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Frequently Asked Questions

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General Information

1. What are collagenases?

Collagenases are metalloproteases originating from the gram positive bacterium Clostridium histolyticum. There are two classes of clostridial collagenase, which are collagenase class I and collagenase class II. They are generated by expression of their respective genes colG and colH.

Collagenases cleave peptide bonds in the triple helical collagen molecule of human or animal tissue in situ. Both classes differ in their activity towards the collagen molecule. It is assumed that collagenase class I cleaves the intact collagen helix into smaller peptides which are a substrate to collagenase class II and that thus both classes act synergistically in efficient digestion of collagen. All Collagenase NB products contain both collagenase classes.

2. What is neutral protease and clostripain?

Neutral protease and clostripain are enzymes originating from the gram positive bacterium Clostridium histolyticum. Both are mostly used in combination with collagenase for tissue dissociation.

The neutral protease is a metalloprotease and cuts diverse peptides in front of hydrophobic amino acids and thereby creates smaller fragments, whereas clostripain, also known as Clostridiopeptidase B or Endoproteinase Arg-C, is a proteinase that cleaves peptide bonds on the carboxyl side of arginine. This protein is a heterodimer composed of a heavy and a light chain that are held together via strong noncovalent forces. 

3. What are the differences between the Nordmark Collagenase NB and Neutral Protease NB products?

Nordmark’s portfolio includes several Collagenase NB and Neutral Protease NB products which can be differentiated according to their purification level (unpurified, purified, highly purified) and field of application (research, clinical).

Collagenase NB 4 Standard Grade (Cat. No. S1745401) and Collagenase NB 4G Proved Grade (Cat. No. S1746501) contain a balanced mix of proteolytic activities. Collagenase NB 4G Proved Grade has a higher collagenase activity (≥ 0.18 PZ U/mg) compared to Collagenase NB 4 Standard Grade (≥ 0.10 PZ U/mg). The proteolytic side activities are also increased accordingly.

Collagenase NB 5 Sterile Grade (Cat. No. N0002779) and Collagenase NB 6 GMP Grade (Cat. No. N0002779) show comparable enzymatic properties to Collagenase NB 4 Standard Grade with a collagenase activity of ≥ 0.10 PZ U/mg and a balanced amount of proteolytic side activities. Collagenase NB 5 Sterile Grade is sterile according to Pharm. Eur. Collagenase NB 6 GMP Grade is manufactured according to cGMP guidelines and is also sterile according to Pharm. Eur. 

Collagenase NB 7D Gentle Grade (Cat. No. S1747401, S1747402, S1747403) is a partially purified collagenase with a collagenase activity of ≥ 0.2 U/mg, between that of the natural blend products and Collagenase NB 8 Broad Range.

Collagenase NB 8 Broad Range (Cat. No. S1745601) is a purified collagenase. Thus it contains higher collagenase activity (≥ 0.9 PZ U/mg) and reduced proteolytic side activities.

Collagenase NB 1 Premium Grade (Cat. No. S1745503) and Collagenase NB 1 GMP Grade (Cat. No. N0002937) are highly purified collagenases. They show a high Collagenase activity (≥ 3.0 PZ U/mg) and nearly completely reduced side activities. The products have comparable enzymatic activities, but Collagenase NB 1 GMP Grade is manufactured according to GMP guidelines.

The animal-free alternatives Collagenase AF-1 GMP Grade (Cat. No. N0003554, N0003855) and Neutral Protease AF GMP Grade (Cat. No. N0003553) can be used instead of Collagenase NB 1 GMP Grade and Neutral Protease NB GMP Grade, respectively, to avoid any contamination from animal-derived components and to simplify the regulatory process.

Neutral Protease NB (Cat. No. S3030111) and Neutral Protease NB GMP Grade (Cat. No. N0002936) have comparable enzymatic activities and have an activity of ≥ 0.5 DMC U/mg. Neutral Protease NB GMP Grade is manufactured according to GMP guidelines.

