Stabilization of biologically active proteins

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19 Dakour, J., Lundblad, A. and Zopf, D. (1987) Anal. Biochem. 161, 140-143 20 Borrebaeck, C. A. K., Soares, J. and Mattiasson, B. (1984) J. Chromatogr. 284, 187-192 21 Renaner, D., Oesch, F., Kinkel, J., Unger, K. K. and Wieser, R. J. (1985) Anal. Biochem. 151,424-427 22 Green, E. D., Brodbeck, R. M. and Baenziger, J.U. (1987) Anal. Biochem. 167, 62-75 23 Swaisgood, H. E. and Chaiken, I. (1985) J. Chromatogr. 327, 193-204 24 Allenmark, S., Bomgren, B., Bor6n, H. and Lagerstr6m, P.O. (1984) Anal. Biochem. 136, 293-297 25 Hermansson, J. (1984) J. Chromatogr. 298, 67-78 26 Glad, M., Ohlson, S., Hansson, L., M~nsson, M.O., Larsson, P-O. and Mosbach, K. (1983) US Patent 44O6792 27 Hagemeier, E., Kemper, K., Boos, K.S. and Schlimme, E. (1983) J. Chromatogr. 282,663-669 28 Small, D. A. P., Atkinson, T. and Lowe, C. (1981) J. Chromatogr. 216, 175-190 []

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29 Kinkel, J. N., Anspach, D., Unger, K. K., Wieser, R. and Brunner, G. (1984) J. Chromatogr. 297, 167-177 30 Rogers, M. E., Adlard, M. W., Saunders, G. and Holt, G. (1985) J. Chromatogr. 326, 163-172 31 Waiters, R. R. (1982) J. Chromatogr. 249, 19-28 32 Kato, Y., Nakamura, K. and Hashimoto, T. (1986) J. Chromatogr. 354,511-517 33 Shimura, K., Kazama, M. and Kasai, K. I. (1984) J. Chromatogr. 292, 369-382 34 Honda, S., Suzuki, K., Suzuki, S. and Kakehi, K. (1988) Anal. Biochem. 169, 239-245 35 Jarrett, H. (1986)J. Chromatogr. 363, 456-461 36 Ernst-Cabrera, K. and Wilchek, M. (1988) Trends Anal. Chem. 7, 58-63 37 Bonnerja, J., Hoare, S. O. M. and Dunnhill, P. (1986) Biotechnology 4, 954-960 38 Hage, D. S. and Walters, R. R. (1987) J. Chromatogr. 386, 37-49 39 Ohlson, S., Gudmundsson, B. M., Wikstr/Sm, P. and Larsson, P.O. (1988) Clin. Chem. 34, 2039-2043 []

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Stabilization of biologically active proteins E. Y. Shami, A. Rothstein and M. Ramjeesingh Recent advances in molecular biology are enabling large quantities of relatively rare, biologically active proteins with a variety of medical and industrial applications to be produced reliably. One drawback to their use is that they are inherently unstable. However, stabilization can be achieved by conformational changes in the protein through mutagenesis or by either covalent or non-covalent modification of the surface chemistry of the protein with specific antibodies. The activity of a protein requires p r o p e r folding to ensure structural and functional integrity of the active d o m a i n 1. This integrity can be irreverE. Y. Shami, A. Rothstein and M. Ramjeesingh are at HYBRISENS Ltd., York University Campus, Farquharson Building, Suite 104, Toronto, Ontario, Canada M3] 1P3. (E. Y. Shami is also at Dept Biology, York University, Toronto; A. Rothstein at Dept Medical Biophysics, University of Toronto, Toronto.) ( ~ 1989, Elsevier Science Publishers Ltd (UK)

sibly disrupted by forces w h i c h are, for example, physical (heat, freezing, radiation), chemical (oxidation, reduction, solvents, metal ions, ionic strength, pH) and biological (enzymatic modification and degradation). The most c o m m o n form of in vivo inactivation of m a n y proteins is e n z y m a t i c modification, particularly e n z y m i c proteolysis 2. The half-life of injected proteins is often relatively short (10-20 minutes) s u c h that clinical application requires multiple injections or m e t h o d s of c o n t i n u o u s

