`
`Second Edition
`
`Pharmaceutical Formulation
`
`
`Development of Peptides
`and Proteins
`
`by
`Edited
`Lars Hovgaard
`Sven Frokjaer
`
`Marco van de Weert
`
`CRC Pren ls an Imprint of the
`
`
`Taylor & Francis Group, an lnformo bu1iness
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`Cl�C Press
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`Library o(Congress Cataloging-ln•Publkation D11ta
`
`Pharmaceutical formulation dcvelopmenl o( peptides and proteins/ edllcrs, Lars
`Hovgaard, Sven Frukjaer. Marco van de Weert. -- 2nd ed.
`p.;cm.
`Includes bibliographical references and index.
`ISBN 978· 1-1398·5388•7 (hardcover: alk. paper)
`I. Hovgaard, Lars, 1962- ll. Frokfrer, Sven, 1947- 111. Weert, Marco van de, 1973-
`[DNI.M: 1. Peptide Blosynthtsis. 2. Chemistry, Pharmaceutical--methods. 3. Protein
`Biosynthesis. QU 681
`
`615.1'9--dc23
`
`2012030490
`
`Visit the Taylor & Francis Web site 11t
`http://www.taylorandfrancis.com
`
`and t.he CRC Press Web site at
`http://www.crcpreu.com
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`

`

`Contents
`
`Prcfnce, .......... ,., ................................ , .• , •...•.•.•.•.••.••• ,, •• ,-,,, .. ,, .. , ........... , .. ,. .........
`vii
`
`
`
`
`
`
`
`
`
`Ed it ors., .... ,, ... ,., ...... , ....................... �.-·�·-·······.,., ............ , .... ,, .... ,, ............. , ................ ,, ix
`
`
`
`
`
`
`
`Contributors ............. , .•...... , ............ , ............... , ........ , ....... , ............. , ........................... xi
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Chapter I Pcplide Synthesis .................................................................................. I
`
`Knud J. Jensen
`
`Chapter 2 Protein E.xpression ... .......................................................................... 17
`
`
`
`Na1111i Din
`
`Chapter 3 Protein Puriticmion ................................
`
`
`
`�---·-·······-··
`
`
`
`
`
`
`
`Lars Hnv,gaard, Lar:. S'kriver, and Sven Frokjaer
`
`Chapter 4 CharacLcriza1ion of Therapeutic Peptides and Proteins ... ................. 49
`
`Marco van de Weert and Tudor Arvi,ite
`
`Chapter 5 Chemical Pathways of Peptide and Protein Degr'Jdarion . ................. 79
`
`
`
`Ter1ma J. SiaJuu.m and Christian SchOneic/r
`
`
`
`Chapter 6 Physical Instability of Peptides and Prolcins ................................... 107
`
`
`
`
`
`Marco van de Weert a,id Theodore W Randolph
`
`Chapter 7 Peptide allld Protein Derivatives .................................................. , ....... 131
`
`
`
`
`
`Kristian Strf>mgaard and Thomas H¢eg-Jensen
`
`Chapter 8 Pep1ides and Proteins as Parenteral Solu1ions ................................. 149
`
`
`
`Michael J. Akers tind Michael R. DeFelippis
`
`
`
`Chupter 9 Peptides ilnd Proteins as Parenteral Suspensions: An Overview
`of Design, Development, and Manufacturing Considerations ......... 193
`
`
`
`
`
`Michael I?. DeFelippis and Michael J. Akers
`
`
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`vi
`
`Contents
`
`
`Chapter
`
`10 Rational Design of Solid Protein
`
`
`
`
`
`Formulations···-··················· ....... 239
`
`
`
`
`
`Bingq1um {Stuart) Wang mu/ MichaeJ J. Pikal
`
`Peptide and Protein Drug Delivery Systems for Nonparenteral
`
`
`Chapter ll
`
`
`
`
`
`
`Routes of Administration�-······················ ············-·········· ............... 269
`
`
`
`Ulrik Lyll Rahbek, Frcmti!ek Hubtifek. and Simon Bjerregcwrd
`
`
`Chapter
`
`12 lmmunogenicity of Therapeutic Proteins .......... , ............................. 297
`
`
`
`
`
`Grzegorz Kijanka, Wim Jiskoor, Melody Sauer/Jorn,
`
`
`
`
`Huub Schellekens, and Vera Brinks
`
`of Peptides and Proteins
`Biosimulation
`Chapter 13
`
`.. 323
`
`
`Ti,e S"eborg, Christian Hove Rasm1use.n
`
`
`Morten Colding-Jprgensen
`
`r Erik Mosekilde. and
`
`
`
`
`
`
`
`
`
`Chapter 14 Registration of Peptides and Proteins.
