Second Edition
`
`Pharmaceutical Formulation
`Development of Peptides
`and Proteins
`
`Edited by
`Lars Hovgoard
`Sven Frokjoer
`Marco van de Weert
`
`CRC Press
`Taylor & Francis Group
`Boca Raton London N ew York
`
`CRC Press Is an imp rint of the
`Taylor & Francis Group, an info rm a business
`
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`CRCPress
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`International Standa rd Book Number: 978-1 -4398-5388-7 (Hardback)
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`Library of Congress Cataloging-in-P ublicatio n Data
`
`Pharmaceutical formulation development of peptides and proteins / editors, Lars
`Hovgaard , Sven Frukjaer, Marco van de Weert. •- 2nd ed.
`p. ;cm.
`Includes bibliographical referencesand inde><.
`ISBN 978 -l -•1398-5388-7 (hardcover : alk, paper)
`I. Hovganrd, Lars, 1962- 11. Frllkjrer, Sven, 1947- UL Weert, Marco von de, 1973 -
`IDNLM : 1. Pq,tidc Blosynthesis. 2. Chemistr y, Pharmaceutical--melhods, 3. Protein
`Biosynthesis. QU 68}
`
`615.1'9 --dc23
`
`2012030490
`
`Visit the Taylor & Francis Web site •t
`http://www.taylorandfrancis.com
`
`and the CRC Press Web site at
`hllp;//www.crcpress.com
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`Contents
`
`Preface ..................................................................................................................... , vii
`Ed it ors ...................... , ............. ···-· ........................................................................... ,. ix
`Contributors ............................................ , .................................... , ....................... , ..... xi
`
`Chapter 1 Peptide Synthesis .................................................................................. I
`
`K1111d J. Jensen
`
`Chapter 2 Protein Expression ...... ........................................................................ 17
`
`Nanni Din
`
`Ch11ptcr 3 Protein Purification ........................................................................... 35
`
`Lars Hovgaard. Lars Skriver, a,1d Sven Frokjaer
`
`Chapter 4 Characterization of Therapeutic Peptides and Proteins .................... .49
`
`Marco van de Weer/ and Tudor Arvittte
`
`Chapter 5 Chemical Pathways of Peptide aod Protein Degradation ................... 79
`
`Teru110 J. Siahuan. and Chris/fan Schoneich
`
`Chapter 6 Physical lnscabilily of Peptides a nd Proteins ................... .............. .. 107
`
`Marco van de Wee rt a,rd Theodore W Randolph
`
`Chapter 7 Peptide and Protein Derivatives . ., ..... r ................................ , .. , .... ., •• 131
`
`Kristian Str¢mgaard and Thomas H¢eg-Jensen
`
`Chapter 8 Peptides and Proteins as Parenteral Solutions .................. _ .............. 149
`
`Michael J. Akers and Michael R. De Felippis
`
`Chupter 9 Peptides and Proteins as Parenteral Suspensions: An Overview
`of Desiga, DevelopmenL. and Manufacturing Considerations ......... 193
`
`Michael R. DeFelippis and Michael J. Akers
`
`II
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`vi
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`Contents
`
`Chapter 10 Rational Design of Solid Protein Formulations ........................... u .. 239
`
`Bingq11a11 (Stuart) Wang and Michael J. Pikal
`
`Chapter 11 Peptide and Protein Drug Delivery Systems for Nonparenreral
`Routes of Administration . ................................................................ 269
`
`Ulrik Lyn Rahbek. Fra11tisek H11balek. and Simon Bjerregcwrd
`
`Chapter 12 lmmunogenicity of Therapeutic Proteins .......... , .............................. 297
`
`Grzegorz K,janka, WimJiskoor, Melody Suuerbom.
