Collagenase I

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition
Issued and maintained by the United States Pharmacopeial Convention (USP)
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SANTASEXYO  FEYLNGUSYT  ELTAIKNIK  WNGINGLY  STORFFGD
KARVQATINA  LOESGRTYTA  NOMKGZETET  EVLRAGFYLG  YYNDGSYL
DANFODKICIP  ΑΝNIAIOKNPN  PKLUTAVODE  VITSUOKLIG  NASANAEVIW
NCVPVLEUGER  ENLIQYAPOY  VESTAVNELE  AGIEFDESGA  AYEXDVKTNP
WYGKIDPFIN  ELKALGLYEN  ITSATEWASD  VGTYYLSKFG  LYSTNINDIV
QSLEKAVERY  EVOKIARVAN  ERITWOYOGI  SINGKKVOND  KFLDDAEKHY
LPKTYTRENS TFIIRAGIEV SEEKIKRLYW ASREVKSOFH RVVGADKALE
VONADOVILTH INSPEEK FATNINGVST ONGGAVIER GTFYTYERTP
QOSIFSLEEL FRHEYTHYLQ ARYLVOGEWG OGPFYERNAL TWFOEGTAEF
FAUSTETSOV LPRESELGYL AKDKVDHRYS LIKTLINSGYD DSDWYNYG
FAVAKRYLYEK OΥΡΤΕΣΚΗΝΗ AILNTOVKSY DEIIKKLSOD ANKNTEYQNH
IQELADKYOG AGIPLVSDOV LEONGYKEAS EVYSEISKAA SLTNTSVTAE
KSQVENTETL RGTYTGETSK GEFKOWDENS KKLOGTLESL AKNSWSGYIKT
LTAYPTNIRV TSUNKVQYDV VFHOULTONA DESANKAPIA KVTGPSTGAV
GANTEPSGAD SADDEDGALTYS YONOFSOGAT SAGKNSVHAY KKAGTYNVTL
EVTODIKGATA TESPTICIAN EDITΤΡΙΤΚΕ ΜΕΡΙΔΟΣKEA NGPIVEGVTV
KEDUNGSODA STFYFDVKED GOVTIELPYS GSSAFTLVY KEGDDDNIA
SGZORNNSAY GTFESTEGAH YVFTYKHOSA SNISYSLNIK GLGNEKLKEK
ENNDSSORAT VIPNENTTHQ GSLLGEDERD VYSFEVKEEG EVNIELDKKD
EFOVTWTLHP ESNINDRITY GOVDGRKYSN KVKLRPGKYY LLVYKYSGSG
MYELRYNE

C₅₀₉₉H₇₇₇₁N₁₃₂₉O₁₆₁₀S₁₄  113,897 daltons (for Beta subtype) CAS RN 9001-12-1.

1 DEFINITION

Collagenase I (EC 3.4.24.3), isolated from Clostridium histolyticum and encoded by colG gene (GenBank accession number BAA77453.1), is a key raw material used in the dissociation or destruction of a broad range of tissue types. Collagenase I is a metalloprotease that acts as an endoprotease and also exhibits a tripeptidylcarboxypeptidase activity. It shows endopeptidic activity with the main cleavage site found in front of the human Collagen duplex amino acids glycine-proline, Hydrolysis takes place near the ends of the triple helical domains of collagen.

Collagenase I is also known as class I collagenase and consists of three subtypes a, l, and y, Collagenase I fl is the full-length enzyme while collagenase 1 a (68,000 Da) and collagenase 1 y (79,000 Da) are thought to be proteolytic degradation products of collagenase 1 caused by other proteases present in C. histolyticum (mainly a trypsin-like enzyme and clostripain/endoproteinase Arg C)

Collagenase i can be provided in a liquid formulation consisting of 5 mM N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid (HEPES) and 1 mM calcium chloride pH 7.5, and stored as a frozen liquid. The specific activity of collagenase I is 0.10-0.60 units/mg of protein. using 4-phenylazobenzyłoxycarbonyl (PZ)-Pro-Leu-Gly-Pro-o-Arg as the substrate described in the Assay. The peak area for collagenase is NLT 90% as determined by HPLC described in the test for Purity. The test for Clostripain Activity is used to assess the activity of the clostripain impurity and the acceptance criterion is NMT 0.5 units/mg of protein. The test for Trypsin Activity is used to assess the activity of the trypsin-like enzyme impurity and the acceptance criterion is NMT 0.5 units/mg of protein.