`
`
`
`. ...... 339
`
`Niamh Kinsella
`
`
`
`
`
`Index ...................................................................................................................... 363
`
`
`
`
`
`
`
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`

`

`Physical I nstability of
`6
`Peptides and Proteins
`
`
`
`Marco van de Weert and Theodore W Randolph
`
`CONTENTS
`
`lntroduclion ............................................................................................ ..... 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 Structure ........................................................................ 108
`6.3 Prorein 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 Pl'Olein Physical Degradation ..................................................................... 114
`6.4.1 Protein Unfolding .......... . ............................................................. 114
`6.4.2 Adsorption .......... _ .....................................
`.... ............................... 117
`6.4.3 Protein Aggregation .......................................................................... 118
`6.4.3.1 Aggregation Mechanisms and Kinetics ............................. 119
`6.4.3.2 Fibrillation: A Special Case of Pro<cin Aggregation ......... 120
`6.4.4 Protein Precipitation ......................................................................... 121
`6.5 Stabilization Strategies ................................................................................. 122
`6.6 Concluding Remarks .................................................................................... 125
`Reference.<...........
`. ............................................................................... .... 126
`
`6.1 INTRODUCTION
`
`The biological function of peptides and prolcins is highly dependent on their three­
`dimensional structure. Chrmgcs in that slructurc. whic.:h may arise due to chemical
`or physical processes, may alter or abolish that runction, or even result in Loxicity.
`Thus. it is of importance that a pharm:1ceu1ical formultttion of therapeutic peptides
`and proteins retains the no1·ma.l (native) structure or those peptides or proteins, or
`1hr11 any changes are fully reversible upon administration to the patient.
`A major difference between proteins and low molecular weight drugs is the com­
`plexity of the three-dimensional structure and concomirnnt sensitivily toward exter­
`nal .;tress factors. The three-dimensional structure of proteins is mostly held together
`by noncovolent interactions. such as hydrogen bonds, salt bridges, and van dcr Waals
`fore�. Any stress factor may alter these noncovalent 1ntemcl1ons, possibly leading to
`new inlrn- or in1ermolecular interactions which may not be reversible upon removing
`the stress factor.
`
`107
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`
`
`108 Pharmaceutical Formulation Development of Peptides and Proteins
`
`
`
`
`
`In this chapter. we will discuss the noncovalent intcraclions that result in the for•
`
`
`
`
`
`
`
`
`
`mation of the specific thrcc•dimensional fold of most proteins, the most important
`
`
`
`
`
`
`stress factors that may cause changes in that protein fold. and the resulling physical
`
`
`
`
`
`
`instability of the protein. It should be noied that we will discuss this physical instabil­
`
`
`
`
`ity as a separate issue to the chemical instability discussed in Chapter
`
`5. In reality, the
`
`
`
`
`
`two are highly imerdepcndern, as chemical destabilization may lead to physical desia.
`
`
`
`
`bilization, and vice versa. This chapter will end with potential stabilization strategics
`
`
`
`
`
`
`to prevent physical instability of proteins in pharmaceutical formulations. Several of
`
`
`
`
`lhese strategics will be discussed with more specific ex1c1mples in other chapters,
`
`
`
`
`
`
`Throughout the chapter, a number of semantic issues wil.l be discussed, which arc of
`
`
`
`
`
`
`importance when reading the literature. The commonly used terminology within the
`
`
`
`
`
`
`field of protein structure, folding, and stability is not always strictly defined. and defini­
`
`
`
`
`
`
`tions may differ over time and depending on the context. Unfortunately, the definitions
`
`
`
`used in a particular scientific paper are often not explicit, which may lead lo confusion
`
`
`
`When the reader is insufficienlly aware of 1he different descrip1ions that are in Use.
