In Vitro Assays for Therapeutic Enzymes

            There are a number of biopharmaceuticals which are enzymes.  A partial list of therapeutic enzymes is shown in Table 1.  The enzymes listed in Table I act in vivo on high-molecular substrates and it is a challenge to develop in vitro methods for accurately assessing activity. 

 There is a commonality among these diverse products in that the conventional method of expressing the potency of these materials is units/mg of biopolymer.   A unit of enzyme activity was defined by the IUBMB (International Union of Biochemistry and Molecular Biology) as 1 μmole of substrate min-1 under specified assay conditions1.   More recent, the term katal (kat) has been adopted by IUPAC (International Union of Pure and Applied Chemistry) as unit of enzyme activity2.  A katal is defined as 1 mole substrate sec-1.  A unit of enzyme activity as defined above is 16.667 x 10-9 kcat3.   Irrespective of nomenclature, the activity of an enzyme is function of affinity for substrate(s), ability convert substrate(s) to product(s) and ability to release product(s)  and is expressed by equation (1)4,5 where v is the observed velocity (reaction rate),

V is Vmax, [S] is substrate concentration, and Km is the Michaelis-Menton constant. 

(1)        v =  V[S]/Km + [S]

This is a simple reaction such as that represented by hydrolysis where the second reactant, water, is in overwhelming molar excess.   While it is clear that the measurement of biological activity and subsequent expression in units is a critical quality attribute6, it is not clear that changes in kinetic constants such as V (V­max­), kcat, kat, or K­m (KS) are useful as quality attributes.    

            While it is relatively easy to obtain kinetic constants for low-molecular substrates where reaction rate can be measured, for example, by colorimetric methods, there is concern about the validity of “non-biological” substrates as surrogate substrates for the biological/therapeutic activity of biopharmaceutical enzymes.   Thrombin is an excellent example of this issue.  Thrombin is one of the oldest biological therapeutic products7 and was used originally (and currently) as a hemostatic agent and more recently as a component in fibrin sealant8 which is a combination product.   The biological or therapeutic effect of thrombin is based on the conversion of fibrinogen to fibrin and somewhat less on the aggregation of platelets which involves the cleavage of a platelet membrane receptor.  The conversion of fibrinogen to fibrin, the formation of a fibrin clot, involves the cleavage of the A(α) chain of fibrinogen with the release of fibrinopeptide A and later the cleavage of the B(β) chain with release of fibrinopeptide B.   The kinetic parameters for these reactions are well-understood9,10.   The activity of thrombin as measured for biological potency as a licensed product is based on the clotting of fibrinogen and is expressed in NIH units or International units11-13.  The most common thrombin assays are based on the clotting of a standard fibrinogen solution and potency is assigned based on a reference standard.   An assay based on the release of fibrinopeptide A has been proposed13 but has not been widely adopted.   Thrombin can also hydrolyze peptide nitroanilide and ester substrates which can provide data inconsistent with fibrinogen clotting14-20; esterase and amidase activity can be retained while fibrinogen-clotting activity is lost20-22.   In the case of thrombin, there would appear to be no effective substitute for fibrinogen-clotting in the measurement of therapeutic thrombin activity; chromogenic substrates may be effectively used in diagnostic tests23-27.

            Hyaluronidase28, earlier known as spreading factor29-32, is being used in cosmetic surgery and as a adjunct for drug delivery33-35.  Hyaluronidase degrades hyaluronic acid, a high molecular-weight glycosaminoglycan found in connective tissue and joint fluids36 by hydrolyzing β-N-acetylhexosaminic bonds37.  The assay methods are complex but can provide good data120; other assay methods are being developed38-45.   A microplate-based assay has been developed44.  The activity of current therapeutic products (from both animal and recombinant sources) which is used both in devices and as a free-standing therapeutic is determined by the amount of undegraded hyaluronic acid remaining after digestion for a predetermined period of time (20 minutes at 37oC)46. Enzyme activity is also used as a measure of molecular integrity of hyaluronidase47,48.

