE; (e) fixing and staining of the gels resulting from step (d); and (f) densitometrical evaluation by fluorescence scanning. The 2D GE method is particularly suited for analyzing and determining the identity, protein and/or peptide pattern and as well the stability of said digestive enzyme compositions or samples containing physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity, with molecular weights of the protein or peptide fractions from about 8 kDa (kilo Dalton) and above. In a variant the invention is particularly suited for analyzing and determining the identity, protein and/or peptide pattern and as well the stability of pancreatin, and in particular of precipitated pancreatin or pancreatin mini-microspheres. Parameters applicable in performing the method variants of the present invention are detailed below in the sections of the description pertaining to "Identifcation of spots using MALDI-TOF MS", "Stress Test Study for Precipitated Pancreatin", "Analytical Procedure for Determining Identity and Protein Pattern of Precipitated Pancreatin Samples by 2D Gel Electrophporesis", and related Tables, and are further illustrated by the Figures given in the context of this Invention. For digestive enzyme samples, e.g. pancreation and in particular precipitated pancreatin or pancreatin mini-microspheres, with molecular weights of the protein or peptide fractions below about 8 kDa (kilo Dalton) the method may be supplemented according to a variant of the Invention by additional application of analytical RP-HPLC method. In an aspect the invention as defined above pertains to analysis of an enzyme mixture of microbially synthesized lipases, proteases and amylases. In another aspect the invention as defined above pertains to analysis of a pancreatin and/or a pancreatin-like mixture of digestive enzymes. In yet another aspect the invention as defined above pertains to analysis of a'pancreatin'a sample* which is a precipitated pancreatin or a pancreatin mini-microspheres. In a further aspect the invention as defined above pertains to an analytical method, wherein the solvent used in step (a) to dissolve the sample is a lysis buffer of 7M urea, 2M thiourea, 4 % (w/v) CHAPS, 1 % (w/v) DTT, and 0.5 % Pharmalyte® at pH 3-10. In a yet further aspect the invention as defined above pertains to an analytical method, wherein the internal standard for quantification of proteins used in step (a) is phosphorylase B, preferably rabbit phosphorylase B, or carbonic anhydrase, preferably bovine carbonic anhydrase. In an additional aspect the invention as defined above pertains to an analytical method, wherein the protease inhibiting agent is Mini Complete and/or Pefabloc. In a further aspect the invention as defined above pertains to an analytical method, wherein the solvent used in step (a) to dissolve the sample is Lp3 composed of 1.5 mg Mini Complete dissolved in 2 ml lysis buffer of 7M urea, 2M thiourea, 4 % (w/v) CHAPS, 1 % (w/v) DTT, and 0.5 % Pharmalyte® at pH 3-10; and :1 mg Pefabloc dissolved in 2 ml lysis buffer; in a ration 1:1 v/v. The invention as defined above pertains also to an analytical 2D GE method, wherein said method is applied for the characterization and quantification of protein and/or peptide fractions with a molecular weight above about 8 kD. In a further aspect the invention as defined above pertains to an analytical method, which comprises determining the identity and/or the protein and/or peptide pattern of pancreatin, preferably of a precipitated pancreatin sample or of a pancreatin mini-microspheres sample. In another aspect the invention as defined above pertains to an analytical method, which comprises the identification of protein and/or peptide spots using in addition MALDI-TOF-MS. In one aspect the invention as defined above pertains to an analytical method, which is performed as a stress or stability test for determining the identity and/or the protein and/or peptide pattern of pancreatin, preferably of a precipitated pancreatin sample or of a pancreatin minimicrospheres sample, and impurities and/or degradanats, and optionally comprising also the quantification said proteins, peptides, impurities and /or degradants. In another aspect the invention as defined above pertains to an analytical method, wherein said method further comprises the characterization and quantification of low molecular weight protein and or peptide fractions with a molecular weight below about 8 kD by RP-HPLC. In still another aspect the invention as defined above pertains also to a solvent composition suitable for characterization and/or specification of a sample of physiologically acceptable enzyme mixtures with lipolytic, proteolytic and amylolytic activity, which are Used in the manufacture of pharmaceutical preparations for the treatment of disorders and/or disorders, by 2 dimensional gel electrophoresis, comprising (a) as solvent suitable for gel electrophoresis and to solve protein materials which solvent is a lysis buffer of 7M urea, 2M thiourea, 4 % (w/v) CHAPS, 1 % (w/v) DTT, and 0.5 % PharmalyteO'at pH 3- 10; (b) an internal standard for quantification of proteins; and (c) and a protease inhibiting agent. In a variant of this solvent, the is a solvent composition, wherein the solvent to dissolve the sample is Lp3 composed of 1.5 mg Mini Complete dissolved in 2 ml lysis buffer of 7M urea, 2M thiourea, 4 % (w/v) CHAPS, 1 % (w/v) DTT, and 0.5 % Pharmalyte® at pH 3-10; and :1 mg Pefabloc dissolved in 2 ml lysis buffer; in a ration 1:1 v/v. List of some Abbreviations and/or Terms used in the following: mms mini-microspheres (pancreatin mini-microspheres) HCI hydrochloric acid API active pharmaceutical ingredient NDA New Drug Application FDA Food and Drug Administration MALDI-TOF MS matrix assisted laser desorption and ionization mass spectroscopy UTLIEF ultrathin-layer isoelectric focusing ESI-MS electrospray ionization mass spectroscopy The analytical methods according to the invention, in particular after validation for pharmaceutical and regulatory purposes, are preferably intended to be used for characterization and specification setting of precipitated pancreatin and may also be applied to pancreatin enteric-coated minimierospheres (pancreatin mini-microspheres). For example, a product specification to be filed for the NDA for the active ingredient precipitated pancreatin and the dosage form pancreatin enteric-coated mms covers the items identification, purity, assay, gastric juice resistance and release of enzymes. State of the art identification is based on enzymatic assays which are used for determining the activity of the enzymes in both, the API and the dosage form. "Purity" also includes the determination of residual solvents (API, mms), fat (API), water (API, mms) and microbiological quality. For taking into account current FDA requirements and expectations based on Q6B Guidance "Specifications, Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products" more detailed characterization is regarded necessary for the drug substance and the dosage form with special attention to identification and quantification of different classes of enzymes, impurities and degradants from these enzymes. Results and methods from characterization will be selected for specification setting. Therefore the present invention proposes for characterization and specification setting the use of 2D Gel Electrophoresis (2D GE), because it was found that, since precipitated pancreatin is a complex mixture of different classes of constituents, two-dimensional gel-electrophoresis is expected to give by far the greatest selectivity for separation of peptides and proteins, i.e. different classes of enzymes, impurities and degradants of proteins. Furthermore, imaging of stained gels permits quantification of the constituents and comparison of the protein and/or peptide patterns in pancreatin samples, samples of precipitated pancreatin or pancreatin mini-microspheres. The present invention shows that identification of the most prominent spots can be performed by spotpicking and MALDI-TOF MS after tryptic digest In general, the separation by the 2D GE method according to the present invention will be performed in the first dimension (step (b) isoelectric focussing) from aqueous buffered solutions of pancreatin samples or mms samples, after desalting of the sample, on gels with a pH gradient from 3 to 10 to cover a broad range of potential constituents or compounds. Focussing is performed on immobiline dry strips. An exemplary gradient to start with is tabulated below: Step (2D GE isoeleetric focussing) 1 2 3 4 5 6 Voltage [V] up to aboout 150 300 600 1200 2400 3500 Time [h] up to about 4, preferably 1 4, preferably 1 3, preferably 1 1 1 8, preferably 7.25 In general, the separation in the second dimension (step (d) SDS-PAGE) of the 2D GE method according to the present invention will be performed on hand-made gels (for example under following condition:! = 13 % , C = 3 %) with e.g. a SDS-GLYCIN-TRIS buffer with an exemplary gradient as tabulated below: Step 1 2 3 Current [mA] about e.g. 80 150 10 Voltage [V] about e.g. Max 45 Max 200 For security In general, staining is performed after fixation with for example ethanol/ acetic acid mixture with a fluorescent dye and subsequent destaining in for