Neutral Protease NB High Active Grade (Cat. No. S3030202) is purified and thus has a higher activity (≥ 5.0 DMC U/mg). It is not available as GMP grade.

Clostripain NB (Cat. No. S2020501) is a highly purified enzyme cleaves peptide bonds on the carboxyl side of arginine. Clostripain exists in two different forms, an oxidized (native condition) and a reduced (activated form) that differ in enzymatic activity.

Cat. No.

Product

Collagenase activity

Proteolytic side activities

S1745401
N0002778
N0002779

Collagenase NB 4 Standard Grade
Collagenase NB 5 Sterile Grade
Collagenase NB 6 GMP Grade

+

++

S1746501

Collagenase NB 4G Proved Grade

++

+++

S1747401Collagenase NB 7D Gentle Grade+++

S1745601

Collagenase NB 8 Broad Range

+++

+

S1745503
N0002937

N0003554

Collagenase NB 1 Premium Grade
Collagenase NB 1 GMP Grade

Collagenase AF-1 GMP Grade

++++

-

4. What is the origin of Collagenase NB, Neutral Protease NB and Clostripain NB?

All Nordmark Collagenase NB, Neutral Protease NB and Clostripain NB products are derived from Clostridium histolyticumC. histolyticum is an anaerobic, gram-positive bacterium, which naturally produces collagenases and neutral protease.

5. Are Collagenase NB 1 and Neutral Protease NB sterile?

Collagenase NB 1 GMP Grade (Cat. No. N0002937), Collagenase NB 1 Premium Grade (Cat. No. S1745503), Neutral Protease NB GMP Grade (Cat. No.N0003553), Neutral Protease NB (Cat. No. S3030111) and Neutral Protease NB High Active Grade (Cat. No. S3030202) are 0.2 µm filtered prior to lyophilisation but they are not manufactured aseptically. Therefore they are not sterile according to Pharm. Eur.

For Collagenase NB 1 GMP Grade and Neutral Protease NB GMP Grade a microbiological examination is performed. The specifications and results for absence of clostridia, total aerobic microbial count (TAMC), and total combined yeast/mould count (TYMC) are stated on the lot-specific certificate of analysis.

Nordmark Collagenase NB 6 GMP Grade (Cat. No. N0002779) and Collagenase NB 5 Sterile Grade (Cat. No. N0002778) are sterile according to Pharm. Eur.

All other Collagenase NB and Neutral Protease NB products are not sterile.

7. Why are Collagenase NB 1, Collagenase AF-1 GMP Grade, Neutral Protease NB and Neutral Protease AF GMP Grade filled according to activity and all other Collagenase NB products according to weight?

Collagenase NB 1, Collagenase AF-1 GMP Grade, Neutral Protease NB and Neutral Protease AF GMP Grade are especially suitable for isolation of islets of Langerhans from human pancreas. Pack sizes for these enzymes were initially designed for digestion of one human pancreas.

Collagenase AF-1 GMP Grade is also available in a smaller pack size (≥ 150 PZ U) for use in multiple additional applications.

Since the other Collagenase NB products are suitable for various applications a special pack size is not required.

Transport, Storage and Stock Solutions

8. How should I store Collagenase NB, Neutral Protease NB and Clostripain NB?

Nordmark Collagenase NB, Neutral Protease NB and Clostripain NB products are provided as a lyophilized powder. Collagenases and Neutral Proteases should be stored at +2 to +8 °C (if not stated otherwise on the certificate of analysis) in a dry environment and Clostripain NB should be kept at -15 °C to -25 °C. 

Under these conditions the products are stable until the expiry date stated on the certificates of analysis. Repeated opening and closing of the vial should be avoided. 

For storage of stock solutions please refer to Q 11.

9. What is the stability of Collagenase NB, Neutral Protease NB and Clostripain NB?

Stability of Collagenase NB, Neutral Protease NB and Clostripain NB is ensured until the expiry date stated on the certificate of analysis. To guarantee a constant performance, the non-GMP grade products are regularly retested and according to the results the expiry date may be prolonged.

