Abstract
The effects of “shear” on proteins in solution are described and discussed. Research on this topic covers many decades, beginning with investigations of possible denaturation of enzymes during processing, whilst more recent concerns are how the quality of therapeutic proteins might be affected by shear or shear related effects. The paradigm that emerges from most studies is that shear in the fluid mechanical sense is unlikely by itself to damage most proteins and that interfacial phenomena are critically important. In particular, moving gas–liquid interfaces can be very deleterious. Aggregation of therapeutic proteins on nanoparticles shed from solid surfaces is a recent concern because of potential consequences on patient safety. It is clear that labeling such damage as “shear” is a mistake as this inhibits clear investigations of, and thinking about, the true causes of damage to proteins in solution during processing.
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References
Baldascini H, Janssen DB (2005) Interfacial inactivation of epoxide hydrolase in a two-liquid-phase system. Enzyme Microbial Technol 36:285–293
Bam NB, Cleland JL, Yang J, Manning MC, Carpenter JF, Kelley RF, Randolph JW (1998) Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interactions. J Pharm Sci 87:1554–1559
Baujard-Lamotte L, Noinville S, Goubard F, Marque P, Pauthe E (2008) Kinetics of conformational changes of fibronectin adsorbed onto model surfaces. Colloids Surfaces B 63:129–137
Bee JS, Stevenson JL, Mehta B, Svitel J, Pollastrini J, Platz R, Freund E, Carpenter JF, Randolph TW (2009a) Response of a concentrated monoclonal antibody formulation to high shear. Biotechnol Bioeng 103:936–943
Bee JS, Chiu D, Sawicki S, Stevenson JL, Chatterjee K, Freund E, Carpenter JF, Randolph TW (2009b) Monoclonal antibody interactions with micro- and nanoparticles: adsorption, aggregation, and accelerated stress studies. J Pharm Sci 98:3218–3238
Bee JS, Nelson SA, Freund E, Carpenter JF, Randolph TW (2009c) Precipitation of a monoclonal antibody by soluble tungsten. J Pharm Sci 98:3290–3301
Biddlecombe JG, Craig AV, Zhang H, Uddin S, Mulot S, Fish BC, Bracewell DG (2007) Determining antibody stability: creation of solid-liquid interfacial effects within a high shear environment. Biotechnol Prog 23:1218–1222
Biddlecombe JG, Smith G, Uddin S, Mulot S, Spencer D, Gee C, Fish BC, Bracewell DG (2009) Factors influencing antibody stability at solid-liquid interfaces in a high shear environment. Biotechnol Prog 25:1499–1507
Bodalo A, Gomez JL, Gomez E, Maximo MF, Montiel MC (2004) Study of L-aminoacylase deactivation in an ultrafiltration membrane reactor. Enzyme Microbial Technol 35:261–266
Brych SR, Gokarn YR, Hultgen H, Stevenson RJ, Rajan R, Matsumura M (2010) Characterization of antibody aggregation: role of buried, unpaired cysteines in particle formation. J Pharm Sci 99:764–781
Caussette M, Gaunand A, Planche H, Monsan P, Lindet B (1998) Inactivation of enzymes by inert gas bubbling—a kinetic study. Enzyme Engineering XIV 864:228–233
Caussette M, Gaunand A, Planche H, Colombie S, Monsan P, Lindet B (1999) Lysozyme inactivation by inert gas bubbling: kinetics in a bubble column reactor. Enzyme Microbial Technol 24:412–418
Charm SE, Lai CJ (1971) Comparison of ultrafiltration systems for concentration of biologicals. Biotechnol Bioeng 13:185–202
Charm SE, Wong BL (1970) Enzyme inactivation with shearing. Biotechnol Bioeng 12:1103–1109
Charm SE, Wong BL (1981) Shear effects on enzymes. Enzyme Microbial Technol 3:111–118
Chou DK, Krishnamurthy R, Randolph TW, Carpenter JF, Manning MC (2005) Effects of Tween 20 (R) and Tween 80 (R) on the stability of albutropin during agitation. J Pharm Sci 94:1368–1381