example ethanol /acetic acid. After washing with water, densitometric scanning is performed. Afterwards, staining with for example colloidal Coomassie blue is performed for identification by MALDI-TOF MS. For this purpose, spots will be picked from the gel and subjected to a tryptic digest. Peptides are eluted from the gel for example with acetonitrile/0.1 % TFA and purification on a C18 ZipTip Column. After cocrystallization with 2i5-dihydroxy benzoic acid, the extracts are pipetted on the target plate. As an example to illustrate the applicability and usefulness of the present invention in the analytical method three batches each of three species of precipitated pancreatin (glands from different countries and different manufacturing processes), including Pancreatin SPL 85 were selected. One sample of each batch was applied on a gel, for one batch of each species the analysis was performed threefold to check reproducibility of the precipitation step, the sample preparation and the separation. The spots were quantified and identification of characteristic spots was performed. As an example to illustrate the applicability and usefulness of the present invention in stability testing, the same batches as used before to illustrate the applicability and usefulness were subjected to stress conditions (temperature, humidity, light) to determine any loss of activity and then to analyze or investigate also differences and, if applicable, identify potential degradants. As indicated above, for digestive enzyme samples, e.g. pancreatin and in particular precipitated pancreatin or pancreatin mini-microspheres, with molecular weights of the protein or peptide fractions below about 8 kDa (kilo Dalton) the method may be supplemented according to a variant of the invention by additional application of analytical RP-HPLC method. Parameters applicable in performing the RP-HPLC method variants of the present invention are detailed below in the section of the description pertaining to "Feasibility of RP-HPLC with MALDI-TOF-MS for Analysis of Pan- • creatin". Identifcation of spots using MALDI-TOF MS", "Stress Test Study for Precipitated Pancreatin", "Analytical Procedure for Determining Identity and Protein Pattern of Precipitated Pancreatin Samples by 2D Gel Electrophporesis", and related Tables, and are further illustrated by the Figures given in the context of this invention. HPLC is a widely automated, well reproducible, highly selective method which is widely used for routine analysis, also in protein analysis. Quantification of compounds is easy and identification of peaks can be performed by LC-ESI-MS. Peptides of lower molecular mass and other lowmolecular compounds can be detected and identified so that the method is complementary to e.g. 2D-gel-electrophoresis or SOS-PAGE. It can therefore be used in particular for fingerprinting, identification purposes and quantification of enzyme classes, impurities and degradants. Usually the HPLC method involves for example an agilent HPLC-equipment consisting of: Autosampler G 1313A; Quat. pump G 1311A; UV-detector G 1314A; Vacuum degasser G 1322A; HP Column Oven G1316A; 1100 control module G 1323A; LAN-interface 35900E; and Chem- Serven or an equivalent system. A typical HPLC column may be as an example a MODULO OCART QS UPTISPHERE 5 WRP, Interchim (UP5WRP$15QS) with a stationary phase of RP 18, 5.0 urn, tubing material of stainless steel with a length of 150 mm and an internal diameter of 3.0 mm; or a comparable equivalent HPLC column. The RP 18, 5.0 urn phase is beneficial for example, as it is possible to operate with 100 % water, and it is suitable for proteins and peptides. Further examples for suitable columns are e.g. Polaris 5 urn C18-A150 x 4.6 mm obtainable from Varian B.V., Middelburg, The Netherlands (article order no. A2000150X046); or e.g. Cogent Bidentate, C( (Octyl), 4 um, 300 A, 150 x 4.6 mm from MicroSolv Technology Corporation, Long Branch, NJ 07740, USA. The HPLC method may be operated under following exemplary conditions: Operating mode Mobile phase Gradient Gradient HPLC mobile phase A mobile phase B water/TFA 0.05 %(v/v) acetonitrile / TFA 0.05 % (v/v) Time [min] 0 75 75.1 80 %A 100 10 100 100 %B 0 90 0 " " 0 linear gradient to linear gradient to isocratic "equilibration" Flow rate Period of analysis Temperature Injection volume 1.0 ml/min 75 min 20 ± 5.0 CC 10 ul For detection for example an UV-detector may be used at a wavelength of 214 nm Identification of Spots using MALDI-TOF MS According to one aspect of the invention the analytical method comprises the identification of protein spots from a 2D gel, e.g. for a sample of precipitated pancreatin. This identification of spots using MALDI-TOF MS is described in more detail in the following paragraphs. The method procedures are described further below in more detail for 2D gel electrophoresis as well as for MALDITOF MS. The protein characterization is performed by establishing a peptide mass fingerprint from a wet gel obtained by performing a 2D gel method, and by additionally applying MALDI-MS/MS for spots which were not unambiguously identified. Peptide mass fingerprint: For peptide mass fingerprinting (PMF) the respective spots are manually cut out of the wet gel us- I'ng a manipulated pipette tip with a diameter of 0.2 cm. Each spot is then transferred into a single 10 tube (0.5 ml). The Coomassie blue-stained spots are destained using a special washing procedure: 1.100 pi of 10 mM ammoniumhydrogen carbonate, shaking for 5 minutes, 2.10 mM ammoniumhydrogen carbonate, 50% acetonitrile, shaking for 5 minutes. This procedure has to be repeated at least 3 times or until all spots are completely colorless. After the last washing step 5 ul of acetonitrile is added to each tube. When the spots are white, 2-6 ul of digestion buffer can be added, depending on the amount of gel within the tubes. The digestion buffer is 10 mM ammoniumhydrogen carbonate containing 0.01 ug/ul modified bovine trypsin (Roche Diagnostics, Basel, Switzerland). The digestion is performed over night at 37°C. After digestion the supernatant is removed, leaving the gel matrix in the tubes, if there is no supernatant left 5- ul of an extraction medium (1%TFA, 50 % acetonitrile) should be added. After 10 minutes of ultrasonification in a Bonification bath the tubes are transferred to a speedvac to remove the acetonitrile. Then the peptides are enriched and the samples are desalted by using a C18 Zip- Tip (Millipore, USA) according to the manufacturer's instructions. The extracted mixtures can now be transferred onto a MALDI-MS target (Applied Biosystems), A quantity of 0.1 ul of the sample is mixed with 0.1 ul of DHBS matrix (2,5 Dihydroxybenzoic acid: 2 Hydroxy-5 rriethoxy-benzoic acid • 9:1). The target is then measured by a MALDI mass spectrometer (Voyager STR, Applied Biosys- . terns, Foster City, CA, USA). As mass range In reflector mode 600-4200 Dalton (Da) was used. The low molecular weight range ( The PMF spectra are labeled and Internally calibrated onto 2 masses of known autotryptic peptides (805.42 Da; 2163.05 Da). The calibrated spectrum normally has an accuracy of less than 50 ppm. The data base searches for the PMFs is performed using the software Propound (Genomic Solutions, USA). Significant hits are reached when the gap between hit 1 and hit 2 is at least e-04 and the most intense masses can be explained or if the sequence coverage is relatively high (> 30%). The results of identification are tabulated below In Table A, and Fig. 1 shows a 2D-gel obtained with the labeling of identified spots from precipitated pancreatin. Furthermore, Fig. 2 depicts the high reproducibility of the method according to the present invention. Three gels were prepared of a single sample of precipitated pancreatin on four different days. MALDI-MS/MS: Protein spots that could not be identified unambiguously by PMF were chosen for MALDI-MS/MS using a Proteomics Analyzer 4700 (Applied Biosystems, Framingham, CA, USA). For this purpose, peptides with certain peak intensity were chosen and fragmented to obtain sequence information. The obtained fragment spectra were used to search the NCBI database (http://www.ncbi.nih.gov/), 11 National Center for Biotechnology Information, National Library of Medicine,Building 38A, Bethesda, MD 20894, USA using the Mascot software (Matrixscience, London, UK). Spectra exceeding a certain Mascot score are significant In questionable cases a manual control is performed. Table A shows the results obtained by MALDI-MS/MS. Stress Test Study for Precipitated Pancreatin ^According to a further aspect of the invention the 2D gel electrophoresis method is used for the monitoring of degradation processes and/or monitoring of the stability samples of precipitated pancreatin. This stability testing is described in more detail in the following test report pertaining to a /stress (stability) test for precipitated pancreatin. According to another aspect of the invention in a stress test the suitability of a 2D gel electrophoretic (2D GE) method was investigated for (a) stability testing when applied to precipitated pan- - creatin, (b) for locating potential degradation products formed during stress testing, and (c) for identifying spots which show degradation. The study revealed significant changes of some spots already identified, which are comparable for both types of precipitated pancreatin included in the study. The method is therefore regarded stability indicating. For the evaluation of the 2D GE method for its suitability for characterization of precipitated pancreatin samples of precipitated pancreatin were put on storage under stress-testing conditions and investigated according to the methods of the present invention. Aliquots of these samples were examined by 2D GE to determine the duration required to perform stress-testing and to look for spots which can be associated to degradation products of pancreatin. The stress testing was performed according to the analytical procedure "Identity and Protein Pattern by 2D Gel Electrophoresis" as described in detail below. For the stress testing the following samples were used: Sample Pankreatin N Pancreatin SPL 85 Storage Condition 40°/75% 40°/75% Time Points/days 0. 