For stability data on GMP-grade products please refer to Q 29.

10. Where do I find the expiry date of Collagenase NB, Neutral Protease NB or Clostripain NB?

A lot-specific certificate of analysis which states the expiry date of the product is delivered with every Collagenase NB, Neutral Protease NB or Clostripain NB.

The expiry date of Collagenase NB 1 GMP Grade, Neutral Protease NB GMP Grade and Collagenase NB 6 GMP Grade is also stated on the label of the vial.

Please contact us if you want to receive a certificate of analysis of a specific lot.

11. Should I reconstitute Collagenase NB enzymes and make stock solution aliquots or take a sample of the lyophilisate every time I need it?

We recommend making a stock solution by dissolving our enzymes in an appropriate volume of buffer, e.g. HBSS, PBS, Tris or Ringer solution. The reconstituted enzyme should be sterile filtered (if not sterile), aliquoted, and stored at -20 °C. Our stability studies show that the enzyme is stable for one year under these conditions, if repeated freeze and thaw cycles are avoided.

If the vial is opened and closed several times the lyophilized enzyme comes in contact with air moisture which might decrease its activity.

12. Can I sterile filter Collagenase NB, Neutral Protease NB and Clostripain NB solutions?

Yes, these solutions can be 0.22 µm filtered. We recommend usage of filters with low protein-binding properties (e.g. cellulose acetate, PVDF, or PES).

13. How should I store the enzyme solution before adding it to the cells?

Collagenase NB, Neutral Protease NB and Clostripain NB solutions should be constantly stored on ice until use since the activity decreases significantly if stored at higher temperatures. In general, the solution should be used as soon as possible.

14. What is the optimum temperature for Collagenase NB and Neutral Protease NB and Clostripain activity?

Collagenase NB, Neutral Protease NB and Clostripain NB activities are at an optimum at 37 °C.

15. What is the optimum pH for collagenase activity?

Collagenase activity is at an optimum at pH 7.4.

16. What is the optimum pH for Neutral Protease and Clostripain NB activity?

Neutral Protease  and Clostripains NB activity is at an optimum in the neutral pH-range.

17. What happens if Collagenase NB, Neutral Protease NB or Clostripain NB comes in contact with air moisture?

It is very important that the enzymes are protected from air moisture. Collagenase and neutral protease are both hygroscopic; therefore the vial should not be opened and closed repeatedly.

If the enzymes come in contact with moisture they could likely lose their activity due to autodigestion.

If the whole product is not used at once it is recommended to prepare a stock solution and to store this at -20 °C. For further information please refer to Q 11.

18. What happens if Collagenase NB, Neutral Protease NB or Clostripain NB gets warm during transport?

Collagenase NB 1 GMP Grade and Neutral Protease NB GMP Grade are shipped on cool packs or dry ice, depending on the destination. Collagenase NB 1 GMP Grade is stable for 2 months at 25 °C or for 7 days at 40 °C. Neutral Protease NB GMP Grade is stable for 6 months at 25 °C or for 7 days at 40 °C. With both products a temperature tracer accompanies the shipment. The tracer indicates if the enzymes were exposed to temperatures of 37 °C or higher.

Clostripain NB is shipped on dry ice including a temperature indicator strip. If the strip is activated during transport, please contact us to receive further information. 

Collagenase AF-1 GMP Grade and Neutral Protease AF GMP Grade are shipped at ambient temperature due to their strong stabillity.

Collagenase NB 6 GMP Grade is shipped at room temperature. For Collagenase NB 6 GMP Grade our data show that it is stable for 3 years at 25 °C and 4.8 months at 40 °C.

For Nordmark GMP products, stability studies according to ICH guidelines were performed. For more information see Q 29. Please contact us if you would like to obtain further information, also on stability data for all other Nordmark Collagenase NB and Neutral Protease NB products.