            Another example is provided by carboxyl proteases49.  Fujiwara and colleagues studies the effect of pressure on the catalytic activity and conformation of two pepstatin-sensitive carboxyl proteases, porcine pepsin and proteinase A from Baker’s yeast, and two pepstatin-insensitive carboxyl proteases, pseudomonapepsin and xanthomonapepsin.  Activity was determined with an octapeptide fluorescent substrate and acid-denatured myoglobin.    Increasing pressure causes a decrease in kcat/Km for pseudomonapepsin and xanthomonapeptide while greater lost of activity was observed with pepsin and proteinase A from Baker’s yeast; there was little effect on the hydrolysis of acid-denatured myoglobin by any of the four proteinases.  Change in conformation was observed with 4th derivative spectroscopy.   The point here is that there is a difference in response between the “biological” substrate and synthetic substrate.

            Larner and colleagues showed that the chain length of the oligosaccharide acceptor influenced the activity of glycogen synthase50.  These investigators used the metrics of S0.5 and Vmax.   S0.5 is a value analogous to KM in that is substrate concentration at ½ Vmax.  S0,5 is usually used for cooperative enzyme systems51-58 but would also be useful for the study of enzymes where there is substrate heterogeneity.   In this case, it would be important to standardize the substrate.  The assay for the presence of prekallikrein activator (activated Hageman Factor, Factor XIIa) is used for the evaluation of plasma protein products59.  While current technology uses a peptide nitroanilide substrate60 with an international standard61.  To the best of my knowledge, this synthetic substrate has not been validated with the biological assay based on kinin release62: this assay (biological assay) required the preparation of a standardized crude substrate from plasma as a source of prekallikrein63.   There is also the issue with enzymes which degrade biopolymers where the substrate is changing during the enzymatic reaction, as for example with cellulose.   Approaches have been developed for the analysis of these reactions64-68.  Heterogeneity is also important for some therapeutic enzyme function (debridement) and for cell dissociation in processing69

            The bulk of the data would suggest that smaller, synthetic substrates while providing for a more facile assay, do not necessarily measure the biological (therapeutic) activity of a biopharmaceutical enzyme.  The use of such a synthetic substrate should be validated against the biological substrate.  In the case of thrombin, fibrinogen clotting activity can be “separated” from esterase/amidase activity by chemical modification with, for example, tetranitromethane70,71. 

It is suggested that the validation of such synthetic substrate occur n the initial stability testing of the product where assay data can be correlated with physicochemical data72-77

The following suggestions are advanced for the characterization of therapeutic enzymes which work on complex substrates.

  • The use of metrics such as S0.5 and Vmax instead of K­M (KS) and kcat may be more valuable in the characterization of biopharmaceutical enzyme. 
  • The complex substrate must be prepared in a reproducible process subject to validation.
  • The use of international standards is recommended whenever possible
  • Use of pharmacopoeial methods is strongly recommended  

Table 1:  Some Examples of Therapeutic Enzymesa


Therapeutic Target

Assay and Units of Activity



Hemostatic agent; fibrinogen-clotting; platelet-aggregation

NIH Unit; International Unit – both based the “clotting” of fibrinogen


DNase (Pulmozyme®)

High-molecular DNA which promotes pathogen colonization in pulmonary abcesses; Cystic fibrosis

The original assay described by Kunitzb was based on hyperchromicityc.    More recent assays have used the hydrolysis of the methyl green complex with DNAd,e


Glucocerebrosidase; imiglucerase


β-D-glucosyl-N-acylsphingosine glucohydrolase

Glucosylceramide (glucocerebroside)

A unit of enzyme activity is amount of enzyme that catalyzes the hydrolysis of one micromole of p-nitrophenyl-β-D-glucopyranoside per minute at 37oC


Blood Coagulation Factor VIIa

Factor VIII inhibitor-bypass activity (FEIBA); hemostatic agent

VIIa is measured in FEIBA units (units of factor VIII inhibitor bypassing activity). More recently, factor VIIa is measured with respect to an International Standardf.