16. 32 0.15.28 Quantities of 20 g of precipitated pancreatin were filled in Petri dishes and were covered with a second Petri dish. The sample was put on storage at40°C/75% r.h. Samples of 100 mg were harvested in the beginning and after 1,2, 4, 8,16 and 32 days. Samples pulled after 0,16 and 32 days 12 were examined by 2D GE. All the samples were stored in a deep freeze, i.e. below -15°C. protected from light and humidity till they are were used for investigation. The results are shown in the Tables A to I and in the Figures 1 to 7 which depict the 2D gels obtained in the stress stability testing of precipitated pancreatin samples. The content of said Tables and Figures is summarized as follows: Contents of Tables A to I: Table A Spots from 2D-GE with Accession No. to NGBI Database (see also Fig. 1) Table B Spot intensities for Pancreatin Batch 1 (t=0 and 16 days) with average and standard deviation and regulation of spot vs. to Table C Spot intensities for Pancreatin Batch 1 (t=0 and 16 days) with average and standard deviation and regulation of spot vs. to Table D Spot intensities for Pancreatin Batch 1 (t=32 days) with average and standard deviation and regulation of spot vs. to Table E Spot intensities for Pancreatin Batch 1 (t=32 days) with average and standard deviation and regulation of spot vs. to Table F Spot intensities for Pancreatin Batch 2 (t=0 and 15 days) with average and standard deviation and regulation of spot vs. to Table G Spot intensities for Pancreatin Batch 2 (t=0 and 15 days) with average and standard deviation and regulation of spot vs. to Table H Spot Intensities for Pancreatin Batch 2 (t=32 days) with average and standard deviation and regulation of spot vs. to Table I Spot intensities for Pancreatin Batch 2 (t=32 days) with average and standard deviation and regulation of spot vs. to Contents of Figures 1 to 7: Fig. 1 2D-Gel obtained with Precipitated Pancreatin with identified spots labeled (Cf. also Data in Table J and K) Fig. 2 Reproducibility of the 2D-gel method with a single sample of precipitated pancreatin; 3 gels performed on 4 different days (IPG 3-1ONL, fluorescence dying; external standard carbonic anhydrase, applied quantity 320 ng) Fig. 3 2D-Gel obtained for Pancreatin batch 1 after Stress Testing (Sypro Ruby) Fig. 4 2D-Gels obtained for Pancreatin Batch 2 after Stress Testing (Sypro Ruby) Fig. 5 Averaged gels (n=3) calculated for Pancreatin Batch 1 Fig. 6 Averaged Gels (n=3) calculated for Pancreatin Batch 2 13 Fig. 7 2D Gel of Precipitated Pancreatin with addition of two internal marker proteins Phosphorylase B and Carboanhydrase, obtained as described in the section in the description pertaining to the Analytical Procedure "Identity and Protein Pattern" Analytical Procedure for determining "Identity and Protein Pattern of Precipitated Pancreatin Samples by 2D Gel Electrophoresis According to one aspect of the invention the 2D get electrophoresis method is used as analytical procedure for determining the identity and the protein pattern of precipitated pancreatin samples by 2D gel electrophoresis. This procedure is described herein in more detail using the following abbreviations: CHAPS 3-(3-cholamidopropyl)dimethylammonid-1-propane-sulfonate DTT Dithiothreitol TRIS Tris(hydroxymethyl)-aminomethane ' APS Ammonium persulfate TEMED N, N, N ",N '-tetramethylethylenediarmne SDS Sodium dodecylsulfate Description of the 2D GE Method applied: Two-dimensional gel elelctrophoresis (2D GE) separation technique (O'Farrell PH, J, Biol. Chem. 250:4007 - 4021 (1975)) takes advantage of the electrophoretic mobilities of individual constituents of a complex mixture of proteins, fractionating according to charge (pi) by isoelectric focusing (IEF) in the first dimension and according to size (Mr) by SDS-PAGE in the second dimension. In general the electrophoresis is performed according to the European Pharmacopeia (Ph. Eur. 2.2.31), and in this context the term "water" without qualification means double distilled or deionized water or water of an equivalent quality. In the method the following Materials and Reagents are applied: - Acetonitrile, e.g. Merck, Art.No. 1033530220 - Acrylamid-Solution, e.g. Serva, Art.No. 10688.02 - Agarose, e.g. VWR International, Art.No. 1.16802.0025 - Ammonium persulfate, e.g. Serva, Art.No. 13 375.01 - Bromphenolblue, e.g. VWR International, Art.No. 1.08122.0005 14 - 2-Butanol, e.g.:VWR International, Art.No. 8.22263.1000 - CHAPS, e.g. Roth, Art.No. 1479.2 - DTT, 1,4-Dithiothreitol, e.g. Roth, Art.No. 6908.2 - Electrode Paper, e.g. Amersham Biosciences, ArtNo. 80-1106-19 - Electrode paper stripes, e.g. Amersham Biosciences, AitNo. 18-1004-40 - Ethanol, e.g. VWR International, Art.No. TC212-9025 - Acetic Acid, e.g. Roth, Art.No. 3738.2 - Immobiline Dry Strips, pH 3-10NL, e.g. Amersham Biosciences, ArtNo. 17-1235-01 - Glycerine, e.g. Serva, Art.No. 23176 - Giyeine, e.g. Roth, ArLNo. 3908.3 - Urea, ag. Roche Diagnostics, Art. No. 1 685 902 - lodacetamide, e.g. Sigma, ArtNo. 1-6125 - PharmalyteTM 3-10, Amersham Biosciences, ArtNo. 17-0456-01 - Protein Test Mixture 4, Serva, Art.No. 39208.01 - Protein Test Mixture 5, Serva, Art.No. 39209.01 - Roti-Blue®-Concentrate,Art.No.,A152.1 - Sample Cups, e.g. Amersham Biosciences, ArtNo. 18-1004-35 - SDS, Sodium dodecyl sulfate, e.g. Serva, ArtNo. 20 763.02 - Silicon Oil, e.g. Serva, Art.No. 35132 - TEMED, e.g. Bio-Rad, ArtNo. 161-0800 - Thiourea, e.g. Fluka, ArtNo. 88810 - TRIS, for Electrode Buffer, e.g. Roth, Art.No. 4855.2 - TRIS, for all other solutions, e.g. Bio-Rad, Art.No. 161-0719 In the method the following Solutions are applied: mLvsis buffer: 7 M Urea, 2 M Thiourea, 4 % (w/v) CHAPS, 1% (w/v) DTT, 0.5% PharmalyteTM pH 3-10 (2} Solvent for Sample Lp3: 1.5 mg Mini Complete dissolved in 2 ml of Lysis buffer 1:1 mg Pefabloc dissolved in 2 ml Lysis buffer (1:1 v/v) (3) Rehvdration Solution: 6 M Urea, 2 M Thiourea, 4 % CHAPS, 0.2% DTT, 0.2% PharmalyteTM pH 3-10, some Bromphenolblue (4) Gel Solution (T=13 %. C=3%V 75 g Glycerol, 425 mL water, 375 mL Separation Gel Buffer (5), 630 mL Acrylamidlosung 15 (5) Separation Gel buffer: Dissolved 181.66 g TRIS, 4 g SDS in 900 ml of water and adjusted with hydrochloric acid R to a pH of 8.8, adjusted to a volume of 1000 ml using water (61 APS-Solution: 10 % (w/v) Ammonium persulfate in water (7) Glvcerol solution: 50% (v/v) Glycerol in water, add some bromophenolblue (8^ Butanol. saturated with water: 2-Butanol, stored above water (9^ Electrode Buffer 19.9 g SDS, 299.6 g Glycin, 58.0 g TRIS are dissolved in 20 1 water MO) DTT-Solution: 1 % DTT in Equilibration Buffer (11) lodacetamide Solution: 4% lodacetamide dissolved in Equilibration Buffer Eaulibration Buffer Dissolve 30 % Glycerol, 6M Urea, 4 % SDS and 33.40 ml Separation Gel buffer (5) in water and adjust the volume to 1000 ml (1 3) Agarose Solution: Dissolve 300 mg Agarose and some Bromophenolblue in 60 ml of Buffer(9) and boil until the solution becomes clear Protein Standard Solution: 10 mg each of Protein Test Mixture 4 and 5 are dissolved in 1 mi Lysispuffer (1). The solution is colored by adding a small qty. of bromophenol blue. Molecular weights of the proteins are: 6.5 kDa, 12.5 kDa, 21 kDa, 29 kDa, 45 kDa, 67 kDa, 97.4 kDa. (15) Ethanol / Acetic acid mixture: 7 % Acetic acid, 10 % Ethanol (16) Svoro Rubv Solution. Biorad Coomassie Solution: Add 180 ml of water and 60 ml of methanol to 60 mL Roti-Blue®-Concentrate while stirring 16 Description of Pefabloc SC: AEBSF 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride; it has the following characteristics: homogeneous in TLC; formula: C8H10N02SF x HCI; molecular weight: Mr = 239.5; specific, potent, and irreversible inhibitor of serine proteases. The inhibitory activity of Pefabloc SC is comparable to PMSF or DFP, however, it is non-toxic. Suggested Starting Concentration is 0.1 -1.0 mg/ml (0.4-4 mM). Description of Mini Complete: Complete Mini Protease Inhibitor Cocktail Tablets; it has the folloing product profile: specificity of inhibitor; mixture of several protease inhibitors with broad inhibitory specificity. Inhibits serine, cysteine, and metalloproteases, as well as calpains. Use for extracts from tissues or cells, including animals, plants, bacteria, yeast, and fungi. Contains both reversible and irreversible proteases. Solubility / Stability: soluble in aqueous buffers, or to be