Cell Isolation Applications

19. For which applications can I use Nordmark Collagenase NB?

Nordmark Collagenase NB products can be applied for cell isolation from all human or animal tissues containing collagen. Efficient cell isolation with high yields of viable cells could be shown e.g. for pancreas, adipose tissue, cartilage, liver, lung, placenta, heart and many more tissue types. Since collagenase is used both for research applications and in the clinical field, Nordmark offers Collagenase NB products as research grades and GMP grades, respectively.

Nordmark Collagenase NB products are available at different purification levels. Collagenase NB 4 Standard Grade, Collagenase NB 5 Sterile Grade, Collagenase NB 6 GMP Grade and Collagenase NB 4G Broad Range are unpurified grades which contain a balanced mixture of collagenolytic and other proteolytic activities such as neutral protease and clostripain. These side activities are required for efficient digestion of many tissue types like adipose tissue, cartilage or liver.

Collagenase 7D Gentle Grade is a partially purified collagenase which has a collagenase activity higher than that of the products mentioned above, but less than that of the purified products. It can be used in multiple applications, e,g, to isolate tumor cells. 

For some applications, such as isolation of islets of Langerhans from rodent pancreas, a collagenase with higher collagenolytic activity and reduced side activities is required. For these applications purified Collagenase NB 8 Broad Range is recommended.

For isolation of human pancreatic islets, Nordmark offers highly purified Collagenase AF-1 GMP Grade, Neutral Protease AF GMP Grade, and Collagenase NB 1 and Neutral Protease NB as GMP and non-GMP grades. The animal-free enzymes are also used to isolate cells from other tissues such as adipose tissues or tumors.

For further information on applications please visit www.nordmark-biochemicals.com or contact us.

20. Why are Collagenase NB 1 and Neutral Protease NB supplied as two separate enzymes?

Since proteolytic side activities are required for efficient tissue dissociation Collagenase NB 1 is usually used in combination with Neutral Protease NB. Both enzymes are especially suitable for isolation of human pancreatic islets of Langerhans. Since the concentration of both enzymes required for isolation depends e.g. on the age of the donor as well as the organ weight and condition, supply of both enzymes separately allows individual dosing according to these variables.

21. Your cell isolation protocol does not work in my lab. What should I do?

The collagenase concentration required depends on tissue type and origin as well as on isolation procedure. Therefore, Collagenase NB concentrations stated in the cell isolation protocols should be considered as starting points. 

You may increase or decrease the digestion time or increase or decrease the Collagenase NB concentration. Progress of the digestion process should be monitored visually in order to determine the optimal conditions.

22. Which buffer should I use for cell isolation?

We do not recommend a specific buffer since we know that several buffer types, including PBS, Tris, and HBSS work for cell isolation with collagenase.

Collagenase depends on calcium, therefore it is recommended to use a buffer with ≥ 2 mM Ca2+. Collagenase is inhibited by EDTA thus absolutely no calcium chelating agents (e.g. EDTA) should be contained in the buffer (see Q 24).

23. How can I stop the digestion process?

The digestion progress can be reduced by cooling down, dilution or washing out of the digestion solution. There is no general recommendation since several methods work and it also depends on tissue and cell type and application.

24. Are there inhibitors or activators of collagenase?

Since collagenase and some of the secondary proteases depend on calcium, it is recommended to use a buffer with ≥ 2 mM Ca2+
Absolutely no calcium chelating agents (e.g. EDTA) should be present at all.

Collagenase is reversibly inactivated at high pH values and irreversibly inactivated at low pH values. Inhibitors of collagenase include cysteine or chelating agents like EDTA.

GMP-Conformant Collagenase NB and Neutral Protease NB Products

25. What does GMP stand for?

GMP stands for Good Manufacturing Practice. A good manufacturing practice is a production and testing practice that helps to ensure a quality product. Many authorities request manufacturing of pharmaceuticals and medical devices according to GMP guidelines.

These guidelines contain requirements on e.g. traceability of the ingredients, manufacturing, quality control and assurance, packaging, storage, and distribution.

26. Who is the manufacturer of Nordmark's Collagenase NB and Neutral Protease NB products?

All Nordmark Collagenase NB and Neutral Protease NB products are manufactured by the German pharmaceutical company Nordmark Pharma GmbH.

