Second Edition
`
`Pharmaceutical Formulation
`Development of Peptides
`and Proteins
`
`Edited by
`Lars Hovgaard
`Sven Frokjaer
`Marco van de Weert
`
`CRC Press is an imprint of the
`Taylor 6t Francis Group, an lnforma business
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`Libr:iry of Congress Catalnging-in -Puhlkalion Data.
`
`Pharmaceutical form ulation development of peptides a nd proteins/ ed itors, Lars
`Hovgaard, Sven Frokjaer, Ma rco va n de Weert. -- 2nd ed.
`p. ;cm.
`Incl udes bibliographica l referenc,esand index.
`ISBN 978 -1-•1398-5388-7 (hardcover: alk, paper)
`I. Hovga.ard, Lars) 1962 - II. Frukjrer, Sven, 1947• LI L \Veert, Marco va n de, 1973 -
`fDNL.M: L (>cptidc Blosynthesis, 2, Chemistry, Pha rmaceutical -- melhod.s, 3. Protei n
`Biosynthesis. QU 68J
`
`615.1'9- dc23
`
`2012030490
`
`Visit the Taylor & Francis Web site 11 1
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`and th e CRC Press Web site a t
`http://www.c rcpress.com
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`Contents
`
`Preface .............. , ........................................................................ , .. ,., .......................... vii
`Editors ................... ................... _ .............................................................................. ix
`Contributors .............................................................................................................. xi
`
`Chapter 1 Peptide Synthesis .................................................................................. I
`
`Knud J. Jensen
`
`Chapter 2 Protein Expression .............................................................................. 17
`
`Nanni Din
`
`Chapter 3 Protein Purification ........................................................... _ .............. 35
`
`!.ars Havgaard. !.ars Skriver, and Sven Frokjaer
`
`Chupter 4 Clrnractcrization of Therapeutic Peptides and Proteins .................... .49
`
`Marco van de Weer/ and Tudor Arvlltte
`
`Chapter S Chemical Pathways of Peptide and Protein Degradation ................... 79
`
`Teru11a J. Sia/,aan and Chris/ian Schorreic/1
`
`Chapter 6 Physical Instability of Peptides and Proteins ................................... 107
`
`Marco van de Weer/ a11d Theodore W. Randolph
`
`Chapter 7 Peptide and Protein Derivatives .................................................... 131
`
`Kristian Str<Jmgaard and Thomas l/9eg•Je11sen
`
`Chapter 8 Peptides and Proteins as Parenteral Solutions ................................ 149
`
`Michael J. Akers and Michael R. DeFelippis
`
`Chapter 9 Peptides and Proteins as Parenteral Suspensions: An Overview
`of Design, Development, and Manufacturing Considerations ......... 193
`
`Michael R. DeFelippis and Michael J. Akers
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`vi
`
`Contents
`
`Chapter IO Rational Design of Solid Protein Formulations ···~··· .. ········· .. ··· ....... 239
`Bingq11t111 (Stuarr) Wang tmd Michael J. Pikal
`
`Chapter U Peptide and Protein Drug Delivery Systems for Nonparcmeral
`Routes of Administration ....................................... .......................... 269
`
`Ulrik Lyu Rahbek, Frm,tisek Hubtilek, and Simon Bjerregcwrd
`
`Chapter 12 lmmunogenicity ol'Therapeutic Proteins ......................................... 297
`
`Grzegorz Kijanka, Wim Jiskoo1, Melody Suuerbon,.