`Huub Schellekens, and Vera Brinks
`
`Chapter 13 Biosimulation of Peptides and Proteins ............................................ 323
`
`Tt1e S¢eborg, Chrisrian Hove Rasmussen, Erik Mosekilde. and
`Morren Coldi11g-J¢rgense11
`
`Chapter 14 Registration of Peptides and Proteins .............................................. 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 Structure ........................................................................ 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 Degradarioo ~ ..................................................................... I 14
`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 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 function of peptides and proteins is highly dependent on their three(cid:173)
`dimensional structuJe. Changes in that strucLUrc, which may arise due to chemical
`or physical processes, may alter or abolish th:n 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 tl1e 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 extcr.
`nal stress factors. The three.dimensional structure of proteins is mostly held log_ethcr
`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 intra- or intermolecular interactions which may not be reversible upon removing
`the stress factor.
`
`107
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`108
`
`Pharrnaceulica.1 Formulation Development of Peptides and Proteins
`
`In this chapter, we will discuss the noncovaJent interactions that result in the for(cid:173)
`mation of the specific three-dimensional fold of most proteins, the most important
`stress factors thut may cause changes in that protein fold, and the resulting physical
`instability of the protein. lt should be noted that we will 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 interdependent, 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 instability of proteins in pharmaceutical formulations. Several of
`these strategies will be discussed with more specific examples in other chapters.
`Throughout the chapter, a number of semantic issues will be discussed, which are of
`importance when reading the literatun:. The commonly used terminology within the
`field of protein structure, folding, and stability is not always strictly defined. and defini(cid:173)
`tions may differ over time and depending on the context. Unfortunately, the definitions
`used in a particula r scientific paper are often not explicit, which may lead to confus.ion
`when the reader is insufliciently aware of the different descriptions that are in use.
`
`6.2 PROTEIN STRUCTURE
`
`6.2.1
`
`PEPTIDES, POLYPEPTIDES, ANO PROTEINS
`
`All peptides, polypeptides, and proteins are considered condensation polymers of
`amioo 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 descriptions,
`rather than definitions, of the difference between peptide and protein alone. The cur(cid:173)
`rently most co1nmon 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 indudc several simple dipeptides (i.e ..
`two amino acids linked together), which can have a biological function. Finally, the
`absence or presence of a well-defined tertiary structure has been used to distinguish
`peptides from proteins. Also, this distinction is not without problems; there arc pro•
`teins that are referred to as "natively unfolded," so called because they do not have
`a specific tertiary structure. ln addition, some " peptides" can form fully reversible
`multimeric structures, such as glucagon (forming trimers) (Formisano et al., 1977),
`whiclJ involves the formation of a defined 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 acids a protein.
`
`6.2.2 PROTEIN STRUCTURE: PRIMARY, SECONDARY,
`TERTIARY, ANO QUATERNARY STRUCTURE
`
`The 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 interchain cross-links are also prevalent, usually through cystcine residues
`(forming a cystine or S- S bridges).
`T he 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:
`helical structures such as the alpha-helix, pleated structures sut:h as the beta-strand
`and beta-sheet, turn structures such as the beta-turns. and loop structures. The lattec
`are often referred to as "random" structures.
`The three-dimensional alignment of the secondary structural elements is known
`as the tertiary structure of a protein. Tbis 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-helix bundles (Orengo and Thornton, 2005).
`Some proteins exist under physiological conditions as specific muhimeric pro(cid:173)
`teins linked through noncovalent interactions. This multimerization is known as the
`quaternary structure of a protein. Examples of proteins with a quaternary structure
`include hemoglnhin, alpha-crystallin, and HIV-I protease. In general, the hiologi(cid:173)
`ca) function of such mullimeric proteins depends on this roullimerization, but some
`proteins may also form specific (and reversible) nmhirners that arc not biologically
`active. Perhaps the best known example of the lallcr is insulin; insulin forms dimers
`and hexamers 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 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(cid:173)
`gest there are multiple "native" structures. Furthermore, artificially created proteins
`(e.g., fusion proteins created by genetic engineering techniques) may assemble 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 the native structure. T he mechanism of protein folding is discussed
`in the following section.