2 IDENTIFICATION

A. It meets the requirements in the Assay.

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the test for Purity,

3 ASSAY

3.1 PROCEDURE

Tris buffer: 0.1 M Tris pH 7.1, prepared as follows. Dissolve 6.05 g of tris(hydroxymethyl)aminomethane (Tris) in 400 ml of water, and adjust with 2 N hydrochloric acid to a pH of 7.1 (at 25 ± 1°). Dilute with water to a final volume of 500 ml..

Substrate solution: Dissolve 10 mg of PZ-Pro-Leu-Gly-Pro-o-Arg in 0.2 ml. of methanol, and dilute this solution with Tris buffer to a final volume of 10 mL (Non-Use a freshly prepared solution only.

Calcium chloride solution: 0.1 M. prepared as follows. Weigh 1.47 g of calcium chloride dihydrate in a volumetric flask, and dilute with water to a final volume of 100 mL

Citric acid solution: 0.025 M, prepared as follows. Weigh 525 mg of citric acid monohydrate in a volumetric flask, and dilute with water to a final volume of 100 mL

Extraction mixture: To one test tube per sample to be assayed, pipette 5.0 mL of ethyl acetate and 1.0 ml. of Citric acid solution. [NOTE-Use a freshly prepared mixture only.]

Drying tube: Into one test tube per sample to be assayed, add 0.35-0.40 g of sodium sulfate anhydrous. Seal the test tube with parafilm.

Standard solution: Dilute USP Collagenase IRS with Tris buffer in the range of 1:50 to 1:100 (v/v). [Νοτε-Avoid freezing and thawing USP Collagenase IRS. After withdrawing USP Collagenase IRS, wipe off the outside of the plastic pipette tips to remove any residual solution.]

Sample solutions: Dilute collagenase I with Tris buffer to an appropriate dilution to achieve the absorbance range of 0.3-0.9 from the Analysis. Prepare in triplicate. [NOTE-Avoid freezing and thawing the collagenase I sample. After withdrawing the collagenase I sample, wipe off the outside of the tip to remove any residual solution.]

3.2 Instrumental conditions.

(See Ultraviolet-Visible Spectroscopy (857).)

Mode: UV

Analytical wavelength: 320 nm

Path length: 1 cm

Temperature: 25"

3.3 Analysis

Samples: Standard solution and Sample solutions.

Transfer 1.0 mL of Substrate solution and 0.2 mL of Calcium chloride solution into a test tube, and equilibrate the test tube to 25".

Start the reaction by adding 0.05 mL of Standard solution or each Sample solution. Prepare a blank by replacing the Standard solution or Sample solution with 0.05 mL of Tris buffer. Mix and incubate for exactly 15 min at 25". Transfer 0.5 mL of the reaction to the test tube containing 6.0 mL of Extraction mixture. Vortex immediately for 20 s. Transfer 3 mL of the ethyl acetate phase (upper layer) into a Drying tube using a glass pipette, and vortex thoroughly. Transfer the supernatant to a disposable, semi-micro cuvette suitable for UV absorbance with a Pasteur pipette. Record the absorbance.

Calculate the activity of collagenase I in units/mL:

Result = (A-A_B) x [V_txV_ε/(εxVxV_RxBxT)] x D

A = absorbance of the Standard solution or Sample solution

A_B = absorbance of the blank

V_t = volume of the reaction mixture, 1.25 ml.

V_ε = volume of ethyl acetate in the Extraction mixture, 5.0 ml.

ε = extinction coefficient for 320 nm, 21 (1 cm² µmol1)

V = volume of the Standard solution or

V_R = volume of the reaction transferred to 0.5 mL

B = absorption path length, 1 cm incubation time, 15 min T

D = dilution factor

OFFICIAL [NOTE-One unit will release the equivalent of 1 µmol of PZ-Pro-Leu from PZ-Pro-Leu-Gly-Pro--Arg per minute under the conditions of the Assay.]

Calculate the specific activity of collagenase I in units/mg of protein:

Result = Activity/C

Activity = activity of collagenase I (units/ml.)

C = protein concentration (mg/mL)

3.4 System suitability

Samples: Standard solution and Sample solutions

Suitability requirements

Average calculated activity: 85%-115% of the value on the label, Standard solution

Absorbance: 0.3-0.9, Standard solution and Sample solutions

Acceptance criteria: 0.10-0.60 units/mg of protein

4 PURITY

4.1 PROCEDURE

Solution A: 20 mM Tris and 1 mM calcium chloride pH 7.5, prepared as follows. Dissolve 2.42 g of Tris and 147 mg of calcium chloride dihydrate in 900 ml of water. Adjust with 2 N hydrochloric acid to a pH of 7.5. Dilute with water to a final volume of 1000 mL

Solution B: 20 mM Tris, 1 mM calcium chloride, and 1 M sodium chloride pH 7.5, prepared as follows. Dissolve 2.42 g of Tris, 147 mg of calcium chloride dihydrate, and 58.44 g of sodium chloride in 900 mL of water. Adjust with 2 N hydrochloric acid to a pH of 7.5. Dilute with water to a final volume of 1000 mL.