added directly to extraction media. Alternatively, prepare 7x stock solutions in 1.5 ml water or 100 mM phosphate buffer, pH 7.0. Stock solution is stable for 1-2 weeks at4 °C or at least 12 weeks at-20°C. All inhibitors in Complete can-be removed via dialysis. Use of a membrane with cutoff > 10 kDa is recommended. Complete can be used in thiol-contain/ng solutions at room temperature. Suggested starting concentration: dissolve one tablet in 10 ml aqueous buffer or water. If very high proteolytic activity is present, use one tablet for 7 ml buffer. The method is performed with the following Equipment or with suitable equivalent equipment known to the skilled artisan: - Ultrasonifier: Bandelin electronics, Sonoplus, HD 2070 - Reswelling Trav: Immobilize DryStrip Reswelling Tray for 7-18 cm IPG strips; Art.No. 80- 6371-84, Arhersham Biosciences - Electrophoresis Apparatus: Multiphor II, Art.No. 18-1018-06, Immobiline Dry Strip Kit, Art/No. 18-1004-30, Amersham Biosciences - Gel caster: DALT Multiple Gel Caster, Art.No. 80-6330-61, Amersham Biosciences - Casting Cassettes: Dalt Gel Cassette, Art. No. 80-6067-27, Amersham Biosciences - Separation Sheets: Separator Sheets, Art. No. 80-6436-63, Amersham Biosciences - Hoefer Dalt-Separation Chamber: IsoDalt Gel Electrophoresis System ID 440-230V; Art.No. 80-6068-98, Amersham Biosciences - Thermostating unit: MultiTemp III Thermostatic Circulator, Art.No. 18-1102-78, Amersham Biosciences 17 - Electrophoresis Power Supply: EPS 3501 XL Power Supply, Art.No. 18-1130-05, Amersham Biosciences - Fluorescence Scanner FLA3000 (Raytest) - ProteomWeaver 2.2 (Definiens AG, 80339 Munchen, Germany) (A) Sample Preparation 10 mg of the sample of precipitated pancreatin to be examined is dissolved in 500ul of Lp3. After shaking for 5 min at ambient temperature, the suspension is centrifuged. The clear supernatant is used for determination of the protein content (Bradford). Two different proteins are used as internal standards, i.e. phosphorylase b (from rabbit) and bovine carbonic anhydrase. These standards are added to the sample solution before separation in the first dimension is performed. To varying amounts K'5-15ul) of the clear supernatant 2ul of internal standard solution 1 and 6.7ul of internal standard solution 2 are added. Preparation of Internal Standard Solution 1 (phosphorylase b)": A solution of phosphorylase b is prepared - lyophilized (Sigma, Prod.No.: P-6635) in Lysis buffer 3 with a concentration of 1(jg / |Jl by shaking at 20°C for 30 minutes. Preparation of Internal Standard Solution 2 (carbonic anhydrase): A solution of carbonic anhydrase is prepared - lyophilized (Sigma, Prod.No.: P-6403) in Lysis buffer 3 with a concentration of 0.03 ug / ul by shaking at 20°C for 30 minutes. (B) Rehydration of Immobiline Dry Strips The ready-cut IPG-strips (Immmobiline Dry Strips (T=4%, C=2,7%, pH 3-10NL) are delivered in a dried and frozen status. Before use the strips are rehydrated over night in the Reswelling Tray. For 18 cm strips 350 uL of Rehydration Solution (3) are used. (C) Isoelectric Focussing (first dimension) (C.1) Preparation of rehvdrated Immobiline Dry Strips: The cooling block of the electrofocusing chamber is thermostated to 20°C. The rehydrated stripes are dipped into water and are placed, gel up, on a sheet of water saturated filter paper (electrode paper). Wet a second sheet of filter paper with water, blot it to remove excess water and put it onto 18 the surface of the IPG gel strips. Blot them gently for a few seconds to remove excess rehydration solution. (0.2) The procedure is performed according to the following steps 1 to 13: 1. The cooling plate is placed into the Multiphor II Electrophoresis unit. 5 ml of silicone oil is pipetted onto the cooling plate followed by positioning of the Immobiline DryStrip tray on the cooling plate. Trapping large air bubbles between the tray and the cooling plate is to be avoided. 2. Connection of the electrode leads on the tray to the Multiphor II unit. 3. Pouring of about 5 ml of silicone oil into the tray. 4. Placing of the Immobiline strip aligner into the tray on top of the oil. 5. Transferring of the rehydrated IPG gel strips (gel-side up and acidic end towards the anode) into adjacent grooves of the aligner in the tray. Aligning of the strips such that the anodic gel edges are lined up. 6. Cutting of two IEF electrode strips or paper strips prepared from 2 him thick filter paper (e.gr WIN 440, Macherey & Nagel, Germany) to a length corresponding to the width of all IPG gel strips lying in the tray. Soak the electrode strips with water, removing ecxessive moisture by blotting with filter paper and placing the moistened IEF electrode strips on top of the aligned strips near the cathode and anode. 7. Positioning of the^electrodes and pressing them gently down on top of the IEF electrode strips. 8. Putting the sample cups on the sample cup bar. Placing of the cups high enough on tiie bar to avoid touching the gel surface. The sample cup bar is put in a position that there is a distance of a few millimeters between the sample cups and the anode (or cathode, in case of cathodic sample application). 9. Moving of the sample cups into position, one sample cup above each IPG gel strip, and finally pressing down the sample cups to ensure good contact with each strip. 10. Once the sample cups are properly positioned, about 50 ml of silicone oil is pourred into the tray so that the IPG gel strips are completely covered. If the oil leaks into the sample cups, suck the oil out, the sample cups are re-adjusted and checked for leakage again. Filling up each sample cup with a few drops of siiicone oil. 11. The samples are pipetted into the cups by underlaying, and again watched for leakage. 12. The lid of the Multiphor II electrophoresis chamber is closed and the run according to the parameters (running conditions) given in the table below is started. For improved sample entry, 19 voltage is limited to low voltages (150-300 V) for the first few hours. Then it is continued to the steady state. Running Conditions for pH 3-1ONL: Step 1 2 3 4 5 6 Voltage [V] 150 300 600 1200 2400 3500 Time [h] 1 1 1 1 1 7.25 13. When the IEF run is completed, the electrodes, sample cup bar and IEF electrode strips are removed from the tray. Clean forceps are use and the IPG gel strips are removed from the tray. Those IPG gel strips which are not used-immediately-for second.dimension run and/or,are.kept for further reference are stored between two sheets of plastic film at -78°C up to several months. (D) Casting of gels for second dimension (SDS PAGE) One day before the casting of gels 1.5 L of gel solution are prepared and degassed and filtered. The solution is stored over night at4°C in a tightly closed flask. The reservoir of the casting chamber is closed with a funnel. 125 ml of glycerol solution are filled in. Immediately before casting 75 uL TEMED and 8 ml of APS-solution are added to the gel solution, while stirring and the solution is fifled into the casting chamber. After complete transfer of the gel solution, the funnel is removed and glycerol solution from the reservoir is added. The gel solution is now located in the casting cassettes of the casting chamber only. The gels are immediately overlayed with butanol, saturated with water, and the gels are polymerized for three hours. (E) Second dimension (SDS-PAGE) The SDS-PAGE procedure is performed according to the following steps 1 to 11: 1. The electrophoresis chamber is filled with Electrode buffer (9) and turned on cooling (13°C). The gels in the casting chamber are overlayed with some water until usage. 2. For each gel, a small strip of filtration paper is soaked with 5 ul of a protein standard solution 20 3. The SDS gel is supported in a vertical position to facilitate the application of the first dimension IPG strips. 4. The IPG gel strips are equilibrated as follows: The focused IPG gel strips are taken out of the freezer and they are placed into individual test tubes. 10 ml of equilibration buffer is added. The test tubes are sealed with Parafilm, they are rocked for 10 min on a shaker and then the equilibration buffer is poured off. 10 ml of iodoacetamide solution is added to the test tube as above and equilibrate for another 10 min on a rocker. 5. After the second equilibration, the IPG gel strip is rinsed with electrode buffer for a few seconds. 6. Excess water is removed from the gel, the IPG gel strip and the filtration paper with protein standard solution are placed besides on top of an SDS gel and overlayed with hot agarose solution (75°C). Carefully the IPG strip is pressed with a spatula onto the surface of the SDS gel to achieve complete contact The agarose is allowed to solidify for at least 5 min. This procedure is repeated for the remaining IPG strips. " . . 7. The gel cassettes are inserted into the electrophoresis apparatus and the electrophoresis is started. 8. The SDS-PAGE gels are run overnight as illustrated in the table below. Step 1 lasts for 50 Vh. Running conditions for second dimension (SDS-PAGE) Step 1 2 3 Current [mA] 80 150 10 Voltage [V] max. 45 max. 200 Last step for security [Vh] 50 Variable, see step 9 9. The run is terminated when the bromophenol blue tracking dye has reached the lower end of the gel. 10. The cassettes are carefully opened with a spatula, and a spatula is also used to remove the agarose overlay from the polyacrylamide gel. 11. The gels are removed from cassette holders and then immersed in water to remove the gel off the glass plate. Then it is continued with fixing, protein staining or blotting. 