0167 - 9430/89/$02.00

40 Janis, L. J. and Regnier, F. E. (1988) J. Chromatogr. 444, 1-11 41 Hansson, L., Glad, M. and Hansson, C. (1983) J. Chromatogr. 265, 37-44 42 Ohlson, S., Lundblad, A. and Zopf, D. (1988) Anal. Biochem. 189, 284-288 43 Clonis, Y. D., Jones, K. and Lowe, C. R. (1986) J. Chromatogr. 363, 31-39 44 Chaiken, I. (1979) Anal. Biochem. 97, 1-10 45 Nilsson, K. and Larsson, P. O. (1983) Anal. Biochem. 134, 60-72 46 Wade, J. L., Bergold, A. F. and Carr, P.W. (1987) Anal. Chem. 59, 1286-1295 47 Anon. (1988) Biotechnol. Bull. 7, 10-11 48 Persson, M., Bergstrand, M. G., Billow, L. and Mosbach, K. (1988) Anal Biochem. 172, 330-337 49 Moks, T., Abrahamsen, L., Osterl6f, B. et al. (1987) Biotechnology 5, 379-382 50 Sellergren, B., Lepiste, M. and Mosbach, K. (1988)]'. Am. Chem. Soc. 100, 5853-5860 51 Ekberg, B. and Mosbach, K. (1989) Trends Biotechnol. 7, 92-96 []

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infusion. Proteins m a d e resistant to proteolysis w o u l d be m o r e effective, allowing use of smaller total a m o u n t s to m a i n t a i n o p t i m u m p r o t e i n bloodlevels. In addition, if resistance to digestion c o u l d be c o u p l e d with resistance to low pH, it c o u l d p r o v i d e the more c o n v e n i e n t o p t i o n of oral administration, either for p e p t i d e drugs or for digestive e n z y m e s in r e p l a c e m e n t t h e r a p y in, for instance, cystic fibrosis. A n o t h e r use of active proteins ( h o r m o n e and growth factors) that could benefit from their stabilization is as s u p p l e m e n t s in m e d i a for m a m m a l i a n cell culture 3. Inactivation of proteins in m e d i a is by proteolysis due to lysosomal enzymes released from lysed cells, m e m b r a n e b o u n d pr0teinases and h o r m o n e specific proteases 4-6. T e m p e r a t u r e stability of e n z y m e s used industrially is an i m p o r t a n t factor in productivity, for proteases, amylases and, recently, lipases 7 used in l a u n d r y detergents, elevated t e m p e r a t u r e s and the p r e s e n c e of an oxidizing agent (bleach) make resistance to heat and o x i d a t i o n desirable. D e p e n d i n g on the system in w h i c h

TIBTECH- JULY 1989 [Vol. 7]

they are used, enzymes in vitro may be exposed to all the forms of inactivation mentioned above (and others), though heat and oxidation are the most common. Resistance to inactivation by organic solvents will add another dimension to use of enzymes in processes where the substrate has extremely low solubility in water 8.

Protein stabilization methods Here, we summarize the existing methods of protein stabilization. We then describe a relatively new approach to stabilization, the use of specific antibodies, and make some comparisons. Selection A traditional approach has been to select 'naturally' stable enzymes from organisms that grow in extreme conditions of temperature, pH, salinity, etc. While this approach has resulted in isolation of several stable enzymes 9, it cannot be considered strictly as a 'method' for stabilization of enzymes. This approach suffers from limitations such as the narrow spectrum of useful proteins that are naturally expressed by microorganisms and the possibility that the expressed enzymes may have different activity and specificity characteristics ~°. It has, however, provided insight into mechanisms of stabilization. Increased stability has been found to be associated with changes in the internal structure of the protein, such as cross-linking via sulfhydryl groups 11'12. In other cases, in vivo stability is conferred by the interaction of other proteins or polyamines with the enzymes, whose inherent stability is unaltered ~2-14. Site-directed m utagen esis A major effort is underway to produce more stable proteins by sitedirected mutagenesis ~5'~6. This approach has considerable promise, but is still largely empirical, based on educated guessing regarding the effective amino acid substitutions. The introduction of additional sulfhydryl groups to achieve stability has had limited success, since the formation of disulfide bonds requires very strict geometry 17. Very recently, large increases, in stability of subtilisin