Clarkson JR, Cui ZF, Darton RC, Clarkson JR (1999a) Protein denaturation in foam—I. Mechanism study. J Coll Int Sci 215:323–332
Clarkson JR, Cui ZF, Darton RC (1999b) Protein denaturation in foam—II. Surface activity and conformational change. J Coll Int Sci 215:333–338
Colombie S, Gaunand A, Lindet B (2001) Lysozyme inactivation under mechanical stirring: effect of physical and molecular interfaces. Enzyme Microbial Technol 28:820–826
Cromwell MEM, Hilario E, Jacobson F (2006) Protein aggregation and bioprocessing. AAPS J 8:E572–E579
Donaldson TL, Boonstra EF, Hammond JM (1980) Kinetics of protein denaturation at gas–liquid interfaces. J Coll Int Sci 74:441–450
Elias CB, Joshi JB (1998) Role of hydrodynamic shear on activity and structure of proteins. Adv Biochem Eng 59:47–71
Fradkin AH, Carpenter JF, Randolph TW (2009) Immunogenicity of aggregates of recombinant human growth hormone in mouse models. J Pharm Sci 98:3247–3264
Ghadge RS, Sawant SB, Joshi JB (2003) Enzyme deactivation in a bubble column, a stirred vessel and an inclined plane. Chem Eng Sci 58:5125–5134
Ghadge RS, Patwardhan AW, Sawant SB, Joshi JB (2005) Effect of flow pattern on cellulase deactivation in stirred tank bioreactors. Chem Eng Sci 60:1067–1083
Gomme PT, Hunt BM, Tatford OC, Johnston A, Bertolini J (2006a) Effect of lobe pumping on human albumin: investigating the underlying mechanisms of aggregate formation. Biotechnol Appl Biochem 43:103–111
Gomme PT, Prakash M, Hunt B, Stokes N, Cleary P, Tatford OC, Bertolini J (2006b) Effect of lobe pumping on human albumin: development of a lobe pump simulator using smoothed particle hydrodynamics. Biotechnol Appl Biochem 43:113–120
Harrington TJ, Gainer JL, Kirwan DJ (1991) Effects of fluid shear on immobilized enzyme-kinetics. Enzyme Microbial Technol 13:610–616
Harrison JS, Gill A, Hoare M (1998) Stability of a single-chain Fv antibody fragment when exposed to a high shear environment combined with air-liquid interfaces. Biotechnol Bioeng 59:517–519
He F, Phan DH, Hogan S, Bailey R, Becker GW, Narhi LO, Razinkov VL (2010) Det ection of IgG aggregation by a high throughput method based on extrinsic fluorescence. J Pharm Sci 99:2598–2608
Imamura K, Shimomura M, Nagai S, Akamatsu M, Nakanishi K (2008) Adsorption characteristics of various proteins to a titanium surface. J Biosci Bioeng 106:273–278
Iyer PV, Ananthanarayan L (2008) Enzyme stability and stabilization—aqueous and non-aqueous environment. Proc Biochem 43:1019–1032
Jabbal-Gill I, Lin W, Jenkins P, Watts P, Jimenez M, Illum L, Davis SS, Wood JM, Major D, Minor PD, Li XW, Lavelle EC, Coombes AGA (1999) Potential of polymeric lamellar substrate particles (PLSP) as adjuvants for vaccines. Vaccine 18:238–250
Jaspe J, Hagen SJ (2006) Do protein molecules unfold in a simple shear flow? Biophys J 91:3415–3424
Jones LS, Kaufmann A, Middaugh CR (2005) Silicone oil induced aggregation of proteins. J Pharm Sci 94:918–927
Joshi JB, Sawant SB, Patwardhan AW, Patil DJ, Kshatriya SS, Nere NK (2001) Relation between flow pattern and de-activation of enzymes in stirred reactors. Chem Eng Sci 56:443–452
Kiese S, Pappenberger A, Friess W, Mahler HC (2010) Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation. J Pharm Sci 99:632–644
Kim MH, Lee SB, Ryu DDY, Reese ET (1982) Surface deactivation of cellulase and its prevention. Enzyme Microbial Technol 4:99–103
Kreilgaard L, Jones LS, Randolph TW, Frokjaer S, Flink JM, Manning MC, Carpenter JF (1998) Effect of Tween 20 on freeze-thawing and agitation-induced aggregation of recombinant, human factor XIII. J Pharm Sci 87:1597–1603
Krstic DM, Antov MG, Pericin DM, Hoflinger W, Tekic MN (2007) The possibility for improvement of ceramic membrane ultrafiltration of an enzyme solution. Biochem Eng J 33:10–15