`Huub Srhel/ekens, and Vern Brinks
`
`Chapter 13 Biosimulation of Peptides and Proteins ........................................... 323
`
`Tue S¢eborg. Chris1ian Hove Ras11111sse11, Erik Mosekilde. and
`Morten Coldi11g-J¢rge11se11
`
`Chapter 14 Registration of Peptides and Protein~ ............................................. 339
`
`Niamh Kinsella
`
`Index ...................................................................................................................... 363
`
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`6 Physical Instability of
`Peptides and Proteins
`
`Marco van de Weert and Theodore W. Randolph
`
`CONTENTS
`
`Introduction .................................................................................................. 107
`6.1
`6.2 Protein Structure ........................................................... ................................ 108
`6.2.1 Peptides. Polypeptides, and Proteins ................................................ 108
`6.2.2 Protein Structure: Primary, Secondary. Tertiary, and
`Quaternary Struciurc .............................................. , .......................... 108
`6.3 Protein Folding: Why Do Proteins Fold? ..................................................... 109
`6.3.1 Role of Water and Stabilizing Interactions ...................................... , 109
`6.3.2 The Energy Landscape of a Protein Molecule ............................ , .... 111
`6.4 Protein Physical Degradation ...................................................................... 114
`6.4. I Protein Unfolding ........... ................................................................ 114
`6.4.2 Adsorption ........................................................................................ l 17
`6.4.3 Protein Aggregation .......................................................................... 118
`6.4.3.1 Aggregation Mechanisms and Kinetics ............................. 11 9
`6.4.3.2 Fibrillation: A Special Case of Protein Aggregation ......... 120
`6.4.4 Protein Precipitation ......................................... ................................ 121
`6.5 Stabilization Strategies ................................................. ................................ 122
`6.6 Concluding Remarks .................................................................................... 125
`References...............................
`................... ...........
`. .......... 126
`
`6.1
`
`INTRODUCTION
`
`The biological funct ion of peptides and proteins is highly dependent on their three·
`djmcnsionaJ structure. Changes in Lha.L structure, which may arise due to chemical
`or physical processes, may alter or abolish that function , or even result in toxicity.
`Thus, it is of importance that a pharmaceutical formulation of therapeutic peptides
`and proteins retains the normal (native) structure of those peptides or proteins, or
`that any changes are fully reversible upon administration to the patient.
`A major difference between proteins and low molecular weight drugs is the com(cid:173)
`plexity of the three-dimensional structure and concomitant sensitivity toward cxtcr.
`nal stress factors. The threc•dimensional structure of proteins is mostly held log.ether
`by noncovalent interactions, such as hydrogen bonds, salt bridges, and van der Waals
`forces. Any stress factor may alter these noncovalent interactions. possibly leading to
`new inlrn• or intermolecular interactions which may not be reversible upon removing
`the stress factor.
`
`107
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`108
`
`Pharmaceutical Formulation Develo1pment of Pe ptides and Proteins
`
`In this chapter, we will discuss the noncovalent ;n1eractions that result in the for(cid:173)
`mation of the specific three-dimensional fold of most proteins, the most important
`stress factors that may cause changes in that protciin fold, and the resulting physical
`instability of the protein. It should be noted that we w ill discuss this physical instabil(cid:173)
`ity as a separate issue to the chemical instability discussed in Chapter 5. In reality, the
`two are highly interdepcndem, as chemical destabilization may lead to physical desta(cid:173)
`bilization, and vice versa. This chapter will end with potential stabilization strategics
`to prevent physical ins1ability of proteins in pharmaceutical formu lations. Several of
`these strategics will be discussed with more specific examples in other chapters.
`Throughout the chapter, a number of semantic issues wil I be discussed, which arc of
`importance when reading the literature. The commonly used terminology within the
`fie ld of protein structure, folding, and stability is not always strictly defined. and dcfini•
`Lions may differ over lime and depending on the cont,ext. Unfortunately, the definitions
`used in a particular scientific paper are often not cxpl.icit, which may lead to confus.ion
`when the reader is insufficiently aware of the different descriptions that are in use.
`
`6.2 PROTEIN STRUCTURE
`
`6.2.1
`
`PEPTLDES, POLYPEPTIDES, AND PROTEINS
`
`All peptides, polypeptides, and proteins are considered condensation polymers of
`amino acids, resulting in a linear backbone of alternating amide, C- C, and C-N
`bonds. However, the distinction between peptide, polypeptide, and protein is rather
`diffuse. One may find at least three different and partly overlapping descript ions,
`rather than definitions, of the difference between peptide a nd protein alone. The cur(cid:173)
`rently most common description refers to any peptide chain of more than 50 amino
`ac ids as a protein. Others refer to peptides as proteins whenever the peptide has
`a biological function. This could then even include several simple dipeptidcs (i.e ..
