International Journal of Pharmaceutics 185 (1999) 129–188
`
`www.elsevier.com:locate:promis
`
`Review
`Instability, stabilization, and formulation of liquid protein
`pharmaceuticals
`
`Wei Wang *
`
`Biotechnology, Bayer Corporation, 800 Dwight Way, Berkeley, CA 94701, USA
`
`Received 21 January 1999; received in revised form 26 April 1999; accepted 28 April 1999
`
`Abstract
`
`One of the most challenging tasks in the development of protein pharmaceuticals is to deal with physical and
`chemical instabilities of proteins. Protein instability is one of the major reasons why protein pharmaceuticals are
`administered traditionally through injection rather than taken orally like most small chemical drugs. Protein
`
`Abbre6iations: ADA, adenosine deaminase; ADH, alcohol dehydrogenase; AcP, acylphosphatase; BDNF, brain-derived neuro-
`trophic factor; BGG, bovine g-globulin; BSA, bovine serum albumin; BSF, bovine serum fetuin; CD, circular dichroism
`spectroscopy; CE, capillary electrophoresis; CMC-Na, carboxymethyl cellulose sodium; rhCNTF, recombinant human ciliary
`neurotrophic factor; rConIFN, recombinant consensus a-interferon; DGK, diacylglycerol kinase; DMSO, dimethylsulfoxide;
`rhDNase, recombinant human deoxyribonuclease; DSC, differential scanning calorimetry; hEGF, human epidermal growth factor;
`aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; rFVIII, recombinant factor VIII; pdFIX, plasma-
`derived factor IX; rFIX, recombinant factor IX; rFXIII, recombinant factor XIII; FTIR, Fourier transform infrared spectroscopy;
`GA, glucoamylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G-CSF, granulocyte colony-stimulating factor; GDH,
`glutamate dehydrogenase; met-hGH, methionyl human growth hormone; pGH, porcine growth hormone; GRF, growth hormone
`releasing factor; GdnHCl, guanidine hydrochloride; HP-b-CD, hydroxypropyl-b-cyclodextrin; IFN-b, interferon-b; IFN-g, inter-
`feron-g; hIGF-I, recombinant human insulin-like growth factor I; IL-1b,
`interleukin-1b; IL-1R,
`interleulin-1 receptor; IL-2,
`interleukin-2; rhIL-1ra, recombinant human interleukin-1 receptor antagonist; IR, infrared spectroscopy; rhKGF, recombinant
`human keratinocyte growth factor; LDH,
`lactate dehydrogenase; phm-MDH, pig heart mitochondrial malate dehydrogenase;
`LMW-UK, low molecular weight urokinase; rhM-CSF, recombinant human macrophage colony-stimulating factor; rhMGDF,
`recombinant human megakaryocyte growth and development factor; MS, mass spectroscopy; rhNGF, recombinant human nerve
`growth factor; NMR, nuclear magnetic resonance spectroscopy; tPA, tissue plasminogen activator; PAGE, polyacrylamide gel
`electrophoresis; hPAH, human phenylalanine hydroxylase; PE40, 40 kD segment of Pseudomonas exotoxin (PE); PEG, polyethylene
`glycol; ml-PEPC, maize leaf phosphoenolpyruvate carboxylase; PFK, phosphofructokinase; huPrP(90-231), recombinant protein
`corresponding to the human prion protein domain (residues 90-231); PVA, polyvinylalcohol; PVP, polyvinylpyrrolidone; rhPTH,
`recombinant human parathyroid hormone; RP-HPLC, reversed phase HPLC; RNase A, ribonuclease A; SDS, sodium dodecyl
`sulfate; SEC-HPLC, size exclusion HPLC; TGF, transforming growth factor; TMAO, trimethylamine N-oxide; TP40, Cys-replaced
`(with Ala in PE40) mutant of TGF-a-PE40; rhTPO, recombinant human thrombopoietin; YEI, yeast external invertase.
`* Tel.: (cid:27)1-510-705-4755; fax: (cid:27)1-510-705-5629.
`E-mail address: wei.wang.b@bayer.com (W. Wang)
`
`0378-5173:99:$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
`PII: S 0 3 7 8 - 5 1 7 3 ( 9 9 ) 0 0 1 5 2 - 0
`
`Ex. 1017 - Page 1 of 60
`
`AMGEN INC.