Kwon YM, Baudys M, Knutson K, Kim SW (2001) In situ study of insulin aggregation induced by water-organic solvent interface. Pharm Res 18:1754–1759
Lee YK, Choo CL (1989) The kinetics and mechanism of shear inactivation of lipase from Candida cylindracea. Biotechnol Bioeng 33:183–190
Lencki RW, Tecante A, Choplin L (1993) Effect of shear on the inactivation kinetics of the enzyme dextransucrase. Biotechnol Bioeng 42:1061–1067
Ludwig DB, Carpenter JF, Hamel JB, Randolph TW (2010) Protein adsorption and excipient effects on kinetic stability of silicone oil emulsions. J Pharm Sci 99:1721–1733
Maa YF, Hsu CC (1997) Protein denaturation by combined effect of shear and air-liquid interface. Biotechnol Bioeng 54:503–512
Mahler HC, Muller R, Friess W, Delille A, Matheus S (2005) Induction and analysis of aggregates in a liquid IgG1-antibody formulation. Eur J Pharmaceut Biopharmaceut 59:407–417
Mahler HC, Friess W, Grauschopf U, Kiese S (2009) Protein aggregation: pathways, induction factors and analysis. J Pharm Sci 98:2909–2934
Mahler HC, Huber F, Kishore RSK, Reindl J, Ruckert P, Muller R (2010) Adsorption behavior of a surfactant and a monoclonal antibody to sterilizing-grade filters. J Pharm Sci 99:2620–2627
McLeod AG, Walker IR, Zheng S, Hayward CPM (2000) Loss of factor VIII activity during storage in PVC containers due to adsorption. Haemophilia 6:89–92
Mohanty M, Ghadge RS, Patil NS, Sawant SB, Joshi JB, Deshpande AV (2001) Deactivation of lipase at gas—liquid interface in stirred vessel. Chem Eng Sci 56:3401–3408
Nakanishi K, Sakiyama T, Imamura K (2001) On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon. J Biosci Bioeng 91:233–244
Narendranathan TJ, Dunnill P (1982) The effect of shear on globular-proteins during ultrafiltration—studies of alcohol dehydrogenase. Biotechnol Bioeng 24:2103–2107
Otero C, Fernandez-Perez M, Perez-Gil J (2005) Effects of interactions with micellar interfaces on the activity and structure of different lipolytic isoenzymes from Candida rugosa. Enzyme Microbial Technol 37:695–703
Paolucci-Jeanjean D, Belleville MP, Rios GM (2001) A comprehensive study of the loss of enzyme activity in a continuous membrane reactor—application to starch hydrolysis. J Chem Technol Biotechnol 76:273–278
Patil NS, Ghadge RS, Sawant SB, Joshi JB (2000) Lipase deactivation at gas—liquid interface and its subsequent reactivation. AICHE J 46:1280–1283
Patro SY, Freund E, Chang BS (2009) Protein formulation and fill-finish operations. Biotechnol Ann Rev 8:55–84
Pereira LGC, Johansson C, Radke CJ, Blanch HW (2003) Surface forces and drainage kinetics of protein-stabilized aqueous films. Langmuir 19:7503–7513
Portugal CAM, Lima JC, Crespo JG (2008) Effect of physicochemical conditions on the ultrafiltration of beta-lactoglobulin: fluorescence probing of induced structural changes. J Membr Sci 321:69–80
Postel C, Abillon O, Desbat B (2003) Structure and denaturation of adsorbed lysozyme at the air-water interface. J Coll Int Sci 266:74–81
Prazeres DMF, Cabral JMS (1994) Enzymatic membrane bioreactors and their applications. Enzyme Microbial Technol 16:738–750
Rathore N, Rajan RS (2008) Current perspectives on stability of protein drug products during formulation, fill and finish operations. Biotechnol Prog 24:504–514
Reese ET, Robbins FM (1981) Denaturation of beta-lactoglobulin by shaking and its subsequent renaturation. J Coll Int Sci 83:393–400
Rosenberg AS (2006) Effects of protein aggregates: an immunologic perspective. AAPS J 8:E501–E507
Ross AC, Bell G, Halling PJ (2000a) Effect of pH on rate of interfacial inactivation of serine proteases in aqueous-organic systems. Biotechnol Bioeng 67:498–503
Ross AC, Bell G, Halling PJ (2000b) Organic solvent functional group effect on enzyme inactivation by the interfacial mechanism. J Mol Catal B: Enzym 8:183–192