`two amino acids linked together), which can have a, biological function. Finally, the
`absence or presence of a well-defined tertiary struc1:ure has been used to distinguish
`peptides from proteins. Also, this distinction is not without problems; there arc pro(cid:173)
`teins that are referred to as "natively unfolded," so called because they do not have
`a specific tertiary structure. In addition, some "peptides" can form fully reversible
`multimeric structures, such as glucagon (forming trimers) (Formisano ct al .. 1977),
`which involves lhc formation of a defined three-dimensional structure. The term
`"polypeptide" generally overlaps with that of "peptide" and "protein." The reader
`may thus encounter a ll three terms used in conn,ection with the same biological
`compound. For practical purposes, we have used the first definition, calling every
`compound with more than 50 amino acids a protein.
`
`6.2.2 PROTEIN STRUCTURE: PRIMARY, 5ECONDARll,
`TERTIARY, AND Q uATERNARV STRUCTURE
`
`T he three-dimensional structure of proteins is often subdivided into four types of
`Structure, referred to as the primary, secondary, tertiary, and quaternary structure.
`The primary structure refers to the amino acid sequence within the polymer chain.
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`109
`
`Intra- or intcrchain c ross-links are also prevalent, usually through cyslcinc residues
`(forming a cystine or S-S bridges).
`The secondary structure refers to the folding of this backbone into specific struc(cid:173)
`tures, which are defined by the bond angles and hydrogen bonding pattern of the
`amide bond. The secondary structure can roughly be subdivided into four classes:
`helic al struct ures such as the alpha-helix, pleated structures such as the beta-strand
`and beta-sheet, turn structures such as the beta-turns. and loop structures. The latter
`are often referred to as "random" struc tures.
`The three-dimensiona l alignment of the secondary structural elemenls is known
`as the tertiary structure of a protein. This alignment often results in an almost ideal
`close packing of the amino acids, particularly in the core of the protein molecule.
`Protein tertiary structures can be described by commonly appearing architectures
`such as barrels or alpha-he lix bundles (Orengo and Thornton. 2005).
`Some proteins exist under physiological conditions as specific multimcric pro(cid:173)
`teins linked through noncovalent interac tions. This multimerization is known as the
`quaternary struc ture of a protein. Examples of proteins with a quaternary struc ture
`include hemoglobin, alpha-crystallin, and HIV-I protease. In general, the biologi(cid:173)
`cal function of such multimeric proteins depends on I.his multimerization, but some
`proteins may also form specific (and reversible) multimers that arc not biologically
`active. Perhaps the best known example of the latter is insulin; insulin forms dimers
`and hcxamers at elevated concentration, especially in the presence of certain diva(cid:173)
`lent metal ions. but is only active as a monomer (Uvcrsky et al .. '2003).
`The ultimate fold of the protein is usually referred 10 as the "native" structure.
`In principle. the latter refers to the functional structure of the protein. However,
`many proteins change structure during their biological function. which would sug(cid:173)
`gest there are multiple "native" struc tures. l'urthermorc, artificially created proteins
`(e.g., fusion proteins created by genetic engineering techniques) may assemble into
`well-defined folds but have unknown levels of func tio n. It may therefore be easier to
`describe the protein structure under physiological conditions (in terms of pH, ionic
`strength, etc.) as the native Slructure. The mechanism of protein folding is discussed
`in the following section.
`
`6.3 PROTEIN FOLDING: WHY DO PROTEINS FOLD?
`
`6.3.1 ROLE OF WATER AND STABILIZING INTERACTIONS
`
`The observation that most proteins are folded into a specific structure 111 simple
`aqueous solutions suggests that fol ding is a thermodynamically favorable process.