`Exhibit 1017
`
`

`

`130
`
`W. Wang :International Journal of Pharmaceutics 185 (1999) 129–188
`
`pharmaceuticals usually have to be stored under cold conditions or freeze-dried to achieve an acceptable shelf life. To
`understand and maximize the stability of protein pharmaceuticals or any other usable proteins such as catalytic
`enzymes, many studies have been conducted, especially in the past two decades. These studies have covered many
`instabilities of
`areas such as protein folding and unfolding:denaturation, mechanisms of chemical and physical
`proteins, and various means of stabilizing proteins in aqueous or solid state and under various processing conditions
`such as freeze-thawing and drying. This article reviews these investigations and achievements in recent years and
`discusses the basic behavior of proteins, their instabilities, and stabilization in aqueous state in relation to the
`development of liquid protein pharmaceuticals. © 1999 Elsevier Science B.V. All rights reserved.
`
`Keywords: Denaturation; Unfolding; Melting; Aggregation; Degradation; Preferential interaction
`
`1. Introduction
`
`The advent of recombinant DNA technology has
`led to a worldwide zeal to develop protein pharma-
`ceuticals in the past two decades. These protein
`pharmaceuticals or pharmaceutical candidates in-
`clude functional regulators and supplements, en-
`zyme
`activators
`and inhibitors, poly-
`and
`monoclonal antibodies, and various vaccines. In
`comparison with small chemical drugs, protein
`pharmaceuticals have high specificity and activity
`at relatively low concentrations. These features
`have made protein pharmaceuticals indispensable
`in combating human diseases.
`Due to advances in analytical separation technol-
`ogy, recombinant proteins can now be purified to
`an unprecedented level (Bond et al., 1998). Highly
`purified protein pharmaceuticals significantly re-
`duce the known and unknown potential side or
`even toxic effects. However, one of the most
`challenging tasks remains in the development of
`protein pharmaceuticals: dealing with physical and
`chemical instabilities of proteins. Protein instability
`is one of the two major reasons why protein
`pharmaceuticals are administered traditionally
`through injection rather than taken orally like most
`small chemical drugs (Wang, 1996). Protein phar-
`maceuticals usually have to be stored under cold
`conditions or even freeze-dried to a solid form to
`achieve an acceptable shelf life.
`In search for ways of stabilizing proteins, scien-
`tists turned their attention to nature for an answer.
`It is well known that certain natural organisms can
`grow well at extreme temperatures. Hyperther-
`mophilic organisms
`(hyperthermophiles)
`such
`as anaerobic, methanogenic, or sulfate-reducing
`
`archaebacteria grow at temperatures near or above
`100°C (Huber et al., 1989; Adams, 1993, 1994).
`Proteins in these organisms function normally at
`high temperatures. For example, enolase and a-glu-
`cosidase in hyperthermophilic Pyrococcus furiousus
`have optimum activity, at \90 and \105°C,
`respectively (Costantino et al., 1990; Peak et al.,
`1994). The most thermostable proteins found so far
`have half-lives in excess of 10 min at 130°C (Daniel
`et al., 1996). The mechanisms responsible for the
`high molecular stability of thermophilic proteins
`include increased hydrophobic interactions, greater
`molecular packing, more H-bonds, more salt-
`bridging, loss of surface loops, more helix-forming
`amino acids, restricted N-terminus mobility, etc.
`(Vieille and Zeikus, 1996; Cowan, 1997; Vogt and
`Argos, 1997). Extrinsic factors (not primary struc-
`ture-
`related) have also contributed to protein stabiliza-
`tion. One of these is the high cellular content of
`sugars, salts, or other organic solutes:osmolytes,
`such as a-glutamate, di-myo-inositol-phosphate
`isomer, b-mannosylglycerate, and di-
`and its
`glycerol-phosphate (Huber et al., 1989; Rupley and
`Careri, 1991; Martins and Santos, 1995; Martins et
`al., 1997; Ramakrishnan et al., 1997).
`The identification of intrinsic and extrinsic fac-
`tors that contribute to the stabilization of ther-
`mophilic proteins has provided valuable infor-
`mation for stabilizing protein pharmaceuticals and
`for designing more stable mutant proteins. Yet the
`structural differences among different proteins are
`so significant that generalization of universal stabi-
`lization strategies has not been successful. Very
`often, proteins have to be evaluated individually
`and stabilized on a trial-and-error basis.