21 (R Fixing and Staining of Gels Each gel is fixed separately. Fixing is carried out with 350 mL of ethanol/acetic acid mixture for 30 minutes. Staining is performed for 3 hours with SyproRuby-solution (350 ml) protected from light. Destaining of the background is accomplished with 350 mL of Ethanol/Acetic acid mixture for 30 minutes, protected from light Before scanning, the gel is washed two times with water. If required, the gel is stained after scanning with colloidal Coomassie-solution over night The coloured gels are shaken the next day in water. When the background Js almost destained, scanning can be performed using the visual scanner. Evaluation of the Analytical 2D Gel Electrophoresis Procedure A typical 2D gel obtained with precipitated pancreatin is shown in Fig. 1 with identified spots labeled. All obtained images for evaluation of the 2D-gels are measured with a Fluorescence Scanner FLA3000 (Raytest). The gels are scanned as 16-bit files with a pixel size of 100(J. Excitation wavelength is 473nm. Evaluation of the tiff-files is done with ProteomWeaver 2.2 (Definiens). An experiment is performed by arranging the images in adequate groups. Then a spot detection over all gels is done, according to the algorithm of the software. The used settings for detection are default settings and are sufficient for almost all observable spots in the gels. After spot detection, all individual gels are rechecked manually to make sure that incorrect detection is reduced to a minimum. At the same time when the spot detection takes place an automatic quantification of the spots is performed by the software, and the quantities are normalized throughout the whole experiment to facilitate a comparison of spot identities of the different spots in the gels. This normalization process is independent of the number of gels in the experiment and can also be performed for those gels which are integrated into the experiment at different time points. A matching process is then started which assigns same spot identities to the identical spots throughout the gels. Furthermore, a typical 2D gel obtained with precipitated pancreatin with addition of two internal marker proteins Phosphorylase B and Carboanhydrase is shown in Fig. 7. Normalization, quantification, and the resulting matching process enables to compare the intensities of one spot referred to the intensities of the two internal marker proteins with a known amount of protein. In an example case according to the present invention phosphorylase B and carbonic anhydrase is spiked to all samples with a characteristic but always constant amount of protein (2ug and 0.2 ug, respectively). It was shown that both proteins perform a constant pattern in the 2D-gel and do not overlap with existing spots of the pancreatic sample. Carbonic anhydrase only results in one single spot, phos22 phorylase B shows up in at least 5 isoforms, but both proteins can be spot-detected and therefore quantified very easily. A direct comparison of the intensities of a spot of interest with the intensities of the two marker proteins leads to an absolute quantification as soon as the intensity of the spot is within the range of the intensity of the marker proteins. To achieve statistically significant results all gels are run in replicates, at least three runs, and the groups of gels are compared with each other. The resulting average intensity of the spots is the basis for the comparison of the spots among each other. Feasibility of RP-HPLC with MALDI-TOF-MS for Analysis of Pancreatin Subject of the feasibility study is to show the usefulness of RP-HPLC with MALDI-TOF-MS for analysis of pancreatin, in particular precipitated pancreatin or pancreatin mini-microspheres. In the following tests the Peptidomics® platform (BioVisioN AG, Hannover) was applied for the characterization of precipitated pancreatin as outlined in detail below incfuding detailed evaluation and validation with regard to specificity of the HPLC method As samples two batches of Pancreatin (batch 1 and batch 2) were used in the HPLC tests. The samples must be stored at 5 +/- 3°C, protected from light and humidity, e.g. in sealed bottles, until they are used for characterization. Before opening, the bottles should be adjusted to ambient conditions, e.g. by storing them at ambient conditions until equilibration is completed. The HPLC method outlined below was used to examine the samples. Splitting into fractions with subsequent characterization by MALDI-TOF-MS was performed according to standard protocols known to the skilled artisan, e.g. protocols according to the Peptidomics® platform. The liquid chromatography is performed according to the European Pharmacopoeia (Ph. Eur., 2.2.29). The protein and/or peptide pattern of characteristic constituents (compounds) of precipitated pancreatin is determined by gradient HPLC on a RP-18 reversed-phase column at the detection wavelength of 214 nm. Quantification is performed according to the Area % method. Generally, Ph. Eur. reagents are indicated by the letter R; quantities weighed or measured are commensurate with the degree of precision indicated in Ph. Eur. (1., General Notices). Furthermore, the term "water" without qualification means deionized water with a resistivity of NLT 0.18 Md m and a TOC of NMT 0.5 mg/ml. 23 The following reagents were used in the test experiments: • Acetonitrile for chromatography R; e.g. Baker, no.: 9017 • Trifluoroacetic acid R • Sodium chloride R • Solvent: Dissolution of 20.00 g of sodium chloride R in 11 of water (2 % NaCI solution) The following instruments or equivalent systems were used in the test experiments: Agilent HPLC-equipment consisting of. • Autosampler G 1313A • ALSTherm G1330A • Quat. pump.G 1311A • UV-detectorG1314A • Vacuum degasser G 1322A • HP Column Oven G1316A • 1100 control module G 1323A • LAN-interface 35900E • ChemServer Heraeus Biofuge 17RS or equivalent system. Column: . Type: MODULO O-CART QS UPTISPHERE 5 WRP, Interchim (UP5WRP$15QS) • Stationary phase: RP-18,5.0 urn • Tubing material: stainless steel • Length: 150 mm • Internal diameter: 3.0 mm The HPLC tests are operated under the following conditions: Operating mode Gradient HPLC Mobile phase mobile phase A water / TFA 0.05 % (v/v) mobile phase B acetonitrile / TFA 0.05 % (v/v) Gradient Time [min] 0 90 90.1 105 %A 100 46 100 100 %B 0 54 0 0 linear gradient to linear gradient to isocratic equilibration 24 Flow rate 1.0rnl/min Period of analysis 95min Temperature 27 ± 2.0 °C Injection volume 10ul Autosampler temperature 4°C ±1°C Detection by UV-detector at a wavelength of 214 nm. Assay Preparation: About 80.0 mg of the precipitated pancreatin to be examined are weighed in a 30 ml beaker. For examination of pancreatin enteric-coated mini-microspheres, the mini-microspheres must be grinded before and 140 mg of the powder are weighed in. The samples are to be dissolved in 10 ml of ice-cold Solvent while stirring at min; 15000 U/min; 4°C; For Heraeus Biofuge 17RS). The clear supernatant is to be injected as the sample solution. Sample Preparation has to be done freshly immediately before the sequence is started. Performance: Different batches of precipitated pancreatin and preparations thereof have already been examined by using the RP-HPLC method described above. The selectivity had been optimized to obtain the greatest number of peaks within a reasonable run-time. A typical chromatogram obtained with pancreatin batch 1 is depicted in the Figures 8 to 11 and Tables J and K.. Contents of Tables J and K: Table J Data for Spot Identification for Pancreatin (cf. Fig. 11): Identifications 20+ Table K Data for Spot Identification for Pancreatin (cf. Fig. 11): Identifications 20+X Contents of Figures 8 to 11: Fig. 8 Typical Chromatogram of Precipitated Pancreatin, Batch 1 Fig. 9 Typical Chromatogram of Precipitated Pancreatin (0-14 min), Batch 1 Fig. 10 Typical Chromatogram of Precipitated Pancreatin (60-90 min), Batch 1 Fig. 11 Annotated RP-HPLC Chromatogram of Precipitated Pancreatin (min 35), Batch 1 With regard to evaluation of specificity and identification of peaks, coupling of LC to ESI-MS was already tried but signals were overlapping. Therefore, by applying the Peptidomics® technology, a sample of precipitated pancreatin was examined. The chromatogram was split automatically in 96 25 fractions, with one fraction corresponding to a run-time of approximately 55.1 seconds. Then, fractions were automatically pipetted together with the matrix of sinapinic acid on a target plate. Each single spot was subjected to MALDI-TOF-MS with multiple desorption and ionization after automatic positioning. The mass range of interest to be considered covers m/z 1 to approximately 60,000. This range was visualized according to standard protocols for the Peptidomics® technology with quantification of single m/z signals. The m/z found were documented along with the corresponding fraction and the original chromatogram. Table A: identified Spots from 20-GE with