`Many decades of research have been aimed at elucidating why. and how. proteins
`fold (Anfinseo, 1973). Altho ugh there arn still several limitations, it is now possible
`to predict with reasonable accuracy how a protein will fold using computational
`methods (Kryshtafovych and Fidel is, 2009). In this section, we will discuss the driv(cid:173)
`ing forces for folding, starting with a protein in the gas phase hcfore moving to the
`more complex situation of a protein in solUlion.
`For a single protein molecule in the gas phase, there are four fundamental forces
`to take into account. The first is the entropy of the amino acid chain. which tends
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`11 0
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`Pharmaceutical Formulation Development of Peptides and Proteins
`
`to disfavor folding. That is, folding of the amino acid chain into a specific structure
`reduces the degrees of freedom for that chain, which results in a loss of entropy. Jn
`contrast, hydrogen bonding and van der Waals forces favor folding. Electrostatic
`interactions may either favorordisfavor folding, depcndingon the sign ofthc charges.
`Experiments with peptides in the gas phase have shown that folding can be spontane(cid:173)
`ous (Chin et al., 2006); hence, at least in some circumstances the entropy loss upon
`foldi ng can be overcome by the enthalpy gain from electrostatic interactions, hydro(cid:173)
`gen bonding, and/or van der Waals forces. Often electrostatic interactions are an
`important driving force for the folding process in the gas phase (Chin el al., 2006).
`Because proteins arc typically found in an aqueous environment, these gas-phase
`experiments offer only limited insights to the understanding of protein folding under
`solution conditions. The high dielectric constant of water means that the strength of
`electrostatic interactions is significantly reduced, and therefore is a much less imper(cid:173)
`Lant driving force for folding. if at all. Moreover, the peptide chain now has the ability
`to form hydrogen bonds with water, as well as to interact with water molecules through
`van der Waals forces. Thus, intramolecular interactions like van der Waals forces and
`hydrogen bonds also are 1101 immediately apparent driving forces for folding.
`And yet, proteins do fold in water, Ao important d riving force of this foldi ng is
`the negative effect of solute- water interactions on the interaction between the water
`molecules themselves. Pure water may be viewed as a collection of oxygen atoms
`suspended in a sea of hydrogen atoms. On average, fou r hydrogen atoms surround
`one oxygen atom in a (imperfect) tetrahedral shape, with two of those hydrogen
`atoms close enough to describe the-bond as covalent a.nd two s lightly fu rther away,
`forming a hydrogen bond. This is, however, a highly dynamic system. and there
`will be a constant exchange between covalently bound and hydrogen bond-linked
`hydrogen atoms. In essence, any solute will negatively affect this dynamic system;
`this is known as the hydrophobic effect (Dill et al., 2005). Whether a solute dissolves
`in water, and how much, is a mailer of accounting: as long as there is a negative
`change in Gibbs free energy for the system as a whole upun dissolution of the solute.
`the compound will d issolve. Thus, the negative energetic contribution by distorting
`the dynamic water network needs to be counterba lanced by the positive contribution
`of the solute dissolving, which includes increased eruropy of the solute upon dis(cid:173)
`solution as well as hydrogen bonding and van der Waals interactions with the water
`molecules. Due 10 the ability to form hydrogen bonds and significant van der Waals
`interactions, polar (hydrophilic) compounds dissolve to a much larger extent in water
`than non polar (hydrophobic) compounds.
`Most proteins contain a significant amount of nonpolar amino acid residues and
`their dissolution in an aqueous environment would be energetically unfavorable.
`In contrast, the dissolution of the polar amino acids would be a favorable process.
`By folding of the amino acid chain such that the hydrophobic amino acids arc hid(cid:173)
`den from the aqueous s urroundings. a protein significantly reduces the hydrophobic
`effect by the nonpolar residues, while maintaining the positive interaction between
`the polar residues and the water molecules. The hydrophobic effect and the result(cid:173)
`ing "hiding" of nonpolar amino acids in the core of the protein is believed to be the
`main driving force for folding (Dill, 1990; Kauzmann, 1959). Further folding and
`specificity of the fold arc then governed by other interactions like hydrogen bonding,
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`Physical Instability of Peptides and Proteins
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`salt bridge formation, and van der Waals interactions between tightly packed resi(cid:173)
`dues (Rose and Wolfenden, 1993). Finally, the ultimalc fold may be stabilized by the
`formation of cystines.