Mobile phase: See Table 1.

Table 1

Storage buffer: 5 mM HEPES and 1 mM calcium chloride pH 7.5, prepared as follows. Dissolve 1.19 g of HEPES and 147 mg of calcium chloride dihydrate in 900 mL of water. Adjust with 4 N sodium hydroxide solution to a pH of 7.5. Dilute with water to a final volume of 1000 mL.

Standard solution: Thaw USP Collagenase I RS at room temperature shortly before use and mix. Store on ice or at 5°. Dilute with Storage buffer to achieve a protein concentration of 5.5 mg/mL. Transfer to an HPLC vial and keep at 5°. Prepare in duplicate, and inject each duplicate once.

Collagenase II solution: Thaw USP Collagenase II RS at room temperature shortly before use and mix. Store on ice or at 5°. Dilute with Storage buffer to achieve a protein concentration of 5.5 mg/mL. Transfer to an HPLC vial and keep at 5". Prepare in duplicate, and inject each duplicate once.

Sample solution: Dilute collagenase I with Storage buffer to achieve a protein concentration of 5.5 mg/mL and keep at 5°.

Blank: Storage buffer

4.2 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 5-mm x 5-cm; 10-µm packing L91

4.3 Temperatures

Column 25°

Autosampler: 5°

Flow rate: 1.5 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

Suitability requirement: The chromatogram from the Standard solution corresponds to the typical chromatogram provided with USP Collagenase I RS.

4.4 Analysis

Sample: Sample solution

The Blank should be considered for integration. Identify the peak corresponding to collagenase II by comparing its retention time to the main peak from the Collagenase II solution. Evaluate the purity of collagenase I using the area-% method but excluding peaks associated with the Blank. All shoulders in the fronting and tailing of the main peak are integrated by dropping a perpendicular line at the inflection points and considered as separate impurities. Disregard any peaks having retention times greater than 25 min.

Acceptance criteria: NLT 90% for the main peak of collagenase I and NMT 3% of collagenase II

5 IMPURITIES

5.1 CLOSTRIPAIN ACTIVITY

Potassium phosphate buffer: 0.1 M pH 7.6, prepared as follows. Dissolve 1.36 g of monobasic potassium phosphate in water, and dilute to 100 mL. Dissolve 2.28 g of dibasic potassium phosphate trihydrate in water, and dilute to 100 mL. Adjust the pH of the second solution to 7.6 with the first solution.

Dithiothreitol solution: 0.194 M, prepared as follows. Dissolve 60 mg of dithiothreitol (DTT) in 2 mL of Potassium phosphate buffer.

Calcium chloride solution: 0.01 M, prepared as follows. Dissolve 147 mg of calcium chloride dihydrate in 100 mL of water.

Substrate stock solution: 38 mM, prepared as follows. Dissolve 13 mg of N-benzoyl--arginine ethyl ester hydrochloride (BAEE-HCI) in 1 mL of Potassium phosphate buffer.

Substrate solution: 0.73 mM BAEE HCL, 7.8 mM DTT, and 0.4 mM calcium chloride, prepared as follows. Transfer 0.5 ml. of Substrate stock solution, 1.0 mL of Dithiothreitol solution, and 1.0 ml of Calcium chloride solution to a 25-ml volumetric flask, and dilute with Potassium phosphate buffer to volume.

Sample solution: Prepare in such a way that 4A/min lies in the 0.02-0.06 range. Dilute with ice-cold Potassium phosphate buffer if necessary.

5.1.1 Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857).)

Mode: UV

Analytical wavelength: 255 nm

Path length: 1 cm

Temperature: 25°

5.1.2 Analysis

Sample: Sample solution

Transfer 3.0 mL of Substrate solution into a cuvette, and equilibrate the cuvette to 25°. Start the reaction by adding 0.05 ml. of Sample solution. Prepare a blank by replacing the Sample solution with 0.05 mL of Potassium phosphate buffer. Mix well. Determine the change in absorbance (4A/min) from the linear range of the reaction. Assay the Sample solution in triplicate.