`
`6.2 PROTEIN STRUCTURE
`
`
`
`ANO PROTEINS 6.2.1 PEPTIDES, POLYPEPTIDES,
`
`
`
`All peptides, polypeptides, and proteins are considered condensation polymers of
`
`
`
`
`
`
`
`
`
`
`
`
`amino acids, rcsuhing in a linear backbone uf altcrnaling urnidc, C-C, and C-N
`
`
`
`
`
`
`bonds. However, the distinction hctween peptide, polypeptide, and protein is rather
`
`
`
`
`diffuse. One may find at least three different and partly overlapping descriptions,
`
`
`
`
`
`
`
`rather than definitions, of the difference between peplide and protein alone. The cur­
`
`rently mos! common description refers to any peptide chain of more than 50 amino
`
`
`
`
`
`
`
`acids 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 linkc<l together), which can have a biological function. Finally, the
`
`
`
`
`absence or presence of a wel l -defined tertiary structure has been used to distinguish
`
`
`
`
`
`peptides from proteins. Also, this distinction is not· wi1hout problems; there arc pro·
`
`
`
`
`
`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 stru{'turcs, such as glucagon (forming trimcrs) (Formisano ct al., 1977),
`
`
`
`
`
`whicb involves the formation of a dcli.ned three•dimensional structure. The term
`
`
`
`
`
`
`"polypeptide" generally overlaps with that of '"peptide" and '"protein." The reader
`
`
`
`may thus encounter all three terms used in connection with the same biological
`
`
`
`
`
`
`compound. For practical purposes, we have used the first definition, calling every
`
`
`compound with more than 50 amino acic.ls a protein.
`
`6.2.2 PROTEIN STRUCTURE: PRIMARY, SECONDARY,
`
`
`
`
`TERTIARY, ANO QUATERNARY STRUCTURE
`
`The thrcc•dimensional structure of proteins ls often subdivided into tour types of
`
`
`
`
`
`
`structure, referred to as the primary, secondary, tertiary. and quaternary s1ructurc.
`
`
`
`
`
`
`The primary structure refers to the amino acid sequence within the polymer chain.
`
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`Physical lnslability of Peptides and Proteins
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`109
`
`Intra- or intcrchain cross-links are also prevalent, usually lhrough cyslcinc residues
`(forming a cysLine or S-S bridges).
`The secondary structure refers 10 the folding of this backbone into specific struc­
`tures, which arc defined by the bond angles and hydrogen bonding pattern of the
`amide bond. The secondary struc.ture can roughly be subdivided into four classes:
`helical structures 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" structures.
`The three-dimensional alignment of the secondary structural clemenls 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 tcniary structures can be described by commonly appearing architectures
`such as barrels or alpha-helix bundles (Orengo and Thornton. 2005).
`Some proteins exisL under physiological conditions as specific muhimeric pro­
`teins linked through noncovalent interactions. This multimerization is known as lhc
`quaternary structure of a pro1eiu. Examples of proteins with a quaternary structure
`include hemoglobin, aJpha-crystallin, and HIV-I protease. In general, the biologi­
`cal func1ion or such multimeric proteins depends on this multimerization, but some
`proteins may also form specific (and reversible) multimers that arc not biologically
`active. Perhaps the best known example of the laucr is insulin; insulin forms dimers
`and hcxamers al elevated concemration, especially in the presence of certain diva­
`lent metal ions, but is only active as a monomer (Uvcrsky et al., 1003).
`The ullirnate fold of the protein is usually referred to 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
`gesl there are multiple "native" slructures. furthermore, artifi.cinlly created proteins
`(e.g., fusion proteins created by genetic engineering techniques) may a�scmble into
`well-defined folds but have unknown levels of function. It may therefore be easier tO
`describe the protein structure under physiological conditions (in terms of pH, ionic
`strength, etc,) as 1hc na1ive structure. The mechanism of protein folding is discussed
`in 1hc following section.
`
`6.3 PROTEIN FOLDING: WHY DO PROTEINS FOLD?
`
`
`
`6,3,1 RotE OF WATER AND STABILIZING INTERACTIONS
`
`The observation that most proteins are folded into a specific structure in simple
`aqueous solu1ions suggests that foldfog is a thermodynamically favorable process.
`Many decades of research have been aimed at elucidating why. and how. proteins
`fold (Anfinscn, 1973). Although there arc still several limitations, it is now possible
`lo 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
`ing forces for folding, starting wilh a pro1ein in the gas phase hcfore moving to the
`more complex situation of a pro1ein in solmion.
`For a single protein molecule in the gas phase, there arc four fundamental forces
`to take into account. The fir!\l is the entropy of the amino acid chain. which tends
`
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`110
`
`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. 1n
`contrast. hydrogen bonding and van der Waals forces favor folding. Electrostatic
`interactions may either favor or disfavor folding, depending on the sign of the charges.
