348 Current Pharmaceutical Biotechnology, 2009, 10, 348-351
1389-2010/09 $55.00+.00电影片名
© 2009 Bentham Science Publishers Ltd.
Mechanisms of Protein Aggregation
John S. Philo * and Tsutomu Arakawa *
Alliance Protein Laboratories, Thousand Oaks, CA, USA
Abstract: Aggregation or reversible lf-association of protein therapeutics can ari through a number of different mechanisms. Five common aggregation mechanisms are described and their relations to manufacturing process to sup-press and remove aggregates are discusd.
Keywords: Aggregation, lf-association, mechanism, nucleation. INTRODUCTION
Protein aggregation can occur through a number of dis-tinct mechanisms or pathways. The mechanisms are not mutually exclusive, however, and more than one can occur for the same product.
While it is certainly not esntial that one understand the aggregation mechanism for a particular protein in order to develop an appropriate manufacturing process, a good formulation, or a method to suppress and remove aggregates, some mechanistic understanding can help point the way to solving aggregation issues (or at least to avoiding excipients and process that are likely to make things wor). Fig. (1) schematically illustrates five impor-tant mechanisms for protein aggregation. How the mecha-nisms and the types of aggregates relate to process and for-mulation development will be discusd in the last ction. M ECHANISM 1: REVERSIBLE ASSOCIATION OF THE NATIVE MONOMER
In Mechanism 1 the tendency to reversibly associate (ag-gregate) is intrinsic to the native form of the protein. The surface of the native protein monomer is lf-complementary so it will readily lf-associate to form reversible small oli-gomers. As illustrated here there may be multiple “sticky” or complementary patches on the monomer surface. Tho can then produce different types of interfaces, potentially pro-ducing multiple conformations for oligomers of the same stoichiometry and different patterns of oligomer growth. As the protein concentration ris and larger and larger oli-gomers form (driven by the law of mass action), over time the larger aggregates often become irreversible (sometimes through formation of covalent bonds such as disulfide link-ages). Insulin is ju
st one example of a therapeutic protein which readily (and normally) associates to form reversible oligomers [1]. Insulin also illustrates that such association can have important conquences for bioactivity, and how manipulation of that association via mutation has produced important new products [2]. Interleukin-1 receptor antagonist (rhIL-1RA) is an example of a product that undergoes re-versible dimerization at high concentrations, followed by formation of irreversible dimers and trimers [3].仔细反义词是什么
*Address correspondence to the authors at the Alliance Protein Laborato-ries, Thousand Oaks, CA, USA;
E-mails: ; M ECHANISM 2: AGGREGATION OF CONFORM A-TIONALLY-ALTERED MONOMER
In contrast to Mechanism 1, for Mechanism 2 the native monomer has a very low propensity to reversibly associate. However after it transiently undergoes a conformational change or partial unfolding the resultant altered conforma-tion of monomer associates strongly (in a manner similar to Mechanism 1). Thus a key difference between Mechanisms 1 and 2 is that in Mechanism 2 the first step is a conforma-tional change to a non-native state, and at any given time the fraction of protein i
n that aggregation-prone non-native state will usually be quite small. For Mechanism 2 aggregation will be promoted by stress such as heat or shear that may trigger the initial conformational change. A further (and im-portant) conquence is that aggregation will be inhibited by excipients or conditions that stabilize the native conforma-tion.
This aggregation mechanism does appear to be the domi-nant one for many proteins and has been discusd in veral reviews [4-6]. Two therapeutics where this mechanism has been reported are interferon- [7] and G-CSF [8, 9]. M ECHANISM 3: AGGREGATION OF CHEM I-CALLY-MODIFIED PRODUCT
Mechanism 3 is really a variant of Mechanism 2 where the change in protein conformation that precedes aggregation is caud by a difference in covalent structure. Usually this difference is caud by chemical degradation such as oxida-tion of methionine, deamidation, or proteolysis. Chemical changes may for example create a new sticky patch on the surface, or change the electric charge in a way that reduces electrostatic repulsion between monomers. In some cas however the chemically different species is not a degradant but rather it is a normal variant within the bulk drug product---for example in glycoproteins there might be a un-glycosylated or under-glycosylated fraction that is prone to aggregation.