Tissue plasminogen activator (tPA); Alteplase (Activase®)

Activation of plasminogen to plasmin in therapeutic fibrinolysis



a See also Vellard, M., The enzyme as drug: application of enzymes as pharmaceuticals, Curr.Opin.Biotechnol. 14, 444-450, 2003

b  Kunitz, M., Crystalline desoxyribonuclease I. Isolation and general properties.  Spectrophotometric method for the measurement of desoxyribonuclease activity, J.Gen.Physiol. 33, 349-360, 1950

c  Plapp, B.V., Moore, S., and Stein, W.H., Activity of bovine pancreatic desoxyribonuclease A with modified amino groups, J.Biol.Chem. 246, 939-945, 1971

d Sinicropi, D., Baker, D.L., Prince, W.S., et al., Colorimetric determination of DNase I activity with a DNA-methyl green substrate, Anal.Biochem. 222, 351-358, 1994

e Lichtinghagen, R., Determination of Pulmozyme® (dornase alpha) stability using a kinetic colorimetric DNase I activity assay, Eur.J.Pharm.Biopharm. 63, 365-368, 2006

f CBER, Summary  Basis of Approval for Novoseven®;


References to Table 1

1.  Lundblad, R.L., Bradshaw, R.A., Gabriel, D., et al., A review of the therapeutic uses of thrombin, Thromb.Haemost. 91, 851-860, 2004

2.  Cheng, C.M., Meyer-Massetti, C., and Kayser, S.R., A review of three stand-alone topical thrombins for surgical hemostasis, Clin.Ther. 31, 32-41, 2009

3. Anderson, C.D., Bowman, L.J., and Chapman, W.C., Topical use of recombinant human thrombin for operative hemostasis, Expert Opin.Biol.Ther. 9, 133-137, 2009

4.  Ayvazian, J.H., Johnson, A.J., and Tillett, W.S., The use of parenterally administered pancreatic desoxyribonuclease as an adjunct in the treatment of pulmonary abscesses, Am.Rev.Tuberc. 76, 1-21, 1957

5. Bryson, H.M. and Sorkin, E.M., Dornase alfa. A review of its pharmacological properties and therapeutic potential in cystic fibrosis, Drugs 48, 894-906, 1994

6. Suri, R., The use of human deoxyribonuclease (rhDNase) in the management of cystic fibrosis, BioDrugs 19, 135-144, 2005

7.  Pentchev, P.G., Brady, R.O., Hibbert, S.R., et al., Isolation and characterization of glucocerebrosidase from human placental tissue, J.Biol.Chem. 248, 5256-5261, 1973

8.  Morales, L.E., Gaucher’s disease: a review, Ann.Pharmacother. 30, 381-388, 1996

9. Cerezyme® NDA 20-362/S-053, Center for Drug Evaluation and Research, FDA,

10.  Hedner, U., Recombinant coagulation factor VIIa: from the concept to clinical application in hemophilia treatment in 2000, Semin.Thromb.Hemost. 26, 363-366, 2000

11. Jurlander, B. Thim, L., Klausen, N.K., et al., Recombinant activated factor VII (rFVIIa): characterization, manufacturing, and clinical development, Semin.Thromb.Hemost. 27, 373-384, 2001

12.  Monroe, D.M., Further understanding of recombinant activated factor VII mode of action, Semin.Hematol 45 (2 Suppl 1), S7-S11, 2008

13.  Karlan, B.Y., Clark, A.S., and Littlefield, B.A., A highly sensitive chromogenic microtiter plate assay for plasminogen activators, Biochim.Biophys.Res.Commun. 142, 147-154, 1987

14. Christodoulides, M. and Boucher, D.W., The potency of tissue-type plasminogen activator (TPA) determined with chromogen and clot-lysis assays, Biologicals 18, 103-111, 1990