`The imrortance of the hydrophobic amino acids for protein folding is also sug(cid:173)
`gested by the relatively conservative changes of the core amino acids for similar pro(cid:173)
`teins across species. That is, even though the overall amino acid sequence may be
`significantly differenl for a given protein isolated from various species, the differences
`in the (usually hydrophobic) amino acids forming the core of the protein are usually the
`smallest (Mirny and Shak.bnovich, 2001), and mutations io these amino ucids ure more
`likely to yield an inactive protein (Guo et al., 2004). As a result, even proteins with a
`mere 30- 40% similarity io amino acid sequence can yield very similar protein folds.
`Considering the above, it should be no surprise that natively unfolded proteins
`generally do not contain such a core of hydrophobic amino acids. In fact, it is likely
`the absence of a significant amount of hydrophobic amino acids, along with many
`charged residues, that allows these proteins to have liule tertiary fold (IJvcrsky and
`Dunker, 2010). However, they often do have a specific seconda ry structure, which
`suggests that for amino acid chains, the intrachain hydrogen bonding is more favor(cid:173)
`able than hydrogen bonding tu water.
`
`6.3.2 THE ENERGY LANDSCAPE Of A PROTEIN MotECULE
`
`As discussed above, proteins may spontaneously folu in aqueous solution. Thal means
`that the change in Gibbs free energy upon folding is negative, that is, t>G, < 0, and
`thus the change in Gibbs free energy of unfolding is positive (tlG. > 0). However,
`due to the complex interaction between protein and solvent, the Gibbs free energy is
`not a simple linear function of temperature (Privalov, 1990; Robertson and Murphy,
`1997). Let us first examine a simple two-state reversible folding process between a
`protein in its unfolded stale (U) und in a folded stale (N) (Scheme 6.l):
`
`U~N
`
`(Scheme 6. 1)
`
`The change in Gibbs free energy for this folding process can be approximated
`using a modi tied form of the Gibbs-Helmholtz equation (Equation 6.1), in which the
`temperature dependence of t>H and t>S are approximated by a constant difference
`in heat capacity between ·.he native and unfolded stated of the protein, t>C,. In this
`equation, Tm is a temperalure where tlG is zero; 6.H, is the enthalpy change upon
`fold ing at this temperature, and t>C,.r is the change in heat capacity upon folding.
`
`(6.1)
`
`Data on Tm, t>C,, and AH, can he Obtained. for example, using differential scan(cid:173)
`ning calorimetry (DSC)•. Plotting this data using Equation 6.1 will yield a parabola
`
`• No1e lhnl in a typical DSC cxp::r1ment the prorcin i5 folded ut the s1art of thecxpcrimcnt. Thus. the till
`and llCP obtained ar-c those for the unfolding proc~ss.
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`112
`
`Pharmaceut,cal formula1ion Developn1e11t or Peplides and Proteins
`
`40
`
`20 1 :;;-
`J
`<I -20
`
`0
`
`\
`
`- Mr>alobln
`
`- - - -~
`
`--IO -'----~---.-----.,;Lys~oz-'1_m_•-.
`240
`320
`280
`T(K)
`FIGURE 6.1 Graphical rcpn:sentalion of lhc 1hcrmodyn11mic stobilily of two model pro(cid:173)
`teins 3S u funclion of temperature a!li: denved from lhc mod1fitd G1bbs-Hclmholt1. equation
`(P.qus\lion 6.1). Figure created using numerical d111a from Anjum et al (2000), wilh r~, =
`340.4 K, All,= -343 kl/mol, and AC.,= -J 1.45 kJ/mol for myoglobln in a pH 6.1 buffer:
`7~"' 335.7 K, AH,= - 372 kl/mol, and AC.,,"' -6.52 kJ/mol for lysn,ymc in a pll 4.8 buffer.
