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

Enzyme

Therapeutic Target

Assay and Units of Activity

Reference

Thrombin

Hemostatic agent; fibrinogen-clotting; platelet-aggregation

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

1-3

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

4-6

Glucocerebrosidase; imiglucerase

(Cerezyme®)

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

7-9

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.

10-12

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

Activation of plasminogen to plasmin in therapeutic fibrinolysis

 

13-15

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®; http://www.fda.gov/cber/sba/viianov032599S.pdf

 

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,

 http://www.fda.gov/cder/foi/label/2002/20367s53lbl.pdf

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

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