Accession No. to NCBI Database: (cf.Fig.1) Spot No.: 156 644 813 1289 1455 1761 4132 5883 5993 1207 12359 1246 126 1261 12998 131-1 13190 1381 1710 1743 1767-1 1767-2 1817 1914 1974 2033 2046 Protein Name alpha-amylase, pancreatic - pig alpha-amylase [Sus scrota] alpha-amylase [Susscrofa] procarboxypeptidase B [Sus scrofa] carboxypeptidase A1 precursor [Sus scrofa] pig alpha-amylase androgen receptor [Sus scrofa] trypsin precursor - pig trypsin-precursor - pig phospholipase A2 (phosphatidylcholine 2-acylhydrolase) - pig Chain B, porcine E-Trypsin alpha-amylase [Scus scrofa] alpha-amylase - pig oviduct-specific glycoprotein precursor [Sus scrofa] triacylglycerol llpase - pig triacylglycerol lipase - pig alpha-amylase [scus scrofa] alpha-amylase, pancreatic - pig procarboxypeptidase B [Sus scrofa] alpha-amylase [Sus scrofa] phosphodiesterase 6B [Mus musculus] elastase, pancreatic - human steroid membrane binding protein triacylglycerol lipase, pancreatic (pancreatic lipase) - pig alpha-amylase [Sus scrofa] type III cytochrome P450 aromatase [Sus scrofa] chymotrypsin-like proteinase - pig Accession N° 67374 6056338 6056338 , 5457422 4336169 1942950 11559518 136429 136429 129436 999627 6056338 : 1942950 2493679 67161 67161 6056338 67374 5457422 6056338 6679255 88301 47522662 6686288 6056338 1762231 89257 Mr /pi 56.1/5.9 57.8/6.5 57.8/6.5 47.8/5.2 47.3 / 5.1 55.9/6.2 97.1/6.0 25.1/7.1 25.1/7.1 14.8/5.6 8.8/6.7 57.8/6.5 55.9/6.2 58.8/9.2 50.6/5.6 50.7/5.6 57.8/6.5 56.1/5.9 47.8/5.2 57.8/6.5 99.6/5.3 30.0/6.4 21.6/4.6 50.9/5.7 57.8/6.5 47.5/8.8 13.4/4.6 Sequence coverage 20% 39% 22% 60% 32% 20% 8% 54% 71% 41% 41% 38% 24% 50% 27% 12% 13% 62% 18% 22% Probability 6.9 e-11 5.6 e.43 1.6 e-04 4.4e-46 1.0 e-11 2.9 e-08 MS Fit 1e-03 4.3 e-14 3.8 e-08 2.0 e-18 1.4e-28 8.8 e-05 4.4e-27 5.4 e-14 6.3 e-05 MS Fit 1.6e-31 1.3 e-03 7.0 e-03 3O 1st V0 1 N> Table A: continued O1en Spot No.: 2066-1 2066-2 2068 223 2322 2380 2420 2524-1 2524-2 2622 2679 2712 278 2783 3316 4105 4318 4488 4488 4780 4790 5027 7 931 Protein Name transforming growth factor neta 1 [Sus scrofa] elastase isoform 2, pancreatic - human pig alpha-amylase ladinin 1 (Lad-1) envelope glycoprotein [Sus scrofa] pig alpha-amylase elastase 1, pancreatic - pig glucose-6-phosphat isomerase [Sus scrofa] N-acetyl-beta-D-glucosaminlde alpha-1,6-fucosy)transferase [Sus scrofa] phospholipaseA2 - pig alpha-amylase [Sus scrofa] pancreatic colipase - pig trypsin [Sus scrofa] elastase 2, precursor - pig alpha-1-antychymotrypsin [Sus scrofa] triacylglycerol (ipase, pancreatic - pig cvtochrome P-450 11A1 fSus scrofal alpha amylase [sus scrofa] alpha amylase [sus scrofa] 17a-hydroxylase cytochrome P450 cytochrome P-450-j [Sus scrofa] Chain C, Porcine E-Trypsin alpha-amylase, pancreatic (1,4-alpha-D-glucan glucanhydrolase) - pig alpha-amylase pancreatic - pig Accession N° 89305 7435612 1942950 12643530 37545606 1942950 355937 47523720 47522688 129436 6056338 1082974 136429 47523026 9968807 6686288 47523912 6056338 6056338 833797 47523896 999628 2811088 67374 Mr/ pi 44.3/8,9 56.0/6.0 57.2/9.7 24.3/8.7 55.9/6.2 29.3/9.1 63.177.9 66.2/7.4 14.8/5.6 57.8/6.5 10.9/5.6 25.1/7.0 28.7/8.3 22.9/5.8 50.1/5.7 60.3/9.1 57.8/6.5 57.8/6.5 56.4/8.9 57.1/8.1 10.5/8.7 56.0/5.8 56.1/5.9 Sequence coverage 14% 18% 21% 35% 14% 6% 41%. 27% 62% 34% 31% 11% 11% 16% 16% 5% 13% 28% 40% Probability 9.2e-fl7 MS Fit 8.5e-05 4.6e-05 MS Fit MS Fit 3.9e-06 1.9e-24 1.86-07 MS Fit 7.1 e-13 MS Fit MS Fit 1.96.07 1.9e-07 MS Fit MS Fit 5.1 e-23 3.7e-52 to -a ee wM tst Table B: Spot intensities for Pancreatin Batch 1 (t=0 and 16 days) to Average 1.1.2 4078.048 3792.744 3932.81 8021.147 6576.846 7263.184 9159.915 8065.862 8595.5 4035.89 4131.425 4949,297 5896.791 5402.312 9260.927 12851.92 10909.661 1868.685 2344.636 2093.177 4011.788 7697.141 5556.915 1990.661 1878.128 10298.58 13972.09 11995.528 5527.63 4269.753 7616.371 7807.967 7711.574 8936.962 9444.953 1112.352 1021.316 4748.027 4694.271 4721.072 963.999 977.224 5848.565 5616.127 5731.168 5586.802 7924.223 6653.65 5113.109 4722.385 4913.865 1 0 = no change during study, + = increase, - = decrease of intensity Degrada tion1 Spot ID 0 1207 12359 1246 + 126 1261 1289 0 12998 131 1381 1455 + 156 0 1767 1817 1914 1974 + 2033 2046 2066 2068 1.1.1 4078.C 8021.1 9159.S 4229.J 4949,: 9260.! 1868.( 4011.1 1771.! 10298 3298. 7616.: 9981 .i 937.7 4748. 990.6 5848. 5586. 5113. i iv uaya Sdv. 3.69 10.44 6.57 2.37 9.15 17.80 12.01 38.51 5.99 16.48 29.46 1.25 5.68 8.91 0.57 1.37 2.05 19.10 4.05 1 nun ayciai 16 days 1.2.1 3650.85 4696.391 215.146 6660.025 5164.268 11221.78 2248.892 4862.033 323.27 14085.23 3220.649 8839.614 4809.418 303.0J47 2172.757 2609.319 2736.'l7 4131.944 2062-239 |C aiiu aian 1.2.2 4045.124 4601.163 162.368 7209.74 5806.186 9950.6 2524.705 5331.125 351.962 15643.25 7817.308 9898.697 4414.818 414.163 2239.186 2329.106 2725.604 7178.561 2733,004 ucuu uwiai i.2.3 4337.619 4965.734 356.896 7165.684 3198.433 10055.62 2731.853 4533.823 455.117 14747.8 6538.588 10648.86 4460.521 428.131 2356.783 1884.798 2949.172 7058.878 2877.695 lun aiiu IGJJU Regulation ReftO 1.02 0.65 0.03 1.70 0.85 0.95 1.19 0.88 0.20 1.23 1.28 1.27 0.48 0.37 0.48 2.31 0.49 0.89 0.52 lauvni vi a}/v>i Average 4001.219 4751.949 231.878 7007.306 4577.331 10393.772 2493.899 4898.169 372.73 14811.739 5480.601 9767.23 4558.238 377.357 2254.968 2254.207 2801.788 5938.031 2531.287 va. iv sdv. 7.34 3.27 38.52 3.67 29.43 5.59 8.32 6.86 15.65 4.39 46.66 7.96 3.89 16.84 3.41 14.43 3.70 29.24 15.77 WO 2005/012911 i i j * ii idO i*.§00 ww to Degrada tion2 ;nsities for Pancreatin Batch 1 to pot ID 223 2380 2524 2679 2712 4105 4132 4488 4780 4790 7 1.1.1 2875.863 957.904 1158.452 1376.759 8153.147 2719.599 1696.162 3712.759 2867.793 1275.707 2530.903 1.1.2 2644.019 1614.924 1236.086 1477.267 7120.373 2687.769 1862.568 3650.984 2769.423 1454.507 2616.458 (t=0 and 16 days) with Average Sdv. 2757.505 1243.761 1196.639 1426.128 7619.281 2703.637 1777.419 3681.742 2818.179 1362.177 2573.325 [%] 4.29 29.84 3.30 3.59 7.01 0.59 4.79 0.84 1.76 6.78 1.68 average and standard deviation and regulation of spot vs. to 16 days Regulation Average 1.2.1 NA 1330.991 1285.804 867.595 8498.175 2014.855 NA 1590.102 NA N.A. 926.152 1.2.2 NA 1187.463 1222.937 754.456 9118.323 1172.338 NA 1855.598 N.A. NA 1065.491 sdv. 1.2.3 ReftO [%] NA NA 1368.273 945.068 9275.288 1170.661 NA 2122.936 NA NA 1093,86 0.00 1.01 1.08 0.60 1.18 0.52 0.00 0.50 0.00 0.00 0.40 NA 1257.18 1290.973 852.062 8957.553 1403.598 N.A. 1843.381 NA NA 1025.805 N.A. 5.87 4.70 9.73 3.86 29.13 NA 12.53 NA N.A. 7.58 % VO Kl VO 2 0 = no change during study, + = increase, - = decrease of intensity oo *>.oo 88 Table D: Spot intensities Degradation^ Spot 0 1207 12359 1246 + 126 1261 1289 0 12998 131 1381 1455 + 156 0 1767 1817 1914 1974 + 2033 2046 2066 2068 for Pancreatin Batch 1 (t=32 days) with 1.3.1 3979.887 5479.002 227.847 9162.12 6256.069 10337.988 1991.713 4112.75 N.A. 15705.897 6908.462 8853.396 5639.384 466.887 959.261 2014.629 3574.896 5628.97 958.091 1.3.2 3986.699 5931.415 424.84 8602.472 3729.149 9444.828 2320.616 4149.592 N.A. 12436.6 7318.195 7161.024 5499.357 492.839 947.507 1459.351 3794.235 4877.957 1250.93 average and standard deviation and regulation I.3.3 Regulation Ref tO 3718.964 3106.072 388.771 7805.051 3971.882 10833.402 2046.659 4294.907 N.A. 13143.237 5965.959 10372.317 6142.168 261.301 1110.288 2233.122 2834.71 3242.119 909.724 0.99 0.64 0.04 2.06 ; 0.84 0.93 1.01 : 0.75 0.00 1.14 1.57 = 1.13 0.61 0.38 0.21 1.92 i 0.59 0.67 0.21 Average sdv[%] 3893.158 4656.116 335.11 8504.817 4525.18 10189.002 2114.909 4185.016 N.A. 13692.694 6706.386 8696.015 5753.805 391.759 1003.04 1872.502 3375.195 4465.107 1029.24 3.29 33.39 31.68 6.82 25.92 5.86 6.88 1.89 N.A. 10.47 8.98 16.39 4.84 33.27 7.47 19.87 13.40 26.33 14.98 I OJo s 00a ' 0 = no change during study, + = increase, - = decrease of intensity Table E: Spot intensities for Pancreatin Batch 1 (t=32 days) with average and standard deviation and regulation of spot vs. to 32 days Degradation4 Spot 223 2380 2524 2679 0 2712 4105 4132 4488 4780 4790 7 1.3.1 N.A. N.A. 684.712 N.A. 8735.452 1273.323 N.A. 792.199 N.A. N.A. 645.021 1.3.2 N.A. N.A. 649.971 N.A. 6751.801 N.A. N.A. 828,132 N.A. N.A. 555.266 1.3.3 N.A. N.A, 721.914 N.A. 7378.296 N.A. N.A. 1044.736 N.A. N.A. 550.648 Regulation Average ReftO 0.00 0.00 0.57 0.00 0.99 0.47 0.00 0.24 0.00 0.00 0.23 •N.A. 'N.A. '684.903 • N.A. 7577.984 1273.323 N.A. • 881.685 .N.A. N.A. ' 582.08 sdv N.A. N.A. 4.38 N.A. 11.28 N.A. N.A. 12.90 N.A. N.A. . 7.54 OtoIo 4 0 = no change during study, += increase, - = decrease of intensity oog s Table F: Spot intensities for Pancreatin to Batch 2 (t=0 and 15 days) with average and standard deviation and regulation of spot vs. to 15 days 3" Spot ID 0 1207 12359 1246 + 126 1261 1289 12998 0 131 1381 1455 1761 1767 1817 1914 1974 ? 2033 2046 2066 2068 1.1.1 70410.727 213088.703 97149.695 151115.266 106723.523 190093.344 38122.777 88975.648 12974.204 314480.219 15691.019 212890.234 186074.484 8234.748 57885.094 19155.631 141966.344 166670.281 41946.742 1.1.2 70104.594 147347 104896.492 158836.781 105456.211 139408.188 35706.887 111207.109 12174.2 289503.844 20691.357 154120.344 199486.219 11394.64 50931.77 31398.879 110510.75 223431.359 50023.508 1.1.3. 