A diagnostic feature of this mechanism is that the aggre-gates will be enriched in the modified form. (Although this is not illustrated in the figure, the modified monomers are sometimes able to recruit normal monomers into the aggre-gates, so the aggregate fraction will not necessarily contain
枯萎的意思
Mechanisms of Protein Aggregation Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 4 349
only modified monomers.) Clearly when Mechanism 3 is operative improving the chemical stability will also reduce aggregation, and converly attempts to improve the con-formational stability of the monomer may not reduce aggre-gation. It is also worth noting that aggregates of chemically altered protein can be particularly immunogenic [10].
M ECHANIS M
4: NUCLEATION-CONTROLLED AGGREGATION Nucleation-controlled aggregation is a common mecha-nism for formation of visible particulates or precipitates [5].
In this mechanism the native monomer has a low propensity
for formation of small and moderately-sized oligomers (the addition of monomers onto the smaller
aggregates is not thermodynamically favored). However if an aggregate of sufficient size manages to form, then the growth of this so-called “critical nucleus” through addition of monomers is strongly favored and the formation of much larger species is rapid. This type of process is similar to growing large crys-tals by adding micro-crystal “eds” to a saturated solution, and thus the critical nuclei are also sometimes called the “eds” or “templates” for aggregate growth.
A characteristic feature of a nucleation-controlled process is that the rate of formation of the large particles or precipi-tates usually exhibits a lag pha. That is, no particles or precipitates can be detected for a long period of time (per-haps months) but then rather suddenly the large species ap-pear and accumulate. The length of the lag pha can vary in
a stochastic manner from one vial to another within a single manufacturing lot, so particles may first appear in individual vials over a wide range of times. Thus far what has been described is called “homogeneous nucleation” where the critical nucleus is itlf a product ag-gregate. In a cond variant of this mechanism the critical nucleus (ed) is not a particle made of the product protein
but rather a particle of an impurity or contaminant. This c-ond variant is called “heterogeneous nucleation”. Two ex-amples of contaminants reported to have rved as eds for
Fig. (1). Shematic illustrations of five common aggregation mechanisms.
350 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 4 Philo and Arakawa
aggregation are silica particles shed by product vials [11] and steel particles shed by a piston pump ud for filling vials [12]. Anecdotal evidence also implicates silicone particles introduced by tubing ud in manufacturing or as lubricants for syringes, and vacuum pump oil particles introduced dur-ing lyophilization.
MECHANISM 5
The last mechanism to be discusd here is surface-induced aggregation (Mechanism 5). This aggregation proc-ess starts with binding of the native monomer to a surface. In the ca of an air-liquid interface that binding would proba-bly be driven by hydrophobic interactions, but for a con-tainer favorable electrostatic interactions might also be in-volved. After this initial reversible binding event the mono-mer undergoes a change in conformation (for example to increa the contact area with the surface). Like in Mecha-nism 2, it is then that conformationally-altered monomer which aggregates, but in this ca that aggregation might occur either on the surface or perhaps after the altered monomer is relead back into the solution. Freeze/thaw damage can also ari from aggreg
ation at the surfaces of ice crystals or crystals of excipients, and thus can occur through Mechanism 5, but freeze/thaw damage can also involve other mechanisms such as changes in pH.
It is interesting to note that Mechanism 4 could be con-sidered a special ca of Mechanism 5 where the surface that induces aggregation is the surface of the critical nucleus. A cond point about this mechanism is that during accelerated stability testing the tests that involve agitation or that try to induce shear forces by moving balls through the liquid (will produce conformational stress and therefore may induce ag-gregation through Mechanism 2), but also may simultane-ously produce significant exposure to surfaces, so it may be unclear which stress is actually inducing the aggregation. WHY DOES MECHANISM MATTER?