The production is carried out by qualified staff using validated and calibrated equipment in dedicated production facilities. This assures consistently high quality and safety of the products.

Nordmark also manufactures collagenase for use as an active ingredient in a finished drug product used for wound healing. The German authorities have granted a licence to Nordmark for the manufacture of pharmaceutical products. In accordance with Article 13 Paragraph 1 of the German drug law (Arzneimittelgesetz - AMG) a permit was granted to manufacture active ingredients of animal and microbial origin.

27. How can I test if Nordmark GMP products work for my application?

For testing Collagenase NB 6 GMP Grade (Cat. Co. N0002779) and establishing protocols we recommend application of Collagenase NB 4 Standard Grade (Cat. No. S1745401). This non-GMP grade has comparable enzymatic properties to Collagenase NB 6 GMP Grade at a more favorable price. After protocol establishment an easy switch from Collagenase NB 4 Standard Grade to Collagenase NB 6 GMP Grade without changing protocol details is possible.

The same is the case for transfer from Collagenase NB 1 Premium Grade (Cat. No. S1745503) and Neutral Protease NB (Cat. No. S3030111) to Collagenase NB 1 GMP Grade (Cat. No. N0002937) and Neutral Protease NB GMP Grade (Cat. No. N0003553) or to Collagenase AF-1 GMP Grade (Cat. No. N0003554) and Neutral Protease AF GMP Grade (Cat. No. N0003553), respectively.

28. Are Collagenase NB and Neutral Protease NB regarded as TSE (Transmissible Spongiform Encephalopathies) safe?

Yes, Nordmark Collagenase NB and Neutral Protease NB are regarded as TSE safe.

For GMP-conformant Collagenase NB and Neutral Protease NB products the safety of the raw materials was certified by the European Directorate for the Quality of Medicines (EDQM) by a “Transmissible Spongiform Encephalopathies - European Pharmacopoeia Certificate” (TSE-CEP).

Upon request a letter of access is issued by the manufacturer Nordmark, which allows the use of the TSE-CEP certificate for filing with authorities. Also a TSE risk statement issued by Nordmark is available for customers of GMP products.

For the non-GMP grade Collagenase NB and Neutral Protease NB products a Certificate of Origin is available upon request from Nordmark. The Certificate of Origin states that the product does not contain and is not derived from specific risk material.

29. What is the stability of GMP grade Collagenase NB and Neutral Protease NB at different temperatures?

For Collagenase NB 1 GMP Grade, Neutral Protease NB GMP Grade and Collagenase NB 6 GMP Grade, stability studies according to ICH guidelines were performed by the manufacturer Nordmark. Upon request, summary reports of these studies are available for customers of GMP products.

Stability studies for the lyophilisate were performed at following conditions: 

Collagenase NB 1 GMP Grade:

Description

Conditions

Stability

Long-term storage (glass vial)

-20 °C
5 °C

3 years
3 years

Short-term storage (glass vial)

25 °C/60 % humidity

2 months

Stress conditions (glass vial)

40 °C/75 % humidity
-85 °C, 5 freeze/thaw cycles

7 days
confirmed

Solubilized in Tris buffer

-25 °C
-85 °C

1 year
1 year

 

Neutral Protease NB GMP Grade:

Description

Conditions

Stability

Long-term storage (glass vial)

-20 °C
5 °C

3 years
3 years

Short-term storage (glass vial)

25 °C/60 % humidity

6 months

Stress conditions (glass vial)

40 °C/75 % humidity
-85 °C
-85 °C, 5 freeze/thaw cycles

14 days
3 years
confirmed

Solubilized in water

-25 °C
-85 °C

1 year
1 year

 

Collagenase NB 6 GMP Grade:

Description

Conditions

Stability

Long-term storage (glass vial)

5 °C

5 years

Short-term storage (glass vial)

25 °C/60 % relative humidity

3 years

Stress conditions (glass vial)