78435.242 175938.297 101615.938 142001.266 130820.516 N.A. 32991.051 75701.594 15987.4 188071.266 13563.425 111851.688 211798.969 7151.643 58662.637 22173.328 71840.992 134839.516 43994.121 Average 72884 176774.938 101170.695 150493.313 113763.445 162789.953 35544.922 90817.508 13617.571 257744.875 16390.914 154247.953 198841,875 8754.921 55715.027 23714.492 104068.852 171240.5 45194.855 Sdv. [%] 5.33 16.26 3.20 4.69 10.40 16.77 6,09 17.08 12.34 25.28 19.14 30.05 5.43 21.54 6.58 23.03 32.48 23.01 7.72 1.2.1 ; 59955.207 187573.031 121943.484 191755.484 95200.484 151575.344 25088J56 68287.352 9131.455 255962.609 13345.886 144958.578 219332.703 7055.372 37022.191 18588.197 157937.969 95422.578 19809.289 1.2.2 62538.777 197302.594 136823.172 185599.875 93413.656 161213.063 23895.512 68784.063 9652.959 274785.219 11320.138 159549.328 204378.953 7289.475 37422.117 12040.256 139401,516 95208.539 23673.643 lKegui.- Average sav. 1.2.3 ReftO [%] 77464.266 190839.766 169640.141 212478.844 119793.773 173787.297 21836.1 96764.992 11400.876 386938.656 8699.047 129887.758 218279.266 6135.638 36630.828 12272.449 124661.922 97034.086 22384.219 0.91 1.09 1.40 1.30 0.90 0.99 0.66 0.85 0.74 1.17 0.67 0.94 1.08 0.78 0.66 0.59 1.35 0.56 0.48 66225.602 191862.766 141454.125 196281.969 102131.867 161938.063 23568.043 76886.266 10016.429 300795.031 10953.602 144289.828 213886.813 6808.09 37023.637 14004.516 140011.156 95884.953 21895.689 11.87 2.12 14.66 5.93 11.97 5.75 5.92 17.66 9.89 19.77 19.28 8.77 3.27 7.76 0.88 22.19 10.15 0.85 7.72 8 '012911 w OH oo •b.ii 5 Degradation: 0 = no change during study, + = increase, - = decrease of intensity Table G: Spot intensities for Pancreatin Batch 2 (t=0 and 15 days) with average and standard deviation and regulation of spot vs. to to 15 days >t> - - - 0 - 0 - - - - - Spot ID 223 2380 2524 2622 2679 2712 4132 4488 4780 4790 7 70134.547 20123.006 14514.978 88859.688 21288.855 154324.95 3 17919.049 35711.895 26554.484 10532.181 20862.508 1.1.1 71777.023 20353.197 14838.783 95788.625 20359.092 171617.51 6 18716.396 36804.625 32128.514 11177.513 20863.723 1.1.2 68743.836 29129.984 15556.002 109042.69 5 17436.951 153426.28 1 21376.762 39441.484 14705.147 7676.267 18351.205 Average 70207.539 22850.125 14963.65 97545.016 19624.281 159575.359 19282.338 37286.891 23236.246 9668.026 19989.762 Sdv. [%] 1.78 18.74 2.93 8.82 8.93 5.28 7.79 4.25 39.48 17.93 6.23 1.2.1 ! 101422.148 31887.355 8014.343 72266.852 9086.'135 123272.273 21209.465 19991.75 26276'.857 9546.;312 12104.132 1.2.2 99083.82 24744.543 8482.458 79633.148 10192.972 131906.54 7 21223.914 20630.113 28980.635 11794.334 11661.612 1.2.3 101165.242 15810.367 7274.182 100843.906 11742.738 128145.391 17571.139 19977.719 22816.873 8648.206 11381.058 Regul. ReftO 1.43 1.02 0.53 0.86 0.52 0.80 1.03 0.54 1.11 1.03 0.59 Average 100551.594 23192.449 7907.841 83411.813 10283.709 127725.688 19924.391 20197.584 25900.758 9911.629 11711.835 sdv. [%] 1.05 33.65 6.55 15.02 11.06 2.81 9.29 1.51 10.31 13.82 2.57 (£ Os O Io 2oo 8 ' Degradation : 0 = no change during study, + = increase, - = decrease of intensity Table H: Spot intensities for Pancreatin Batch 2 (1=32 days) with average and standard deviation and regulation of spot vs. to Degradation7 0 - - + - - - 0 - - - - - - - 7 - - Spot 1207 12359 1246 126 1261 1289 12998 131 1381 1455 1761 1767 1817 1914 1974 2033 2046 2066 2068 32 days 1.3.1 90339.25 113074.078 44936.344 209982.422 123483.938 156977.578 18334.098 105044.617 3661.021 225169.469 5739.493 75451.969 197082.078 5876.411 7621.188 31489.441 69711.344 73859.352 19679.99 1.3.2 82992.906 155135.547 36451.109 165129.609 89137.664 102686.602 18471.773 90399.398 N.A. 190251.938 N.A. 131763,344 134650.938 5032.68 6571.003 17034.555 130779.367 109141.953 13170.204 1.3.3 ; 90244.383 118655. 8 39889.012 213440.969 90827.406 99013.48-4 13933.649 98988.914 N.A. 170228.453 N.A. N.A. ;' 170172.078 N.A. ; 5024.076 31166.553 41067.059 71584.461 23304.211 Regulation ReftO 1.20 0.72 0.40 1.29 0.88 0.72 0.47 1.08 0.00 0.75 0.00 0.65 0.83 0.62 0.11 1.08 0.51 0.49 0.40 Average 87790.164 127679.484 40276.703 194877.594 99991.453 116864.313 16773.01 97958.461 N.A. 193920.438 N.A. 99708.594 165290.641 5438.207 6313.015 25569.85 53505.512 83254.039 18211.705 sdv 4.05 14.93 8.95 12.45 16.12 23.27 14.02 6.37 N.A. 12.19 N.A. 32.15 16.98 8.06 18.82 33.27 30.29 21.15 27.04 eno w hd a 5oo cB 7 0 = no change during study, + = increase, - » decrease of intensity Table!: Spot intensities for Pancreatin Batch 2 (t=32 days) with average and standard deviation and regulation of spot vs. tO 32 days Regulation Average sdv Degrada tion8 - .- - 0 - 0 - - - - Spot 223 2380 2524 2622 2679 2712 4132 4488 4780 4790 7 1.3.1 19150.346 N.A. 4161.9 94891.992 N.A. 153619 9918.519 7064.731 6016.258 N.A. 6380.595 1.3.2 16766.926 N.A. 5392.458 97860.477 N.A. N.A. 6933.719 5263.715 10940.175 N.A. 2987.297 1.3.3 17665.701 N.A. 5381.154 .93523.508 N.A. 153772.938 5683.137 5626.913 N.A. N.A. 4538.519 ReftO 0.25 0.00 0.33 0.98 0.00 0.96 0.38 0.16 0.35 0.00 0.22 17834.201 , N.A. 4942.932 1 95408.242 . N.A. 153695.953 7311.397 • 5936.804 . 8112.886 : N.A. j 4422 . 67 [%] 5.62 N.A. 12.93 1.91 N.A. 0.05 25.91 13.43 34.85 N.A. 36.39 ooe no MK > VO W 8wt o ' 0 = no change during study, + = increase, - = decrease of intensity I Table J: Data for Spot Identification for Pancreatin (cf. Fig. 11): Identifications 20+ Ul O fracb'on 17 36 47 48 51 52 SA l>4 54 55 5'>61 ProJefn Trypiin activation peptlde alpha-Amylase PSncrcntic Ribomdease component PhcephoBpaseAS, major enzyme alplia-Am^ase Colipase TrypsFn aJpha-Arti^ase Adijsoaeclln Tr>psfn CbKpase ActMa (SwissProfl P00761 POQ690 P00671 POOS92 PQ0690 P02703 P00761 P00690 QTYRFfl (TrEMBD P00761 P02703 Posten Mr 493-5TQ 92-124 (expi, kD^ 0,95 JjDa 1,9 kDa low abundant 43-146 (complete) 291-310, 422-444 23-95 67-103 290-310,285-310 225-243 67-TD2 23-104 13, 2/|OkDa;2,6kDa 7,9fcDa 4,04 Pa 2,46 kDa; 2, 2,18 kDa 3,94 kOa 9,03 ItDa Table K: Data for Spot Identification for Pancreatin (cf. Fig. 11): Identifications 20+X Fraction 62-64 62-64 67 69 69 70-71 70-71 72 73 75 76-78 03 65 Protein Trypstnogen Trypsin Elas1asfs2 TrtoeyJglycerol lipase Chymatrypsin A$ Chyrficrfrypsift C Chymotrypsn A/B Oiymotrypsirt M? Elastasel Coltpara A' alpha-Amyfasc. Cuboxypepfidase H Carboxypepfidase AT Acc.Na (SwissFVoO Postion Pno76i 1-231 (complete) .PGQ761 9-231 (tMrnplete)- P0.8419 23-269 (complete) ' POOS91 1 -450-{coinpleie) Q9ER05 (JV-ISOB motlSfi) 127-136,163-173 Q9ER05 CMSDB mouse} 163-173 q^EROS (MSD8 mouse) 56-68 P00772 27-266 fottnpfete) P027G3 23-108 (COmpJpH PG069Q 17-511 P0995S a6-40t P099S4 113-41ST, 115-419 Mriexp., kDa) 24.4 )cDa 23.5 kDa 25,8 kDa S0rl kDa 26,0-27,5 kDa 26,0- 27^1^ 26,0- 27,5 kOa 25,90 fcDa 9,28 IcDa 55.4 kDa 34.5 kDft 34,4 kDa, 33,9 kDa We claim 1. A two dimensional gel electrophoresis method for analysing and determining the identity, protein, peptide patterns and stability of a digestive enzyme mixture with lipolytic, proteolytic and amylolytic activity, characterized in that said method comprises the steps of: (a) preparing the protein sample by dissolving the sample in a solvent composition for gel electrophoresis comprising a solvent suitable to dissolve protein materials, an internal standard for quantifying proteins, and a protease inhibiting agent; (b) defining the first dimension of the gel electrophoresis by isoelectric focussing, and applying a gradient to separate protein fractions; (c) subsequently re-buffering the protein fractions; (d) transferring the protein fractions from step (c) to the second dimension of the gel electrophoresis and separating components of the fractions by SDS-PAGE; (e) fixing and staining SDS-PAGE gels resulting from step (d); and (f) evaluating the gels densitometrically by fluorescence scanning. 2. A process as claimed in claim 1, wherein the enzyme mixture is a mixture of microbially synthesized lipases, proteases and amylases. 3. A process as claimed in claim 1, wherein the enzyme mixture is pancreatin and/or a pancreatin-like mixture of digestive enzymes. 4. A process as claimed in claim 3, wherein the sample is precipitated pancreatin or pancreatin mini-microspheres. 5. A process as claimed in claim-1, wherein the solvent used in step (a) to dissolve the sample is a lysis buffer of 7M urea, 2M thiourea, 4 % (w/v) CHAPS, 1% (w/v) DTT, and 0.5% Pharmalyte® at pH 3-10. 6. A process as claimed in claim 1, wherein the internal standard for quantification of proteins used in step (a) is phosphorylase B, preferably rabbit phosphorylase B, or carbonic anhydrase, preferably bovine carbonic anhydrase. 7. A process as claimed in claim 1, wherein the protease inhibiting agent is Mini Complete and/or Pefabloc. 8. A process as claimed in claim 7, wherein the solvent used in step (a) to dissolve the sample is Lp3 composed of 1.5mg Mini Complete dissolved in 2 ml lysis buffer of 7M urea, 2M thiourea, 4 % (w/v) CHAPS, 1% (w/v) DTT, and 0.5 % Pharmalyte® at pH 3-10; and 1 mg Pefabloc dissolved in 2 ml lysis buffer; in a ratio of 1:1 w/v. 9. A process as claimed in claim 1, wherein the sample is a protein and/or a peptide fraction with a molecular weight above 8 kD. 10. A process as claimed in claim 9, optionally comprising characterizing and quantifying protein and/or peptide fractions with a molecular weight below 8 kD by RP-HPLC.