The principal advantage of an understanding of the mechanism of aggregation is that this can help guide process development and/or the formulation effort. There are a num-ber of ways such understanding might help with either up-stream or downstream process development. When proteins are marginally stable against partial or complete unfolding, care must be exercid to avoid both mechanical stress and exposure to air or solid surfaces that may lead to adsorption-induced unfolding. In such cas, process that minimize the surface exposure should reduce aggregation. Certain pro-teins are nsitive to mechanical stress such as agitation and hence may aggregate d
uring chromatography or filtra-tion due to shear strain, requiring extra care during the operations. Aggregation during production steps can also sometimes be prevented by adding appropriate stabilizing co-solutes, provided that such additives do not interfere with the purification process.
An understanding of aggregation mechanisms is particu-larly uful during formulation, with respect to the addition of “generic protein stabilizers” such as sucro, polyols and certain amino acids and salts. The co-solutes increa the stability of native protein structure against various environ-mental stress that cau unfolding. The co-solute interac-tions with the protein surface are thermodynamically unfa-vorable, which favors a minimal surface area and hence the native structure (e Chapter 4.1 for further discussion of this stabilization mechanism). Such co-solutes should therefore reduce aggregation that is caud by Mechanism 2.
Mechanism 2 is certainly an important one that has been reported for a number of proteins [7, 9, 13, 14], and one which has received particular emphasis by John Carpenter, Ted Randolph and co-workers at the University of Colorado [5]. Antimicrobial prervatives added to multi-do formu-lations often increa protein aggregation, and it was shown that benzyl alcohol drives aggregation through Mechanism 2 by promoting transitions to partially-unfolded states [15]. However many scientists do not realize that addition of “ge-neric structure stabilizers” is not a universal cure for aggr
e-gation problems, and in fact will usually significantly in-crea aggregation arising via Mechanisms 1, 4, and 5. As already noted the co-solutes tend to drive the protein to a state of minimum surface area expod to the co-solute. One way to minimize the surface area per monomer is for the native monomer to lf-associate (Mechanisms 1 and 4). An alternative way to minimize protein surface area expod to co-solute is to leave the solution by adsorbing to a surface (Mechanism 5).
Converly, for mechanisms 1, 4 or 5 adding co-solutes that weakly bind to the protein may reduce aggregation. Co-solutes that strongly bind to the protein would also reduce aggregation via this mechanism, but may also destabilize the folding and hence enhance aggregation via Mechanism 2. Such destabilizing co-solutes, e.g., urea, guanidine hydro-chloride or strong detergents, are strong protein solubilizing compounds, as briefly described in chapter 4.1. Among weakly binding co-solutes, arginine is especially effective in reducing protein aggregation that occurs due to Mechanisms 1, 4 and 5. The mechanism of aggregation suppression by arginine is described in detail in chapter 4.2 and 4.3.
This discussion of mechanisms also helps in understand-ing whether addition of a surfactant will help to reduce ag-gregation. It is fairly obvious that surfactants should reduce aggregation through Mechanism 5 by reducing exposure of the protein to the surfaces. Surfactants may also help reduce
nucleation-controlled aggregation (Mechanism 4) by cover-ing the surface of the critical nuclei. Typically however sur-factants are not helpful for Mechanisms 1, 2, or 3 (and in-deed the impurities commonly found in polysorbates may increa chemical degradation and therefore drive Mecha-nism 3).
A third way in which mechanism may matter is that some forms of aggregates may be wor than others. We e that for Mechanisms 2-5 the aggregates are primarily made from non-native monomers. This makes it more likely they will have altered potency as well as altered immunogenicity (be-cau the altered monomers prent different epitopes). On the other hand, becau Mechanism 1 aggregates are native-like, if their larger size does induce an immune respon it is more likely tho antibodies will cross-react with (and poten-tially neutralize) the native monomer. While the formulation or process development scientist may not be able to control which type of aggregates are dominant for a particular pro-营销培训
Mechanisms of Protein Aggregation Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 4 351
tein, such considerations might be important when different pathways dominate under different formulation conditions.