15. Koley, K., Owen, W.G., and Machovich, R., Dual effect of synthetic plasmin substrates on plasminogen activation, Biochim.Biophys.Acta 1247, 239-245, 1995



1. Racker, E. for the National Academy of Sciences-National Research Council, International Unit of Enzyme Activity, Science 128, 19-20, 1958

2. Dybkaer, R., Unit “katal” for catalytic activity (IUPAC technical support), Pure Appl.Chem. 73, 927-931, 2001

3. Dybkaer, R., The tortuous road to the adsorption of katal for the expression of catalytic activity by the General Conference on Weights and Measures, Clin.Chem. 48, 586-590, 2002

4.  Dixon, M. and Webb, E.C., Enzymes, Academic Press, New York, NY, USA, 1964

5. Contemporary Enzyme Kinetics and Mechanism, ed. D. Purich,  Academic Press, New York, NY, USA, 1981

6.  ICHQ8   Pharmaceutical Development Revision 1, International Conference on Harmonization,

7.  Lundblad, R.L., Bradshaw, R.A., Gabriel, D., et al., A review of the therapeutic uses of thrombin, Thromb.Haemost. 91, 851-860, 2004

8. Spotnitz, W.D. and Prabhu, R., Fibrin sealant tissue adhesive - - review and update, Long Term Eff.Med.Implants 15, 245-270, 2005

9.  Lewis, S.D., Shields, P.P., and Shafer, J.A., Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin, J.Biol.Chem.260, 10192-10199, 1985

10. De Cristofaro, R. and Castagnola, M., Kinetics aspects of release of fibrinopeptides AP and AY by human α-thrombin, Haemostasis  21, 85-90, 1991

11. Baughman, D.J., Thrombin Assay, Methods Enzymol. 19, 145-157, 1970

12. Lewis, S.D. and Shafer, J.A., A thrombin assay based upon the release of fibrinopeptide A from fibrinogen: definition of a new thrombin unit, Thromb.Res. 35, 111-120, 1984

13. Gaffney, P.J. and Edgell, T.A., The International and “NIH” units for thrombin - - how do they compare?, Thromb.Haemost. 74, 900-903, 1995

14.  Ronwin, E., The relationship between the peptidase, esterase and clotting activity of thrombin, Acta Haematol. 23, 129-139, 1960

15.  Lundblad, R.L. and Harrison, J.H., The differential effect of tetranitromethane on the proteinase and esterase activity of bovine thrombin, Biochem.Biophys.Res.Commun. 45, 1344-1349, 1971

16. Lottenberg, R., Hall, J.A., Fenton, J.W.,2nd, and Jackson, C.W., The action of thrombin on peptide p-nitroanilide substrates: hydrolysis of Tos-gly-pro-arg-pNA and D-phe-pip-arg-pNA by human α and γ and bovine α and β-thrombins, Thromb.Res. 28, 313-332, 1982

17. Jenny, N.S., Lundblad, R.L., and Mann, K.G., Thrombin, in Hemostasis and Thrombosis.  Basic Principles and Clinical Practice, 5th edn., ed. R.W. Colman, V.J. Marder, A.W. Cloves, J.N. George and S.Z. Goldhaber, Lippincott, William & Wilkins, Philadelphia, PA,  Chapter 10, pps 193-213,  2007

18. Lundblad, R.L., Kingdon, H.S., and Mann, K.G., Thrombin, Methods Enzymol. 45, 156-176, 1976

19. Lundblad, R.L. and Workman, E.F., Jr., Thrombin, CRC Handbook on Clinical Science, Section I, Hematology 3, 149-169, 1980.