--Table I

Thermostability of enzyme-antibody complexes Activity half-life Enzyme Alpha-amylase Glucoamylase Subtilisin

Temperature

Free Enzyme

70°C 66°C 65°C

5 min 3 min 4 min

have been accomplished through a combination of site-directed mutagenesis and in vitro random mutagenesis 18. Covalent conjugation Conjugation of macromolecules such as polyethylene glycol (PEG) 19 or albumin 2° to enzymes can increase resistance to proteolysis and increase circulatory half-life. This approach, and all other stabilization methods requiring random covalent coupling, may result in modification of the active enzyme site and consequent loss of biological activity. Another approach in this category involves the elimination, by chemical modification, of unfavorably charged (repulsive) groups that promote unfolding, from the surface of the protein. Considerable stabilization has been achieved in some cases 2~ using this technique. Multipoint covalent immobilization or crosslinking Multipoint-immobilization or crosslinking of several enzymes has resulted in impressive increases in stability. For example, immobilized trypsin was 1000 times more stable than its soluble form, while immobilized lipase was 140 times more stable than soluble lipase 22, Protein-protein non-covalent association Protein-protein interactions can confer stability by excluding water from the interaction area on the protein surface, thereby reducing the free energy and driving the reaction towards the folded state 23. Certain thermophilic organisms confer stability on their enzymes by elaborating /l~rotective macromolecules such / as peptide and poly-

Enzyme-antibody 16 h 3h >3 h

amines 12-~4, and multimers or enzyme aggregates are often more stable and active than the constituent monomers 24. Another form of stabilization through protein-protein association has been reported for several enzymeinhibitor complexes. The transition (folded to unfolded state) temperature for subtilisin-inhibitor complex was 20°C higher than for the free enzyme 25. However, this high degree of protection was not useful because the enzyme-inhibitor complex was not biologically active.

Active protein stabilization by antibodies The observations on stabilization by protein-protein interactions led us to examine the immune system, a natural mechanism which can elaborate proteins which interact specifically with many overlapping surface features of a folded target protein. Some antibodies might interact at sites where protein unfolding is initiated, or where proteolytic digestion occurs, thereby stabilizing the protein. Is the concept viable? For the average affinity antibodyantigen binding (108 M-l}, the interaction could reduce the free energy of the antigen by about 10 Kcal tool -1 (Ref. 26). This is sufficient (in a general thermodynamic sense) to confer increased stability since the differences in free energy between the folded and unfolded states for active proteins are i n the range of 5-15 Kcal mo1-1 (Ref. 27). The interaction between chicken egg lysozyme and the antigenbinding fragments (Fab) of three monoclonal antibodies (Mab), and the interaction of neuraminidase of

TIBTECH- JULY 1989 [Vol. 7]

Fig. 1 100"

~" 90influenza virus with a Fab of one of its monoclonal antibodies, were studied by X-ray crystallography (recently reviewed by Davies et a].28). It was concluded that the interactions were tight, such that all water molecules are excluded from the area of contact, which measures around 700 A 2 (Ref. 29). If the size of contact area is typical of antibody-antigen interaction, it will dictate an epitope comprising more than one continuous oligopeptide3°; an epitope consisting of several discontinuous oligopeptides or residues crosslinked by the antibody reaction. As many as three salt links, ten hydrogen bonds and 74 Van der Waals interactions are involved in the antibodylysozyme interaction 29. Such crosslinking supported by hydrophobic interactions should stabilize the folded structure of the antigen. While the specificity of the antibody will be determined primarily by the individual side chain reactions, hydrogen bonding and Van der Waals interactions, the affinity of the association and the reduction in free energy of the antigen is primarily due to hydrophobic interactions 26. Although most enzymes can be inhibited by certain antibodies, it should be possible to generate noninhibitory protective monoclonal antibodies since numerous monoclonal antibodies with overlapping specificities 31 should interact with areas on the enzyme surface that are not directly involved in the active centers. Indeed, earlier studies (late 1960s and early 1970s), concerned primarily with enzyme and isoenzyme characterization, revealed an increase in the heat resistance of several enzymes in the presence of specific polyclonal antibodies 32,33. More recently, a monoclonal antibody was used to block the single trypsin cleavage site of placental alkaline phosphatase 34. However, since cleavage at this site does not inactivate the enzyme (R. Jemmerson and T. Stigbrand, pers. commun.), protection against proteolytic inactivation was not demonstrated.