`Experiments with peptides in the gas phase have shown that folding can be spontane­
`ous (Chin et al., 2006): hence, at least in some circumstances the entropy loss upon
`folding can be overcome by the enthalpy gain from electrostatic interactions, hydro­
`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. 1hcse gas•phasc
`experiments offer only limited insights to the understanding of protein folding under
`solUlion conditions. The high dielectric com;tanl of water means that the strength of
`electrostatic interactions is signiJlCantly reduced, and therefore is a much less impor•
`tant driving force for folding. if at all. Moreover, the peplidechain now has the ability
`to form hydrogen bonds with water. as well as lO interact with water molecules through
`van der Waals forces. Thus, intramolecular interactions like van der Waals forces and
`hydrogen bonds also are not immediately apparent driving forces for folding.
`And yet, proteins do fold in water, An important driving force or this folding 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, four 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 and two slightly further 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. (n essence, any solulc 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 matlcr of accounting: as long as there is a negative
`change in Gibbs free energy for the system as a whole upon dissolution of the solute ..
`the compound will dissolve. Thus. the negative energetic contribution by distorting
`the dynamic water net work needs to be counterbalanced by the positive contribution
`of the soluce dissolving, which includes increased e1ltropy of the solute upon dis­
`solution as well as hydrogen bonding and van dcr Waals interactions with the water
`molecules. Due to the ability 10 form hydrogen bonds and significant van dcr Waals
`inte1w.:1iuns, polar (hydrophilic) (.:Ornpuumls dissolvt: tu a much huger cxtem in wutt:I'
`than nonpolar (hydrophobic) compounds.
`Most proteins contain a significant amount of nonpolar amino acid residues and
`their dissolution in an aqueous environment would be energetically unfavordble.
`I n contrast, the dissQlution 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
`<len from the aqueous surroundings, 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·
`ing "hiding" of nonpolar amino acids in the core of the prolein 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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`Phys,cal lnstabohty of Peptides and Proteins
`
`111
`
`salt bridge formation. and van der Waals intcrnctions between l1ght..ly packed resi­
`due.< (Rose ond Wolfenden. 1993). Finally, the ultimote fold mny be stabilized by the
`formation o( cys1incs.
`The imponance of the hydrophobic om,no oci<b for protein folding is also sug­
`gested by 1he relallvcly conserviJtive. changes of the core amino oc1ds for similar pro­
`That is, even 1hough the overall
`teins ocross species.
`
`ammo acid sequence mny be
`significantly different for a given protein i'°l:ncd from various �pccics, the differences
`in the (usually hydrophobic) amino acids forming the core of the protein are usually the
`smallest (Mirny and Shakbnovich, 2001), and mutations io thc,c amino acids ure more
`likely 10 yield an inactive protein (Guo et al., 2004). As a result, even proteins with a
`mere 30- 40% similarity i11 amino acid sequence Cfln yield very similar protein folds.
`Considering the above, it should be no surprise that natively unfolded proteins
`generally do not contain such n core of hydmphohic amino ncidR. ln fact, it is likely
`the absence of n significant amount of hydrophobic amino ucids, ulong with many
`charged residues, that allows these proteins 10 have little tertiary fold (Uversky and
`Dunker, 2010). However, they often do hove o specific secondary structure. which
`suggests that for amino acid chains, the intrachain hydrogen bonding is more favor•
`able than hydrogen bonding to water.
`
`
`
`6.3.2 TH, ENncv LANDSCAPE o, A PaornN Moucuu
`
`As discussed above, proteins may spontaneously fold ia aqU<."OUS >0Ju1iot1. Thal means
`that the change m Gibbs free energy upon folding is ncgath·e. that is. 6G, < 0, and
`thw. the change in Gibbs free energy of unfolding ,s pos111vc (6G, > 0). However,
`due lo the complex mteraction 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 eJ<.amine a simple two-stale reversible folding procc.s between a
`proteia in ,ts unfolded state (U) and in a folded slate (N) (Scheme 6.1).
`
`U ,= N
`
`(Scheme 6.1)
`
`The change in Gibbs free energy for this folding process con be opproxima1ed
`using a modified form of the Gibbs-Helmholtz equution (Equatiou 6.1), in which the
`tempe1::nure dependence of Alf nnd AS are op11roximated by a constant <liffcrcnce
`in heat capacity between the native and u11folded stated of 1hc 1>roh.�i11, 6.C". In this
`
`equation. T. is a temperalure where AG is tern; OH, i.s the cmhulpy cha11gc upon
`
`folding at this temperature. am.I 6CP., is the change in ht:at capaci1y upon foldfog.