`
`Ex. 1017 - Page 2 of 60
`
`

`

`W. Wang :International Journal of Pharmaceutics 185 (1999) 129–188
`
`131
`
`To understand and maximize the stability of
`protein pharmaceuticals or any other usable
`proteins such as various catalytic enzymes, many
`studies have been conducted in the past
`few
`decades. These studies have been reviewed with
`emphasis on general protein stability (Jaenicke,
`1991; Kristja´nsson and Kinsella, 1991), mecha-
`nisms of chemical and physical
`instabilities of
`proteins (Manning et al., 1989), mechanisms and
`prevention of major protein degradation path-
`ways (Cleland et al., 1993), and various means of
`stabilizing proteins in aqueous or solid state and
`under various processing conditions
`such as
`freeze-thawing or drying (Gianfreda and Scarfi,
`1991; Arakawa et al., 1993; Timasheff, 1993;
`Manning et al., 1995; Wong and Parascrampuria,
`1997). This article reviews these investigations and
`achievements in recent years and discusses the
`basic behavior of proteins, their instabilities, and
`stabilization in aqueous state in relation to the
`development of liquid protein pharmaceuticals.
`
`2. Basic protein behavior and properties
`
`Protein pharmaceuticals, unlike small drug
`molecules, have high molecular weight (\5 kD).
`Their large size, compositional variety, and am-
`phipathic characteristics constitute specific behav-
`ior such as folding, conformational stability, and
`unfolding:denaturation. Understanding proteins’
`basic
`behavior may
`help
`toward
`their
`stabilization.
`
`2.1. Protein folding and its related forces and
`stability
`
`Biologically active proteins are properly folded.
`The number of possible conformations of a folded
`polypeptide chain with an average domain size is
`about 1080 (Jaenicke, 1991). The three-dimen-
`sional folded state of a protein is a fluctuating
`state of a limited number of preferred conforma-
`tions (Tang and Dill, 1998). The most stable (least
`energy) conformation of a protein is usually the
`native state (Darnell et al., 1986). Under native
`conditions the vast majority of protein molecules
`exist in their unique native state (N). A tiny
`
`fraction must also occupy all possible higher en-
`ergy states as dictated by the Boltzmann relation-
`ship (Bai and Englander, 1996).
`
`2.1.1. Protein Folding Process
`Folding of newly-synthesized polypeptides in
`cells requires the assistance of so-called molecular
`chaperone proteins (Hendrick and Hartl, 1995).
`These proteins bind unfolded or partially folded
`polypeptides in their central cavity and promote
`folding by ATP-dependent cycles of release and
`rebinding. The molecular chaperone GroEL facili-
`tates protein folding by preventing protein aggre-
`gation and correcting protein misfolding (Golbik
`et al., 1998). Protein folding is generally a highly
`cooperative process, in which only the native and
`unfolded states are stable (Goto and Fink, 1989).
`When a protein folds, about 80% of nonpolar
`side chains (Ala, Val, Ile, Leu, Met, Phe, Trp,
`Cys) are buried in the
`interior of protein
`molecules out of contact with water. For example,
`folding of RNase T1, a compact globular protein
`(104 aa), removes about 85% of nonpolar residues
`from contact with water (Thomson et al., 1989).
`More than 80% of amino acids in globular
`proteins exist in a-helix:b-sheet or in the turns
`connecting them (Pace et al., 1996).
`The rate of protein folding is usually high.
`Many small proteins can fold in milliseconds or
`less (Dobson and Hore, 1998). Some may fold at
`a slower rate. For example, the folding of a
`bacteria protein MerP (72 aa) by diluting the
`protein solution containing 3 M guanidine hy-
`drochloride (GdnHCl)
`shows
`two exponential
`phases. The initial phase is fast with a rate con-
`stant of 1.2:s, but the second phase is slow with a
`rate constant of 0.053:s, accounting for about
`20% of the folding signal (Aronsson et al., 1997).
`Folding of proteins involving Pro isomerization is
`also relatively slow.
`
`2.1.2. Major forces in6ol6ed in protein folding
`Many forces are involved in protein folding.