The relationship between thermostability and resistance to proteolysis Protection against proteolytic in-

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activation is related, to a certain degree, to protection against inactivation by unfolding. In tightly 'folded' proteins, because few or no specific proteolytic sites are exposed, proteolytic cleavage is limited. Cleavage does not necessarily result in inactivation, as shown by placental alkaline phosphatase 34 and some enzymes with multiple active sites a5. This is because catalytic domains are highly organised and more stable, while the peptide loops that link the domains are relatively unorganized and more susceptible to proteolytic digestion. In unfolded proteins, on the other hand, all of the cleavage sites are exposed and extensive degradation and loss of biological activity c a n o c c u r 36. There is a correlation between thermal stability and resistance to proteolysis. Thermostable enzymes from thermophilic bacteria, for example, are more resistant to proteolysis than are similar enzymes from mesophilic organisms 37. Enzymes with higher 'melting' temperatures (Tm) a r e more resistant to proteolysis than those with lower Tm36'37. Therefore, processes that confer thermal stability may also confer a significant degree of protection against inactivation by proteolytic enzymes.

Experiments with enzyme-antibody complexes To test this hypothesis we used a number of model enzymes and complexed them with specific antibodies (polyclonal antibodies for alpha-amylase, glucoamylase and subtilisin, and selected monoclonal antibodies either singly or in mixtures for L-asparaginase). The enzymatic activity of the complexes and the free enzymes after exposure to several inactivation methods was monitored 38. Briefly, pure enzymes were complexed with increasing concentrations of specific antibodies in order to find the optimal antibodyenzyme ratio for protection (found to be between 1 and 2.5). Then, the complexed and the free enzyme were preincubated for different times, exposed to various inactivating conditions. Stability was assessed by determining the residual activity of the enzyme. Some initial loss of activity (1015%) occurred only with glucoamylase even though polyclonal antibodies (which may have contained inhibitory antibodies) were used in most of the studies. Similar observations have been noted elsewhere 32,33. The absence of significant inhibition could be explained by a combination of:

TIBTECH - JULY 1989 [Vol. 7] --

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• the active site of the enzyme used being located in a 'blind' spot for the immune system; • the inhibitory antibodies being of lower affinity or quantity and their binding to the enzyme active site being sterically hindered by adjacent high affinity antibodies; • the low molecular weight synthetic substrates used in the assays being less susceptible to steric interference imposed by inhibitory antibodies. The temperature stability of the enzyme complexes was much higher than that of free enzymes; activity half-lives were as much as 200 times as long (Table 1 and Fig. 1). To evaluate chemical disruption, the enzymes were treated with low pH, NaOC1 (oxidant) or alcohol: • pH: L-asparaginase exposed to pH 3.0 lost 98% of its activity in 50 minutes while antibody-protected enzyme retained close to 40% activity at 50 minutes and 25% for the next 17 hours; • NaOCI: subtilisin exposed to 0.05% NaOC1 for 30 minutes retained only 25% of its original activity while the antibody-protected enzyme retained close to 80% of its activity (Fig. 2); • Alcohol: glucoamylase preincubated with 2.5% alcohol for 25 hours

retained only 10% of its original activity while the antibody-protected enzyme retained 98% of its activity. The model used to evaluate biological inactivation was proteolytic treatment with trypsin of the antileukemic enzyme L-asparaginase. This enzyme is extremely sensitive to proteolytic enzymes yet a mixture of three selected MABs conferred good protection (Table 2). Even a single MAB (not one of the original three) affords almost complete protection. The protected trypsin-treated enzyme was virtually as active as the trypsin-untreated control, whereas the trypsin-treated unprotected enzyme displayed little activity. The binding site of the antibody appears to be close to the single tryptic cleavage site (E. Y. Shami et al., unpublished). Another model for biological inactivation is the self digestion of trypsin; this can be largely inhibited by its antibodies 38. Current status and future developments Substantial protection has been demonstrated against enzyme inactivation by high temperature, proteolysis, low pH, oxidation (NaOC1)