`
`(6.1)
`
`Data on T •• ac •. and 6H, can be obtained. for example, using differential scan­
`ning calorimetry (DSC)•. P1011ing this data using Equation 6.t wtll yield a parabola
`
`NOleltu.t inalyi,teal DSCcxpcr1menl 1hcprocc1n II folckd al lhCstar1 ol lhecxpcnmcnL rhus. the.Ml
`
`
`
`
`and 6C• obtained are those for the ■nfoldi
`qg PfUCCN
`
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`112
`
`Pharmaceutical Formulation Dev�loprnent of Peptides and Proteins
`
`40
`
`20
`
`�
`
`a
`
`<t -20 -Y - Lysozyme
`
`--40 ��--�--�--�
`240 280
`T(K)
`
`360
`320
`
`stability of two model pro­
`FIGURE 6.1 Graphical representation of the thcrmodyn1mic
`
`
`as derived from the modified Gibbs-Helmholtz equation
`
`
`teins as a function of temperature
`using numerical da1a from Anjum et al. (2000),
`with r.1 ==
`
`(Equation 6.1). Figure created
`340.4 K, 6Hr = -343 kJ/mol, and 6C,., == -11.45 kJ/mol for myoglobin in a pH 6.1 buffer;
`1;., = 335.7 K, 6-Hr= -372 kJ/mol, and 6C11 • .,;::; - 6.52 kJ/mol for lysozymc in a pl·l 4.8 buffer.
`Biophy.,. Acta 1476, 2000.)
`(Data from Anjum et al.. Biochim.
`
`(Figure 6.1). which is known as the protein stability curve. It has been observed that
`many proteins have their highest thermodynamic stability around 283 K, indepen­
`dent of their melriog temperatures (Rees and Robertson, 2001). This is significantly
`below physiological temperatures for many orgilnisms, probably because some struc­
`LUral flcxjbility is required for activity.
`Figure 6.1 shows there are two crossings where 6.G = 0, suggesting that proteins
`can unfold due 10 both increased as well as decreased temperatures. The latter, cold
`denaturation (Privalov, 1990), usually occurs at temperatures below 270 K and thus
`is less likely to be observed in standard analytical techniques due to ice formation.
`Furthermore, the kinetics of unfolding slows down with decreased 1emperature,
`which may result in kinetic trapping of the protein in its folded structure. Finally, it
`is important to note that under physiological conditions, the magnitude of 6.Gr.,n;i� is
`relatively small, typically ca. IU-50 kJ/mol. This is a rather weak stabilizing inLerac­
`tion, con.idering that a typical hydrogen bond conrribures about 5-30 k.J/niol
`The pathway from unfolded to folded stare is, for m11ny proteins, likely not as sim•
`pie as suggested by Scheme 6.J. Unfolded proteins may assume an enormous number
`of conformational states; indeed, a simple calculation ,hows thar in a typical sample
`of unfolded protein molecules, each molecule is likely to be found in a different co11-
`fon11ational state.• Yet. proteins can spontaneously fold to their native conformation
`within a second. This suggests that each protein molecule musl necessarily follow a
`slightly different palhway 10 the folded state. This complex folding process can be
`conctplUalizcd in terms of a biased random walk. wherein proteins fold via a large
`number of small conformational changes, with 1hc likelihood of any conformational
`change occurring befog biased toward those that lower the overall free energy of the
`protein (Bryngelson et al., 1995). The collection of all possible conformational trajec­
`tories and associated free energies forms an °energy landscape." To belier visualize
`
`coofM­
`and allow each s.mino ncid only two different
`
`
`
`• Tilke, for example, a protein of 100 amino acids,
`
`matio"s. This already yields 1100 = IQ.'-> different p01:en1ial conformat
`
`This exceeds ,he nurnbtr of
`ions.
`
`molcct1Jcs of 3 specific pro1ein on Earth
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`Physical Instability of Peptides and Protein5
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`113
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`the high-dimensional space represented by this enormous collection of conformational
`states and energies, the energy landscape is often conceptualized as a "folding fun­
`ael," wherein the vertical position on the Funnel is represenr:uivc of the free energy
`of a gjven conformatfon, and the circumference of the funnel is reprcscn1a1ive of the
`number uf states having a given free energy (Bryngelson et al., 1995). Thus, the large
`number of unfolded states would be found al the top of lhe funnel, and the �ingular
`native state conformation would be found at the funnel bottom (Figure 6.2a).