`(D:uo Fron, Anjum et al .. Biochim. Biophys. Acea 1476, 2000.)
`
`(Figure 6.1). which is known as the protein stability curve. It hos been observed thar
`many proteins have their highes1 thermodynamic stability around 283 K, indepen(cid:173)
`dent of their melting temperorures (Recs and Robertson, 2001). Thi& is significantly
`below physiological temperatures for many organisms, probably because some struc(cid:173)
`tural llcxibility is required for :ictivity.
`Figure 6.1 shows there are two crossings where AG = 0, suggesting 1ha1 proteins
`can unfold due 10 both increased as well as decrea.ed 1emperntures. The laucr, cold
`denaturation (Privalov, 1990), usually occurs at temperatures below 270 K and 1hus
`is less likely 10 be observed in standard analytical techniques due 10 ,ce formation.
`Funhermore, the kinetics of unfolding slows down with decreased temperature,
`which may result in kine1ic trapping of 1hc protein in its foldled s1ruc1ure. Finally, i1
`is important to no1e that under physiological conditions, the magnitude of lJ.G,..,., is
`relatively smaJJ, typically ca. 10-50 kJ/mol. This 1s a n11her weak stabilizing interac(cid:173)
`tion, con<idering that a typical hydrogen bond conmbures about 5-30 kJ/mol
`The pathway from unfolded to folded state is, for mnny protein•, likely not as sim(cid:173)
`ple as suggested by Scheme 6.J. Unfolded proteins mn)' assume an enormous number
`of conformational states; indeed, a simple calculation shows that in a typical sample
`of unfolded protein molecules, each molecule is likely to be found in u different co11-
`for111a1ional state.• Yet, proteins can spontaneously fold lo their native conformation
`within a second. This suggests that each protein molecule must ntcessarily follow a
`slightly different pathway to the folded state. Thi, complex folding process can be
`conccptuolizcd in terms of a biased random walk, wherein proteins fold via a lnrge
`number of small conformational changes, with the likdihood of any conformational
`clumgc occurring being biased 1oward tho,e rhat lower the overall free energy of the
`protein (Bryngelson el al., 1995). The collection ot all possible cooforma11onal trajec(cid:173)
`tories and associa1ed free energies forms an "tnergy landscape" To belier visualize
`
`• T;ake, ror example, a prot~in of 100 amino acids, and aUow each a.mmo ricld only 1wo diffcren1 coofor(cid:173)
`maIK>fl:S Thr:salrcady yiclds1• = 10-• diffcrcnt potenual conform:ulon&. This exceeds the numbered
`mob:-11.ks of a specific proeein on Earth.
`
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`Physical Instability of Peptides and Proteins
`
`113
`
`the high-dimensional space represented by this enormous col leclion of conformational
`stales and energies, the energy landscape is often conceptualized as a "folding fun(cid:173)
`nel," wherein the vertical position on the funnel is represemativc of the Free energy
`of a given conformation, and the circumference of the funnel is representative of the
`number of stales having a given free energy (Bryngelson et al., 1995). Thus, the large
`number of ui1foldcd stales would be found al the lop of the funnel, ant! the singular
`native state conformation woultl be found at the funnel boltom (Figure 6.2a).
`
`Entropy
`
`(a)
`
`Amorphous
`"ggregatcs
`
`Fibril,
`(bl
`
`FIGURE 6.2 Energy landscape of n protein. (a) (See color insert .) An idcnlizcd folding fun
`nel for a single protein molecule. At the high Gibbs free energy end (top of picture), the protein
`molecule can adopt. many different confonnations: the widlh of the funnel can be viewed as a
`mca5ure of the conformational entropy. At the bottom of the funnel a singular folded state with
`very limllodconformational enlropy is present. (b) A t00rc realistic two•dimcnsional rcprcscnta•
`lion of the energy land~cnpe of a protein. On the left.hand side. the tolding of a single protein i,;;
`shown; as depicted, there may be several folds with almost the same low Gibbs f rec energy. Also,
`there may be a folding iolcrmcdimc(s) and mi,;;foldcd species with higher energy which cnn be
`significantly popul.-tc:c.J due to kim:lic ba1rit:fs. Jn the middle. and on tlH: right•hund side possible
`energy states are shown for ensembles of protein molecules, resulting in various aggregated
`species (oligomers, fibrils1 and amorphous aggregates). Thcso may haw lower Gibbs free energy
`than lhe native protein. hut may also be populated. due to a large kinetic barrier toward refolding.