5.1.3 System suitability

Sample: Sample solution

Suitability requirement: 0.02-0.06 for AA/min

Calculate the activity of clostripain in units/ml, in the portion of collagenase I taken:

Result = [V_t/(ε x V x B)] x ΔA/min x D

V_t = volume of the reaction mixture, 3.05 mL

ε = extinction coefficient for 255 nm, 0.81 (1 cm² mmol)

V = volume of the Sample solution, 0.05 mL

B = absorption path length, 1 cm

ΔA/min = change in absorbance from the linear range of the reaction

D = dilution factor

Calculate the specific activity in units/mg of protein:

Result = Activity/C

Activity activity of clostripain (units/mL)

C = protein concentration (mg/ml.)

Acceptance criteria: NMT 0.5 units of clostripain activity per mg of protein

5.2 TRYPSIN ACTIVITY

Buffer: 0.1 M Tris and 0.02 M calcium chloride pH 8.0, prepared as follows. Dissolve 6.05 g of Tris and 1.45 g of calcium chloride dihydrate in 400 mL of water. Adjust with 2 N hydrochloric acid to a pH of 8.0 (at 25±1°). Dilute with water to a final volume of 500 mL.

Substrate stock solution: Dissolve 10 mg of carbobenzoxy-valyl-glycyl-arginine-4-nitril-anilide acetate, accurately weighed, in 1.5 mL of water. Store on ice. [NOTE-Use freshly prepared solution only.)

Substrate solution: Prepare a solution by mixing 9.2 mL of Buffer and 1.0 mL of Substrate stock solution. Store on ice. [Non-Use freshly prepared solution only.)

Sample solution: Undiluted collagenase I solution

5.2.1 Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857).)

Mode: Vis

Analytical wavelength: 405 nm

Path length: 1 cm

Temperature: 25

5.2.2 Analysis

Sample: Sample solution

Transfer 1.02 ml. of Substrate solution into a polystyrene, semi-micro cuvette, allow the temperature to stabilize, equilibrate the cuvette to 25", and wait for 10 min. Start the reaction by adding 0.10 ml. of Sample solution, Start recording the absorbance and continue for at least 5 min after the addition of the Sample solution. Determine the change in absorbance (AA/min) from the linear range of the reaction. Assay the Sample solution in triplicate. [NOTE-Use polyethylene pipette tips to transfer the Sample solution. The pipette tip should not be wet before transfer, and each pipette tip should be used only for transferring one sample. After withdrawing the Sample solution, wipe off the outside of the tip to remove any residual solution. After adding the Sample solution to the Substrate solution, rinse the tip by pipetting the solution up and down 2-3 times, discard the tip, and mix.]

5.2.3 System suitability

Sample: Sample solution

Suitability requirement: 0.01 for AA/min

Calculate the activity of trypsin in units/mL in the portion of collagenase I taken

Result = (V_t /(ε x V_u x B)] x ΔA/min x D

V_t = volume of the reaction mixture, 1.12 mL

ε = extinction coefficient for 405 nm, 10.4 (1 cm² mmol)

V = volume of the Sample solution, 0.10 mL

B = absorption path length, 1 cm

ΔA/min = change in absorbance from the linear range of the reaction

D = dilution factor

Calculate the specific activity in units/mg of protein:

Result = Activity/C

Activity = activity of trypsin (units/mL)

C = protein concentration (mg/ml)

Acceptance criteria: NMT 0.5 units of trypsin activity per mg of protein

6 SPECIFIC TESTS

6.1 PROTEIN CONTENT

Sample solutions: Dilute collagenase I in water. Prepare at least in triplicate. [Nors-Prepare the dilution using plastic pipette tips and not glass pipettes. Carefully wipe off the outside of the tip to remove any residual solution.]

Blank solution: Water

6.1.1 Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857)

Mode: UV

Analytical wavelength: 280 nm\

Path length: 1 cm

6.1.2 System suitability

Samples: Sample solutions

Suitability requirement: Absorbance is in the range of 0.10-1.00.

6.1.3 Analysis

Samples: Sample solutions and Blank solution

Determine the net absorbance of Sample solutions by subtracting the absorbance of the Blank solution from the absorbance of each Sample solution. Determine the average net absorbance of Sample solutions.

Calculate the protein concentration in mg/mL:

Result = A_u x D/ε

A_u = average net absorbance of the Sample solutions

D = dilution factor

ε = extinction coefficient (A, 0.1%/cm) for collagenase, 1.4

BACTERIAL ENDOTOXINS TEST (85); NMT 50 USP Endotoxin Units/mg of protein

MICROBIAL ENUMERATION TESTS (61): The total bacterial count is NMT 100 cfu/ml.

7 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Store in closed containers at -15° to -25"

LABELING: The labeling states that the material is derived from Clostridium histolyticum along with the lot number, product or catalog number, and storage conditions.

USP REFERENCE STANDARDS (11)

USP Collagenase R

USP Collagenos

LISP Endotoxin