20.  Lanchantin, G.F., Presant, C.A., Hart, D.W., and Friedman, J.A., A comparison of the esterase (TAMe) and clotting activities of human and bovine thrombin preparations, Thromb.Diath.Haemorrh. 14, 159-175, 1965

21. Lundblad, R.L., Uhteg, L.C., Vogel, C.N., et al., Preparation and partial characterization of two forms of bovine thrombin, Biochem.Biophys.Res.Commun. 66, 482-489, 1975

22. Lundblad, R.L., Nesheim, M.E., Straight, D.L., et al., Bovine α- and β-thrombin. Reduced fibrinogen-clotting activity is not a consequence of reduced affinity for fibrinogen, J.Biol.Chem. 259, 6991-6695, 1984

23.  Rijkers, D.T., Hemker, H.C., and Tesser, G.I., Synthesis of peptide p-nitroanilides mimicking fibrinogen- and hirudin-binding to thrombin.  Design of slow reacting thrombin substrates, Int.J.Pept.Protein Res. 48, 182-193, 1996

24. Meddahi, S., Bara, L., Fessi, H., and Samama, M.M., Standard measurement of clot-bound thrombin by using a chromogenic substrate for thrombin, Thromb.Res. 114, 51-56, 2004

25. Nowak, G., Lange, U., Wiesenburg, A., and Bucha, E., Measurement of maximum thrombin generation capacity in blood and plasma using the thrombin generation assay (THROGA), Semin.Thromb.Hemost. 33, 508-514, 2007

26. Devreese, K., Wijns, W., Combes, I., et al., Thrombin generation in plasma of healthy adults and children: chromogenic  versus fluorogenic thrombogram analysis, Thromb.Haemost. 98, 600- 613, 2007

27. Gardiner, C., Machin, S.J., and Mackie, I.J., Measuring thrombin generation based sensitivity to activated protein C using an automated coagulometer (ACL 9000), Int.J.Lab.Hematol. 30, 261-268, 2008

28. Menzel, E.J. and Farr, C., Hyaluronidase and its substrate hyaluronan: biochemistry, biological activities and therapeutic uses, Cancer Lett. 131, 3-11, 1998

20. Bergquist, S. and Packalen, T., The bacteriological origin of a spreading factor present in the nasal secretion, Acta Pathol.Microbiol.Scand. 25, 255-258, 1948

30. Woodin, A.M., Hyaluronidase as a spreading factor in the cornea, Br.J.Ophthalmol. 34, 375-379, 1950

31. Bensley, S.H. Histological studies of the reactions of cells and intercellular substances of loose connective tissue to the spreading factor of testicular extracts, Ann.N.Y.Acad.Sci. 52, 983-988, 1950

32.  Farrar, G.E.,Jr., The spreading factor, Clin.Ther. 11, 705-706, 1989

33.  Girish, K.S. and Kemparaju, K., The magic glue hyaluronan and its eraser hyaluronidase, a biological overview, Life Sci. 80, 1921-2007, 2007

34. Frost, G.I., Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration, Expert Opin.Drug Deliv. 4, 427-440, 2007

35. Anwer, K., Formulations for DNA delivery via electroporation in vivo, Methods Mol.Biol. 423, 77-89, 2008

36. Gandhi, N.S. and Mancera, R.L., The structure of glycosaminoglycans and their interactions with proteins, Chem.Biol.Drug.Des. 72, 455-482, 2008

37. Vercruysse, K.P., Lauwers, A.R., and Demeester, J.M., Absolute and empirical determination of the enzymatic activity and kinetic investigation of the action of hyaluronidase on hyaluronan using viscosimetry, Biochem.J. 305, 153-160, 1995

38. Menzel, E.J. and Farr, C., Hyaluronidase and its substrate hyaluronan: biochemistry, biological activities and therapeutic uses, Cancer Lett. 131, 3-11, 1998

39. Kinoshita, M., Okino, A., Oda, Y., et al., Anomalous migration of hyaluronic acid oligomers in capillary electrophoresis: correlation to susceptibility to hyaluronidase, Electrophoresis 22, 3458-3465, 2001

40. Courel, M.N., Maingonnat, C., Transchepain, F., et al., Importance of hyaluronan length in a hyaladherin-based assay for hyaluronan, Anal.Biochem. 302, 285-290, 2002