Sanchez-Ruiz JM (2010) Protein kinetic stability. Biophys Chem 148:1–5
Sandwick RK, Schray KJ (1987) The inactivation of enzymes upon interaction with a hydrophobic latex surface. J Coll Int Sci 115:130–138
Sauerborn M, van Beers M, Jiskoot W, Schellekens H (2008) Immunogenicity of therapeutic proteins: a ‘classical’ break of tolerance? Wien Klin Wochenschr 120:123
Schellekens H (2002) Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clin Ther 24:1720–1740
Schellekens H, Jiskoot W (2006) Role of aggregation in immunogenicity of recombinant human proteins. Nephrol Dial Transplant 21:318
Serno T, Carpenter JF, Randolph TW, Winter G (2010) Inhibition of agitation-induced aggregation of an IgG-antibody by hydroxyl-beta-cyclodextrin. J Pharm Sci 99:1193–1206
Sharma B (2007) Immunogenicity of therapeutic proteins. Part 2: impact of container closures. Biotechnol Adv 25:318–324
Shire SJ. (2005) Impact of reversible protein self-association on manufacturing, formulation and delivery of protein pharmaceuticals. Abs Pap Am Chem Soc 229:194
Shire SJ, Shahrokh Z, Liu J (2004) Challenges in the development of high protein concentration formulations. J Pharm Sci 93:1390–1402
Siedlecki CA, Lestini BJ, KottkeMarchant K, Eppell SJ, Wilson DL, Marchant RE (1996) Shear-dependent changes in the three-dimensional structure of human von Willebrand factor. Blood 88:2939–2950
Sluzky V, Klibanov AM, Langer R (1992) Mechanism of insulin aggregation and stabilization in agitated aqueous solutions. Biotechnol Bioeng 40:895–903
Thirumangalathu R, Krishnan S, Ricci MS, Brems DN, Randolph TW, Carpenter JF (2009) Silicone oil- and agitation-induced aggregation of a monoclonal antibody in aqueous solution. J Pharm Sci 98:3167–3181
Thomas CR, Dunnill P (1979) Action of shear on enzymes—studies with catalase and urease. Biotechnol Bioeng 21:2279–2302
Thomas CR, Nienow AW, Dunnill P (1979) Action of shear on enzymes—studies with alcohol dehydrogenase. Biotechnol Bioeng 21:2263–2278
Tirrell M, Middleman S (1975) Shear modification of enzyme kinetics. Biotechnol Bioeng 17:299–303
Tyagi AK, Randolph TW, Dong A, Maloney KM, Hitscherich C, Carpenter JF (2009) IgG Particle formation during filling pump operation: a case study of heterogeneous nucleation on stainless steel nanoparticles. J Pharm Sci 98:94–104
Tzannis ST, Hrushesky WJM, Wood PA, Przybycien TM (1996) Irreversible inactivation of interleukin 2 in a pump-based delivery environment. PNAS 93:5460–5465
van der Veen ME, van Iersel DG, van der Goot AJ, Boom RM (2004) Shear-induced inactivation of alpha-amylase in a plain shear field. Biotechnol Prog 20:1140–1145
van Reis R, Zydney A (2007) Bioprocess membrane technology. J Membr Sci 297:16–50
Virkar PD, Narendranathan TJ, Hoare M, Dunnill P (1981) Studies of the effects of shear on globular-proteins—extension to high shear fields and to pumps. Biotechnol Bioeng 23:425–429
Walstra P (2001) Effect of agitation on proteins. In: Dickinson E, Miller R (eds) Food colloids: fundamentals of formulation. Royal Society of Chemistry, Cambridge, pp 245–254
Wierenga PA, Egmond MR, Voragen AGJ, de Jongh HH (2006) The adsorption and unfolding kinetics determines the folding state of proteins at the air-water interface and thereby the equation of state. J Coll Int Sci 299:850–857
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The authors sincerely thank Lindsay Schmiedel for critical review and insightful comments and the Formulation Development Group at Merck & Co., Inc. for their support of the manuscript.
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Thomas, C.R., Geer, D. Effects of shear on proteins in solution. Biotechnol Lett 33, 443–456 (2011). https://doi.org/10.1007/s10529-010-0469-4
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DOI: https://doi.org/10.1007/s10529-010-0469-4