`
`6.3 PROTEIN FO LDING: WHY DO PROTEINS FO LD?
`
`6.3.1 RO LE OF W ATER AND STABILIZING I NTERACTIONS
`
`The observation that most proteins are folded into a specific structure in simple
`aqueous solutions suggests that foldjng is a therruodynarnically favorable process.
`Many decades of research have been aimed at elucidating why, and how, proteins
`fold (Anfinsen, 1973). Although there are 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 before moving to the
`more complex siwation of a protein in solution.
`For a single protein molecule in the gas phase, there are four fundamental forces
`to take into account. The fi rst is the entropy of the amino acid chain, which tends
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`Pharmaceutical Formulation Development of Peptides and Proteins
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`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. ln
`contrast, hydrogen bonding and van der Waals forces favor folding. Blectrostatic
`interactions may either favor or d isfavor folding, depending on the sign of the charges.
`Experiments with peptides in the gas phas.e have shown that folding can be spontane(cid:173)
`ous (Chin er al., 2006); hence, at least in some circumstances rhe entropy loss upon
`folding 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 et al., 2006).
`Because proteins are typically fouod in an aqueous environment, these gas-phase
`experiments offer only limited insights to the understanding of protein folding under
`solmi.on conditions. The high dielectric constant of water means that the strength of
`electrostatic interactions is signiiicantly reduced, and therefore is a much less impor(cid:173)
`tant driving force for folding, if at all. Moreover, the peptide chain now has the ability
`to form hydrogen bonds with waler, 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 not immediately apparent driving forces for folding.
`And yet, proteins do fold in water, Ao important driving force of 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 el(change 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 (Dil l 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 d issolve. Thus, the negative energetic contribution by distorting
`the dynamic water network needs to be coumerbalanced by the positive contribution
`of 1he solute dissolving, which includes incccased er\tropy of the solute upon dis(cid:173)
`solution as well as hydrogen bonding and van der Waals interactions with the warer
`molecules.Due to the ability to form hydrogen bonds and significant van dcr Waals
`interactions, polar (hydrophilic) compounds dissolve to a much larger extent in water
`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 unfavorable.
`In contrast, the d issolution of the polar amino acids would be a favorable process.
`By folding of the amino acid chain such that the hydrophobic amino acids are hid(cid:173)
`den 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 resuH(cid:173)
`ing " hiding" of nonpolar amino acids io 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 Protelns
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`111
`
`salt bridge formation, and van der Waals interactions between lightly packed resi(cid:173)
`dues (Rose and Wolfenden, 1993). Finally, the ultimate fold may be stabilized by the
`formation of cystines.
`The importance 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. Thal is, even though the overall amino acid sequence may be
`significantly different 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 Shakhnovich, 2001), and mutations iu these amino acids are more
`likely to yield an inactive protein (Guo et al., 2004). As a result, even proteins with a
`mere 30- 40% similarity in amino acid sequence cnn 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 hydrophohic amino acids. In foct, it is likely
`the absence of a significant amount of hydrophobic amino acids, along with many
`charged residues, that allows these proteins to have little tertiary fold (Uversky and
`Dunker, 2010). However, tbey often do have a specific secondary structure, which
`suggests that for amino acid chains, the intrachaio 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 fold in aqueous solution. That means
`that the change in Gibbs free energy upon folding is negative, that is, 6G, < 0, and
`thus the change in Gibbs free energy of unfolding is positive (6G0 > 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) and in a folded state (N) (Scheme 6.L):
`
`(Scheme 6.1)
`
`The change in Gibbs free energy for this folding process can be approximated
`using a modified form of the Gibbs-Helmholtz equation (Equation 6.1), in which the
`temperature dependence of l:i.H and l:i.S are approximated by a constant <lifference
`in heat capacity between :.he native and unfolded stated of the protein, l:i.CP. In this
`equation, T,. is a temperature where l:i.G is zero; l:i.Hf is the enthalpy change upon
`folding at this temperaLure, and t.Cp.r is the change in heal capacity upon folding.