40 °C/75 % relative humidity

4.8 months

Solubilized in Tris buffer

-25 °C
-85 °C

1 year
1 year

30. Which documents are available for GMP grade products?

For customers of Collagenase NB 1 GMP Grade, Collagenase NB 6 GMP Grade or Neutral Protease NB GMP Grade, the following documents are available upon request:

Certificate of analysis
GMP Certificate of Nordmark
Manufacturing permit for Collagenase
EDQM Certificate for TSE safety (TSE-CEP)
Summary of viral safety study
Stability summary reports

Additional Information

31. I have used collagenase from another supplier. Now I want to use Nordmark Collagenase NB. How do I choose the right product?

It is difficult to compare the collagenase products from different suppliers since every collagenase manufacturer has its own production and purification process. In addition suppliers state the collagenase activity in different units.

One option is to choose the suitable Nordmark Collagenase NB product according to the application. For information on recommended products and application data, please visit our homepage or contact us.

Another option is to convert activity units of the collagenase used previously into PZ units (see Q 33).

32. What is the unit definition of PZ (collagenase activity)?

The activity of Nordmark Collagenase NB products is stated in PZ units according to Wuensch. 
1 PZ U catalyzes the hydrolysis of 1 µmol 4-phenylazobenzyl- oxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine (PZ) per minute at 25 °C, pH 7.1

(Wünsch, E. & Heidrich, H.G. (1963) Hoppe-Seyler's Z. Physiol. Chem. 333, 149-51).

33. How do I convert activity units from other suppliers into PZ units and vice versa?

Collagenase activity is usually stated in PZ units (according to Wuensch), collagen digestion units (CDU, also known as Mandl units) or FALGPA units. There are no exact conversion factors, but the following approximate conversion factors can be applied:

1 PZ U ~ 3.9 FALGPA U
1 PZ U ~ 1000 Mandl U or CDU

In addition, suppliers offer collagenases at different levels of purification, which makes it even more difficult to compare collagenases from different suppliers. An option is to choose the suitable Collagenase NB product and concentration according to the application. For information on recommended products and application data, please visit our homepage or contact us.

34. What is the definition of DMC U (neutral protease activity)?

1 DMC Unit catalyzes the cleavage of 1 µmol peptide bond from dimethylcasein (DMC) per minute at 25 °C, pH 7.0, expressed in terms of newly formed terminal amino groups, determined with TNBS

(Lin, Y.-C. et al. (1969) J. Biol. Chem. 244, 789-93).

35. Are your products tested for mycoplasma?

No, Nordmark Collagenase NB and Neutral Protease NB products are not tested for mycoplasma.

However, all media used in the fermentation process of Collagenase NB are autoclaved prior to the manufacturing which deactivates all possible mycoplasma present in the process. 

36. Are your products tested for endotoxins?

Endotoxins are lipopolysaccharides which are major constituents of the outer cell membrane of gram-negative bacteria.

The following products are currently tested for endotoxins:

Collagenase AF-1 GMP Grade (Cat. No. N0003855, N0003554)
Collagenase NB 1 GMP Grade (Cat. No. N0002937)
Collagenase NB 1 Premium Grade (Cat. No. S1745503)
Collagenase NB 6 GMP Grade (Cat. No. N0002779)
Collagenase NB 8 Broad Range (Cat. No. S1745601)
Neutral Protease AF GMP Grade (Cat. No. N0003553)
Neutral Protease NB GMP Grade (Cat. No. N0003553)
Neutral Protease NB High Active Grade (Cat. No. S3030202)

37. What is a certificate of analysis?

A certificate of analysis is a lot-specific document that is provided with every Collagenase NB and Neutral Protease NB shipment. On the certificate of analysis, lot-specific data such as collagenase activity and shelf life are stated.

You can find CoAs for our technical products here and for GMP CoAs, please contact us

38. Can I apply Collagenase NB or Neutral Protase NB products directly in humans?

Nordmark Collagenase NB and Neutral Protease NB products are not intended for direct application in humans, e.g. for injections or ointments.