`
`(a)
`
`Native ,tate(s)
`
`Amorphous
`aggregates
`Ftbrlls
`(bl
`
`FIGURE 6.2 Energy landscape of o. pro1ein. (a) (See color insert.) An idenlizcd folding
`
`
`
`
`fun­
`
`
`
`
`nel for a single protein molecule. At the high Gibbs free energy end (top of picture), lhc protein
`
`
`can adopt rnany diffcrenl conformations; the width oflhc funnel can be viewed as a
`molecule
`
`
`
`
`
`measure of the conformational entropy. Al the bo!tom of the funnel a singular folded state wi1h
`
`
`
`
`
`very 1im1tcdconformationo.l entropy is present. (b) A morcrealisL1c two-dimensional representa­
`
`
`
`
`
`
`tion of the energy landJ;cape or ::i protein. On the left-hand side, the tokling of a single protein i1;
`
`
`
`
`shown; as depleted, there may be several folds with almost the same low Gibbs free energy. Also,
`
`
`
`
`
`there may be II folding intcrmcdiotc(s) and mi,;foldcd species with higher energy which can he
`In the middle and on the right-hand
`
`
`signific:intly populeted due to kinl!licbarriers.
`side possible
`
`
`
`
`
`
`energy states are shown for cmcmbles or protein molecules, resulting in various aggregated
`
`
`
`
`
`
`species (oligomers, fibrils, and amorphow aggregates). These may ha� lower Gibbs free energy
`hul may also be populated
`
`
`due to a large kinetic barrier toward refolding.
`
`than the native prolein,
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`114
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`Pharmaceutical Formulation Development of Peptides and Protei11s
`
`The energy landscape of proteins can probably be better conceptualized as a
`jagged Funnel (Figure 6.2b), where the protein [old, or a subpopulation of protein
`molecules, may be kinetically trapped in local minima. rather than in the lhcrmo­
`dynam.ically lowest energy state. Nore that the aggregated states, in particular the
`fibrillar state, are shown in Figure 6.2b as being equally or even more thermody­
`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 fibrillar state (Baldwin el al., 20l l ). In
`the human body, various regulatory processes have developed that are designed to
`degrade and eliminate improperly folded proteins, and thus prevent and/or reduce
`the rate of fibril formation. In a pharmaceutical formulation, there are no processes
`thal remove rnisfolded protein, meaning that aggregation and fibri.llation can occur
`for protejns that are not known to aggregate or fibrillate in vivo, or can occur much
`faster 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 all'er the re1ative magnitudes of 1hc local minima relative
`to the global minimum, possibly resulting in a new global minimum. This may also
`occur upon binding of a protein to a l igand or to its receptor, if this involves signifi­
`cant changes in protein structure. The energy barriers between the various states will
`likely also change, and may either increase or decrease. This will affect the kinetics
`of the physical degradation processes taking 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 bioactivc protein that docs
`not involve formation or breakage of cJ1emical bonds and is sometimes also referred
`to as denaturation. It can be subdivided in four, often interrelated processes: unfold­
`ing, adsorption, aggregaiion, and precipitation.
`
`6.4.1 PROTEIN U NFOLDING
`In the previous section. the spontaneous folding of a protein into a specific three•
`dimensional sLructurc was discussed. Here, we look al the reverse prm:ess: the
`spontaneous unfolding
`of a protein. sometimes also referred to as denaturation. As
`discussed above, under physiological conditions, the most thermodynamically stable
`state for a single protein molecule (usually) is the folded state. Any deviation from
`physiological conditions. for example, a change in temperature, pH, or ionic strength,
`will change the intramolecular interactions within the protein, as well as the inter•
`actions between protein and water. Thus, oac may expect a change in the protein
`folding stability upon changing 1J1e environment of the protein. As long as those
`changes are fully reversible upon removing the stress factor or upon administration
`to the patient, this may appear irrelevant for a therapeutic protein in a formulation.
`However, as will be discussed in more detail in Section 6.4.3, (parliul) unfolding is
`commonly the first step in protein aggregation, which is often irreversible. Moreover,
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`Physical Instability of Peptides and Proteins
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`115
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`(partial) unfolding followed by subsequenl refolding may trap the protein in a non•
`
`
`
`
`
`
`
`
`
`
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`n::i.live and thus inactive conformation. Finally, unfolded proteins are often more sus­
`
`
`
`
`
`
`ceptible to chemical degradation. Protein unfoliling is thus, in general, delr