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`114
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`Pharmaceutical Formulation Development of Peptides and Proteins
`
`The energy landscape of proteins can probably be better conceptualized as a
`jagged funnel (Figure 6.2b), where 1hc protein fold, or a subpopul ation of protein
`molecules, may be kinetically trapped in loca l minima, rather than in lhc thermo(cid:173)
`dynamically lowest energy state. Note that the aggregated states, in particular the
`fibrillar state, are show n in Figure 6.2b as being equa lly or even more thermody(cid:173)
`namically stable than the native state. That is, the protein fold observed for native
`proteins may well be considered a metastable state, with kinetic barriers preventing
`rapid population of the aggregated and/or fibr ill ar state (Baldwin et al., 20ll ). In
`the human body, various regulato ry processes have developed that are designed to
`degrade and eliminate improperly folded proteins, and thus prevent and/or reduce
`the rate of fibri l formation. In a pharmaceutical formulation, there are no processes
`that remove misfolded protein, mea!ling that aggregation and fibrillation can occur
`for proteins that are not known to aggregate or fibri llate in vivo, or can occur much
`faste r than observed in vivo. Aggregation and fibrillation is further discussed in
`Section 6.4.3.
`The jagged funnel depicted in Figure 6.2b should not be seen as static; changing
`the solution conditions will alter the relative magnitudes of the local minima relative
`io the global minimum, possibly resulting in a new global minimum. This may also
`occur upon binding of a protein ID a ligand or 10 its receptor, if this involves signi fi(cid:173)
`ca nt changes in protein structure. The energy barriers between the various Slates will
`likely also change, and may either increase or decrease. This will affect the ki netics
`of the physical degradation processes talcing place. As a result, even small changes in
`solution conditions can have a major impact on the main degradation route.
`
`6.4 PROTEIN PHYSICAL DEGRADATION
`
`The physical degradation of proteins refers to any loss in bioactive protein that docs
`not involve formation or breakage of chemical bonds and is sometimes also referred
`to as dena1ura1ion. ll can be subdivided in four, often interrelated processes: unfold(cid:173)
`ing, adsorption , aggregation, and precipitation.
`
`6.4.1
`
`PROTEIN UNFOLDING
`
`In the previous section, the spontaneous folding of a protein into a specific three(cid:173)
`dimensional structure was discussed. Here, we look at the reverse process: the
`spontaneous 1111[0/ding of a protei n, sometimes also referred to as denaturation. As
`discussed above, under physiologica l conditions, the most thermodynamica lly stable
`state for a single protein molecule (usua lly) is the folded state. Any deviation from
`physiological conditions, for example, a change in temperature, pH, or ionic strength,
`will change the intramolecu lar interactions with in the protein, as well as the inter(cid:173)
`actions between protein and water. Thus, one may expect a change in the protein
`fold ing stabi lity upon changi ng !lie environment of the protein. As long as ll1ose
`changes are fully reversible upon removing the stress factor or upon admi nistration
`ID the patient, this may appear irrelevant for a therapeutic protein in a fo rmulation.
`However, a~ wi ll be discussed in more detail in Section 6.4.3, (partial) unfoldiJ1g is
`commonly the first step in protein aggregation, which is often irreversible. Moreover,
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`(partial) unfolding followed by subsequent refolding may trap lhe protein in a non(cid:173)
`native and thus inactive conformation. Finally, unfolded proteins are often more sus(cid:173