41. He, D., Zhou, A., Wei, W., et al., A new study of the degradation of hyaluronic acid by hyaluronidase using quartz crystal impedance, Talanta 53, 1021-1029, 2001

42. Schulze, C., Bittorf, T., Walzel, H. et al., Experimental evaluation of hyaluronidase activity in combination with specific drugs applied in clinical techniques of interventional pain management and local anaesthesia, Pain Physician 11, 877-883, 2008

43. Yavav, G., Prasad, R.L., Jha, B.K., et al., Evidence for inhibitory interaction of hyaluronan-binding protein 1 (HABP1/p32/gClqR) with Streptococcus pneumonia hyaluronidase, J.Biol.Chem. 284, 3897-3905, 2009

44. Frost, G.I. and Stern, R., A micro-titer assay for hyaluronidase activity not requiring specialized reagents, Anal.Biochem. 251, 263-269, 1997

45. Muckenschnabel, I., Bernhardt, G., Spruss, T. et al., Quantitation of hyaluronidase by the Morgan-Elson reaction: comparison of the enzyme activities in the plasma of tumor patients and healthy volunteers, Cancer Lett. 131, 13-20, 1998

46. Bailey, L.C. and Levine, N.A., Optimization of the USP assay for hyaluronidase, J.Pharmaceut.Biomed.Analy. 11, 285-292, 1993

47. Gupta, G.S. and Sharma, P.K., Molecular inactivation of testicular hyaluronidase in solid state after proton irradiation: a study based on target size, substrate binding and thermodynamic analysis of heat denaturation, Indian J.Biochem.Biophys. 32, 266-271, 1995

48. Maksiemenko, A.V., Schechilina, Y.V., and Tischenko, E.G., Resistance of detran-modified hyaluronidase to inhibition by heparin, Biochemistry 66, 456-463, 2001

49. Fujiwara, S., Kunugi, S., Oyama, H., and Oda, K., Effects of pressure on the activity and spectroscopic properties of carboxyl proteinases.  Apparent correlation of pepstatin-insensitivity and pressure response, Eur.J.Biochem. 268, 645-655, 2001

50.  Larner, J., Takeda, Y., and Hizukuri, S., The influence of chain size and molecular weight on the kinetic constants for the span glucose to polysaccharide for rabbit muscle glycogen synthase, Molec.Cell.Biochem. 12, 131-136, 1976

51. Neet, K.E., Cooperativity in enzyme function: Equilibrium and kinetic aspects, in Contempory Enzyme Kinetics and Mechanism, ed. D.L. Purich, Chapter 11, Academic Press, New York, NY, USA, 1983

52. Lawlis, V.B. and Roche, T.E., Regulation of bovine kidney α-ketoglutarate dehydrogenase complex by calcium ions and adenine nucleotides. Effects on S0.5 for α-ketoglutarate, Biochemistry 28, 2512-2518, 1981

53. Tippett, P.S. and Neet, K.E., Interconversions between different sulfhydryl-related kinetic states in glucokinase, Arch.Biochem.Biophys. 222, 285-298, 1982

54. Richards, E.W., Hamm, M.W., and Otto, D.A., The effect of palmitoyl-CoA binding to albumin on the apparent kineics behavior of carnitine palmitoyltransferase I, Biochim.Biophys.Acta 1076, 23-28, 1991

55. Suganuma, T., Maeda, Y., Kitahara, K., and Nagahama, T., Study of the action of human salivary α-amylase on 2-chloro-4-nitrophenyl α-maltotrioside in the presence of potassium thiocynate, Carbohy.Res. 303, 219-227, 1997

56. Buchbinder, J.L., Luong, C.B., Browner, M.F., et al., Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the stie of regulatory phosphorylation, Biochemistry 36, 8039-8044, 1997

57. Akowski, J.P. and Bauerle, R., Steady-state kinetics and inhibitor binding of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tryptophan sensitive) from Escherichia coli, Biochemistry 36, 15817-15822, 1997