`
`(6.1)
`
`Data on Tm, l:i.Cr, and ti.Hr can he obtained. for example, u.~ing differential scan(cid:173)
`ning calorimetry (DSC)*. Plotting this data using 8quatlon 6.1 will yield a parabola
`
`• Note lhnt in a typical DSC cxp,rimenl the protein is folded111 the start of thecxpcrimcnl. Thus. the ti.fl
`ond <!IC, obtnincd are those for the unfolding process.
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`112
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`Pharmaceutical Formulation Development of Peptides and Proteins
`
`40
`
`i 20
`~ 0
`J
`<I - 20
`
`240
`
`280
`T (I<)
`
`320
`
`360
`
`FIGURE 6.1 Graphical representation of the thcrmodyn~mic s1ability of two model pro(cid:173)
`teim as a function of temperature as derived (rom the modified Gibbs-Helmholtz equation
`(Equation 6.1). Figure created using numerical data from Anjum el aL (2000), with T,.:
`340.4 K, t:.Hr = -343 kJ/moJ, and t:.C.,,: -1 1.45 kJ/mol for myoglobin in a pH 6.1 buffer:
`7~ = 335.7 K, AH,"' -372 kJ/mol, and t.C9 _., = -6.52 kJ/mol for lysozyme in a pH 4.8 buffer.
`(Data from Anjum el al., Biochim. Biophy.,. Acla 1476, 2000.)
`
`(Figure 6.1), which is known as the protein stability curve. It has been observed lha1
`many proteins have their highest tbermodynamk stability around 283 K, indepen(cid:173)
`dent of their melliog temperatures (Rees and Robertson, 2001). This is significantly
`below physiological temperatures for many organisms, probably because some struc(cid:173)
`tural flexibility is required for activity.
`Figure 6 .1 shows there are two crossings where AG= 0, suggesting that p roteins
`can unfold due to both increased as well as decreased temperatures. The latter, cold
`denaturation (Privalov, 1990), usua.lly occurs at temperatures below 270 K and thus
`is less likely to be observed io standard aoalylical techniques due Lo ice formation.
`Furthermore, the kinetics of unfolding slows down with decreased temperature,
`which may result in kinetic trapping of the protein in its folded structure. Finally, it
`is important 10 note that under physiological conditions, the magnitude of AGr .• .,, is
`relatively small, typically ca. l0-50 kJ/mol. This is a rather weak stabilizing interac(cid:173)
`tion, considering that a typical hydrogen bond conlributes about 5-30 kJ/moL
`The pathway from unfolded to fold\:d stare is. for mnny proteins, likely not as sin,(cid:173)
`pte as suggested by Scheme 6.1. Unfolded proteins may assume an enormous number
`of conform ational states; indeed, a simple calculation shows that in a typical sample
`of unfolded protein molecules, each molecule is likely to be found in a different con(cid:173)
`formational state.* Yet, proteins can spontaneously fold to Lheir native conformalion
`within a second. This suggests that each protein molecule must necessarily follow a
`slightly different pathway to the folded stale. This complex folding process can be
`conceptualized in terms of a biased random walk, wherein proteins fold via a large
`number of small conformational changes, with the likelihood of any conformational
`change occurring being biased toward those that lower the overall free energy of the
`protein (Bryngelson et al., 1995 ). The collection of all possible conformational trajec(cid:173)
`tories and associated free energies forms an "e.nergy landscape." To better visualize
`
`• Take.for example, n protein of 100 amino acids. and allow each amino aoid only two differenl coofor•
`mations. This already yields 2''° = IO'° different potenliat conforma1ions. This exceeds 1hc nurnbe.r of
`molec.ulcs of a sped fie protein o n Ear1h.