Documents:

979-DELNP-2006-Abstract-(02-06-2009).pdf

979-DELNP-2006-Abstract-(08-07-2009).pdf

979-delnp-2006-abstract.pdf

979-DELNP-2006-Claims-(02-06-2009).pdf

979-DELNP-2006-Claims-(08-07-2009).pdf

979-delnp-2006-Correspondence Others-(06-07-2012).pdf

979-delnp-2006-correspondence-others 1.pdf

979-DELNP-2006-Correspondence-Others-(02-06-2009).pdf

979-delnp-2006-correspondence-others.pdf

979-DELNP-2006-Description (Complete)-(02-06-2009).pdf

979-delnp-2006-description (complete).pdf

979-DELNP-2006-Drawings-(02-06-2009).pdf

979-delnp-2006-drawings.pdf

979-DELNP-2006-Form-1-(02-06-2009).pdf

979-DELNP-2006-Form-1-(08-07-2009).pdf

979-delnp-2006-form-1.pdf

979-delnp-2006-form-18.pdf

979-DELNP-2006-Form-2-(02-06-2009).pdf

979-DELNP-2006-Form-2-(08-07-2009).pdf

979-delnp-2006-form-2.pdf

979-DELNP-2006-Form-3-(02-06-2009).pdf

979-delnp-2006-form-3.pdf

979-delnp-2006-form-5.pdf

979-DELNP-2006-GPA-(02-06-2009).pdf

979-delnp-2006-gpa.pdf

979-DELNP-2006-Others-Domnuts-(02-06-2009).pdf

979-delnp-2006-pct-210.pdf

979-delnp-2006-pct-237.pdf

979-delnp-2006-pct-304.pdf

979-DELNP-2006-Petition-137-(02-06-2009).pdf

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