and alcohol. Each of the four enzymes tested could be protected. This high success rate (albeit based on a small sample) encourages the view that protection might be afforded to any protein. From the range of antibodies that bind to overlapping epitopes over the surface of a 'target' protein, it should be possible to screen for those that are noninhibitory and result in stabilization. On the negative side, the production of monoclonal antibodies via mammalian cell culture is more costly by at least two orders of magnitude than 'target' protein production in microorganisms, thereby excluding on financial grounds many in vitro and some in vivo uses of enzyme-antibody complexes. Production of monoclonal antibodies in microorganisms could be the solution to this problem. Recently, several groups have reported success in obtaining fully functional recombinant antibody fragments; Fv (variable region fragments-), Fab, and fused, single-chain antibodies, all produced in E. coli. The subject was recently assessed by Wetze139 and, although the process is not problem free (low yield, some improper folding, low solubility in some cases), progress is encouraging. The single chain antibodies are of special interest as protecting agents. In principle, they could be either co-produced with their 'target' antigen or fused via a cross-linker peptide to form a chimeric protein. Thus a protected and stabilized protein could be produced by a single organism.

m Table 2

Resistance of asparaginasemonoclonal antibody complex to proteolysis

Sample Control free enzyme Free enzyme + trypsin M a b - e n z y m e + trypsin

Conversion time a (min) 18 690 20

aTime for the conversion of 50% of a 1 mM L-asparagine preparation to L-aspartic acid

TIBTECH- JULY 1989 [Vol. 7]

A second potential p r o b l e m for in vivo use is antigenicity. Most m o n o clonal antibodies are n o n - h u m a n (mouse) i m m u n o g l o b u l i n s and are, therefore, antigenic. This might restrict the clinical use of antibodyp r o t e c t e d proteins to cases w h e r e r e p e a t e d administration is not required. However, the problem of immunogenicity in non-human m o n o c l o n a l antibodies is shared with all other in vivo applications of m o n o c l o n a l antibodies and is being actively investigated. For example, r e d u c t i o n s in a n t i b o d y size (Fab fragments or single chain antibodies) m a y r e d u c e antigenicity. Chimeric antibodies c o m p r i s e d of m o u s e variable d o m a i n s and h u m a n constant d o m a i n s 4° could also offer solutions.

Comparison with other stabilization methods Comparisons of different stabilization m e t h o d s are difficult. T h e technologies are in an early stage of d e v e l o p m e n t and the a p p r o a c h e s are still largely empirical. T h e m o d e l systems, the destabilizing forces used, and the conditions of test are not uniform. Data are s o m e t i m e s i n c o m p l e t e because of confidentiality considerations. A tentative, general c o m p a r i s o n can be m a d e with reference to a series of desirable criteria: • stability s h o u l d be increased by at least one to three orders of magnitude; • protection s h o u l d be conferred against different inactivating forces biological, physical and chemical; • there s h o u l d be no r e d u c t i o n in biological activity or changes in specificity; • m e t h o d s s h o u l d be suitable for all classes of 'target' proteins; • m e t h o d s s h o u l d be usable in vitro or in vivo; • there s h o u l d be potential for e c o n o m i c a l scale-up, usually involving p r o d u c t i o n by r e c o m b i n a n t technology. T h e m e t h o d s involving r a n d o m covalent modifications (e.g. attachm e n t of PEG) fall short of these criteria although, in specific instances, they m a y prove useful. R a n d o m or site-directed mutagenesis (protein engineering) and selected

antibody p r o t e c t i o n are general technologies that could meet the stated criteria. The former has the advantages that stabilization can potentially o c c u r by changes in internal bonding forces whereas the latter involves stabilizing the surface. Surface stability may, however, be more important in protection against proteolytic cleavage. Mutations result in protection w i t h i n the 'target' protein, whereas the a n t i b o d y proc e d u r e requires p r o d u c t i o n of a s e c o n d protein. This might be less of a drawback if the same organisms can p r o d u c e both, or if a single protected, chimeric protein can be constructed. While the p r o d u c t i o n of protecting antibodies is relatively quick and simple, mutagenesis, with its trial and error requirements, is more time consuming. Only time and experience will d e t e r m i n e w h i c h procedure will be the more useful.

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