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`Physical Instability of Peptides and Proteins
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`11 3
`
`the high-dimensional space represented by this enormous collection of conformational
`states and energies. the energy landscape is often conceptualized as a "Colding fun(cid:173)
`oel," wherein the vertica I position on the fonnel is representative of the free energy
`of a given conformation. and the circumference of the funnel is representative of the
`number of states having a given free energy (Bryngelson et al., l995). Thus, the large
`number of unfolded states would be found at the top of the funnel, and the ~ingular
`native state conformation would be found at the funnel bottom (Figure 6.2a).
`
`Entropy
`
`(a)
`
`Native state(~)
`
`Amorphous
`Rggregate~
`
`Fibril~
`(b)
`
`FIGURE 6.2 Energy landscape of a protein. (a) (See color insert.) An idealiicd folding fun
`ncl for a single protein molecule. At tbe high Gibbs free energy end (lop of' picture), the protein
`molecule can adopt many different confonnations: the width of the funnel can be viewed as a
`measure of the conformational entropy. At the bonom of the funnel a singular folded state with
`very limilod conformational entropy is present. (b) A more realistic two-dimensional representa(cid:173)
`tion of the energy landscape cf a protein. On the left-hand side. the folding of a single protein is
`shown; as depicted, there may be several fold~ with almost the same low Gibbs free energy. Also,
`there may be a folding intermcdiate(s) and misfoldcd species with higher energy which cnn be
`significantly populated due tokinetie barriers. In the middle and on Lhe right-hand side possible
`energy states are shown for ensembles of protein molecules, resulting in various aggregated
`species (oligomers, fibrils, and amorphous aggregates). These may have lower Gibbs free energy
`than the native protein, but may also be populated due to a large kinetic barrier toward refolding.
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`Pharmaceutical Formulation Development of Peptides and Proteins
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`The energy landscape of proteins can probably be bener conceptualized as a
`jagged funnel (Figure 6.2b), where the protein fold, or a subpopulation of protein
`molecules, may be kinetically trapped in local minima, rather than in the thermo(cid:173)
`dynamically lowest energy state. Note that the aggregated states , in particular the
`librillar state, are shown in Figure 6.2b as being equally 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 fibrillar state (Baldwin et al., 20ll). Jn
`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. ln a pharmaceutical formulation, there are no processes
`that remove misfolded protein, meaning that aggregation and fibrillation can occur
`for proteins 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
`SecLion 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
`to the global minimum, possibly resulting in a new global minimum. This may also
`occur upon binding of a protein to a ligand or to its receptor, if this involves signifi(cid:173)
`cant changes in protein structure. The energy barriers between the variou. states will
`likely also change, and may either increase or decrease. This win affect the kinelics
`of the physical degradation prm:esses 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 bioactive protein that docs
`not involve formation or breakage of chemical bonds and is sometimes also referred
`to as denaturation. It 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 pro1ein into a specific three(cid:173)
`dimensional structure was di cussed. Here, we look al the reverse process: the
`spontaneous 1111/olding 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 intramolecu lar in teractions within the protein, as well as the inter(cid:173)
`actions between protein and water. Thus, one may expect a change in the protein
`folding stability upon changing U1e 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, (partial) unfolding is
`commoo.ly the first step in. protein aggregation, which is often irreversible. Moreover,
`
`Bausch Health Ireland Exhibit 2009, Page 12 of 27
`Mylan v. Bausch Health Ireland - IPR2022-01102
`
`

`

`Physical Instability of Peptides and Proteins
`
`115
`
`(par tial) unfolding followed by subsequent refolding m ay trap the protein in a non(cid:173)
`native and thus inactive conformation. Finally, unfolded proteins are often more sus(cid:173)
`ceptible to chemical degradation. Protein unfoldi