58. Swieca, A., Rybakowska, I., Nagel-Starczynowska, G., et al., AMP-deaminase from human term placenta, Mol.Cell.Biochem. 252, 363-367, 2003

59. Tanaka, K., Sawatani, K., Dias, G.A., et al., High quality human immunoglobulin G purified from Cohn fractions by liquid chromatography, Braz.J.Med.Biol.Res. 33, 27-30, 2000

60. Snape, T.J., Griffith, D., Vallet, L., and Wesley, E.D., The assay of prekallikrein activator in human blood products, Dev.Biol.Stand. 44, 115-120, 1979

61. Kerry, P.J., Curtis, A.D., Paton, C.J., and Thomas, D.P., Standardization of prekallikrein activator (PKA): the 1st British reference preparation of PKA, Brit.J.Haematol. 52, 275-281, 1985.

62. Tankersley, D.L., Fournel, M.A., and Schroeder, D.D., Kinetics of activation of prekallikrein activator, Biochemistry 19, 3121-3127, 1980

63. Lundblad, J.L., In vitro assay for prekallikrein activator (PKA), Develop.Biol.Standard. 44, 107-114, 1979

64. Mansfield, S.D., Mooney, C., and Saddler, J.N., Substrate and enzyme characteristics that limit cellulose hydrolysis, Biotechnol.Prog. 15, 804-816, 1999

65. Zhang, S., Wolfgang, D.E., and Wilson, D.B., Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis, Biotechnol.Bioengineer. 66, 35-41m 1999

66. Gan, Q., Allen, S.J., and Taylor, G., Kinetic dynamics in heterogeneous hydrolysis of cellulose: an overview, an experimental study and mathematical modeling, Process Biochem. 38, 1003-1018, 2003

67. Gutierrez, O.A., Chavez, M., and Lissi, E., A theoretical approach to some analytical properties of heterogeneous enzymatic assays, Anal.Chem. 76, 2664-2668, 2004

68.  Drissen, R.E.T., Maas, R.H.W., Van Der Maarie, M.J.E.C., et al., A generic model for glucose utilization from various cellulose sources by a commercial cellulase complex, Biocatalysis and Biotransformation 25, 419-429, 2007

69. Chen, R.L. and James, R.F., Characterization of an important enzymatic component in collagenase that is essential for the effective digestion of the human and porcine pancreas, Cell Transplant. 10, 709-716, 2001

70. Lundblad, R.L. and Harrison, J.H., The differential effect of tetranitromethane on the proteinase and esterase activity of bovine thrombin, Biochem.Biophys.Res.Commun. 45, 1344-1349, 1971

71. Lundblad, R.L., Noyes, C.M., Featherstone, G.L. et al., The reaction of bovine α-thrombin with tetranitromethane.  Characterization of the modified protein, J.Biol.Chem. 263, 3729-3734, 1988

72. Martin, M., Streptokinase stability pattern during storage in various solvents and at different temperatures, Thromb.Diath.Haemorrh. 33, 586-596, 1975

73. Linhardt, R.J., Cooney, C.L., Tapper, D., et al., An immobilized microbial heparinase for blood deheparinization, Appl.Biochem.Biotechno. 9, 41-55, 1984

74.  Johnson, C., Royal, M., Moreadith, R., et al., Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography, Biomed.Chromatog. 17, 335-344, 2003

75. Dedrick, S.C. and Ramirez-Rico, J., Potency and stability of frozen urokinase solutions in syringes, Am.J.Health Syst.Pharm. 61, 1586-1589, 2004

76. Lichtinghagen, R., Determination of Pulmozyme (dornase alpha) stability using a kinetic colorimetrc DNase I activity assay, Eur.J.Pharm.Biopharm. 63, 365-368, 2006

77. Luthra, S., Obert, J.P., Kalonia, D.S., and Pikal, M.M., Investigation of drying stresss on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer, J.Pharm.Sci. 96, 61-70, 2007