Understanding aggregation mechanisms may also help developing an optimal chromatography step to remove ag-gregation (Chapter 5.1, 5.2, 5.3 and 5.4) or for pressure-induced aggregate disruption (Chapter 5.5). When high hy-drostatic pressure is ud to dissociate aggregates, under-standing the aggregation mechanism may help in designing optimal solvent and temperature conditions, as high pressure mainly disrupts hydrophobic interactions. A lightly cha-otropic co-solvent may enhance dissociation conferred by pressure. It is obvious that chromatography chon to re-move aggregates should not generate new aggregates and hence understanding the mechanism should be helpful; e.g., aggregate removal chromatography that generates shear strain should not be ud when the protein is susceptible to shear stress. In addition, knowing the type of aggregates may help design a better chromatography method for aggregate removal. Hydrophobic interaction chromatography may be most effective, for example, when conformational change and hence exposure of hydrophobic surface that cau Mechanism 2 aggregation are extensive.
REFERENCES
[1] Pekar, A.H. and Frank, B.H.(1972) Biochemistry,11(22), 4013-6. [2] Brems, D.N.; Alter, L.A.; Beckage, M.J.; Chance, R.E.; DiMarchi,
R.D.; Green, L.K.; Long, H.B.; Pekar, A.H.; Shields, J.E. and广东二本
Frank, B.H.(1992) Protein Eng.,5, 527-33.
[3] Alford, J.R.; K endrick, B.S.; Carpenter, J.F. and Randolph, T.W.
(2007) J. Pharm. Sci.,97(8), 3005-21.
[4] K rishnamurthy, R. and Manning, M.C.(2002) Cur r. Phar m. Bio-
technol.,3(4), 361-71.
[5] Chi, E.Y.; Krishnan, S.; Randolph, T.W. and Carpenter, J.F.(2003)
Pharm. Res.,20(9), 1325-36.
[6] Wang, W.(2005) Int.J. Pharm.,289(1-2), 1-30.
[7] K endrick, B.S.; Carpenter, J.F.; Cleland, J.L. and Randolph, T.W.
(1998) Proc. Natl. Acad. Sci. USA,95(24), 14142-6.
[8] K rishnan, S.; Chi, E.Y.; Webb, J.N.; Chang, B.S.; Shan, D.X.;
Goldenberg, M.; Manning, M.C.; Randolph, T.W. and Carpenter,
J.F.(2002) Biochemistry,41(20), 6422-31.
[9] Raso, S.W.; Abel, J.; Barnes, J.M.; Maloney, K.M.; Pipes, G.;
Treuheit, M.J.; King, J. and Brems, D.N.(2005) Protein Sci.,14(9),
2246-57.
[10] Hermeling, S.; Schellekens, H.; Maas, C.; Gebbink, M.F. B.G.;
Crommelin, D.I. A. and Jiskoot, W.(2006) J. Pharm. Sci.,95(5),
1084-96.
水晶头颅
[11] Chi, E.Y.; Weickmann, J.; Carpenter, J.F.; Manning, M.C. and
Randolph, T.W.(2005) J. Pharm. Sci.,94(2), 256-74.
[12] Tyagi, A.K.; Randolph, T.W.; Dong, A.; Maloney, K.M.;
Hitscherich, C., Jr. and Carpenter, J.F.(2008) J. Phar m. Sci.,93,
费斯切拉1390-1402.
[13] Chi, E.Y.; K rishnan, S.; K endrick, B.S.; Chang, B.S.; Carpenter,
J.F. and Randolph, T.W.(2003) Protein Sci.,12(5), 903-13.
[14] Manning, M.C.; Matsuura, J.E.; K endrick, B.S.; Meyer, J.D.;
Dormish, J.J.; Vrkljan, M.; Ruth, J.R.; Carpenter, J.F. and Shefter,
E.(1995) Biotechnol. Bioeng.,48(5), 506-12.
[15] Thirumangalathu, R.; Krishnan, S.; Brems, D.N.; Randolph, T.W.
and Carpenter, J.F.(2006) J. Pharm. Sci.,95(7), 1480-97.
40字日记
Received: November 30, 2008 Revid: February 18, 2009 Accepted: February 18, 2009