`These include hydrophobic interactions, electro-
`static interactions (charge repulsion and ion pair-
`ing), hydrogen bonding,
`intrinsic propensities,
`and van der Waals forces. Hydrophobic interac-
`tions are repulsive interactions between water and
`
`Ex. 1017 - Page 3 of 60
`
`

`

`132
`
`W. Wang :International Journal of Pharmaceutics 185 (1999) 129–188
`
`non-polar residues in proteins, leading to minimal
`hydration of the hydrophobic core. These interac-
`tions are strongly disfavored and associated with
`a large increase in heat capacity (Dill, 1990). A
`hydrogen bond is the strong dipole–dipole attrac-
`tion between covalently-bonded hydrogen atoms
`and other strongly electronegative atoms such as
`oxygen and nitrogen. It is primarily a linear ar-
`rangement of donor, hydrogen, and acceptor. Hy-
`drogen bonds between amide hydrogen and
`carbonyl oxygen make up 68% of the total hydro-
`gen bonds in globular proteins (Pace et al., 1996).
`Among all the forces involved in protein fold-
`ing, the apparent dominant force is hydrophobic
`interaction (Jaenicke, 1990; Kristja´nsson and Kin-
`sella, 1991). The dominant opposing force is the
`loss of non-local conformational entropy (Dill et
`al., 1989; Dill, 1990). The difference in heat capac-
`ity of a protein in the native folded and denatured
`states (DCp) is a measure of hydrophobic stabi-
`lization of the protein, and the large DCp on
`thermal denaturation of proteins supports the ma-
`jor role of hydrophobic interactions in protein
`stabilization (Wang et al., 1996a). Recently, it was
`found that the contribution of electrostatic inter-
`actions in proteins at neutral conditions to the
`free energy difference between the folded and
`unfolded states is close to 0, indicating that the
`main driving forces for protein folding under
`these conditions are hydrophobic and hydrogen-
`bonding interactions
`(Dimitrov and Crichton,
`1997).
`albeit dominant,
`interaction,
`Hydrophobic
`needs to be balanced to maintain protein activity.
`In studying the stability of mutant Rop proteins,
`Munson et al. (1996) demonstrated that under-
`packing the hydrophobic core with small amino
`acids like Ala not only loses protein activity but
`also decreases protein stability. On the other
`hand, overpacking the hydrophobic core with
`only large amino acids like Leu stabilizes proteins
`but protein activity is lost. This indicates that
`both favorable steric interaction and burial of
`sufficient hydrophobic volume and surface area
`are important to stabilize a protein.
`Protein molecular packing may be the most
`applicable factor that leads to the unique struc-
`tures of most globular proteins (Richards, 1997).
`
`The packing in native proteins is so well arranged
`that all solvent molecules are essentially excluded
`and the protein interior is more like a crystalline
`solid than a non-polar liquid (Jaenicke, 1991).
`Glu, Lys, and Arg, with three or four rotatable
`bonds, are almost
`invariably located on the
`protein surface and use their flexibility to help
`ensure exposure of their charged groups to the
`solvent. However, buried polar residues do not
`necessarily destabilize a protein if they can form
`stable hydrogen bonds (Pace et al., 1996) and:or
`form stable intramolecular salt bridges, enhancing
`the structural rigidity (Vieille and Zeikus, 1996).
`Interaction forces in proteins can also be di-
`vided into two types: local (short-ranged or sec-
`ondary) and non-local (long-ranged or tertiary)
`(Chan and Dill, 1991). Long-range interactions
`happen between two residues that are separated
`by at least ten residues (Doszta´nyi et al., 1997).
`Long-range interactions such as parallel and an-
`tiparallel sheets are mainly responsible for poly-
`mer collapse to compact states whereas local
`forces are mainly responsible for helix formation
`such as hydrogen bonding. It seems that long-
`range interactions (non-local forces) control over-
`all protein stability. The dominant
`role of
`long-range interactions in protein stability is sup-
`ported by recent identification of so-called ele-
`ments of stabilization center (SC) in proteins. By
`long-range interactions, these SCs, mainly consist-
`ing of hydrophobic residues that are less flexible,
`tend to connect more sheets to each other, and are
`primarily responsible for stabilization of protein
`structures (Doszta´nyi et al., 1997). If different
`proteins have similar structural folds, their con-
`formational stability may still be very different
`due to the difference in their primary sequences
`(Chiti et al., 1998).
`
`2.1.3. Protein folding and free energy change
`The folded state of proteins has conformational
`stability, which is defined as the free energy
`change, DGf(cid:147) u (or simply DG), for the unfolding:
`denaturation reaction under physiological condi-
`tions (Pace, 1990). The larger the DGf(cid:147) u, the
`more stable the protein. The folded state of pro-
`teins is only marginally more stable than the un-
`folded state since its DGf(cid:147) u is small. The DGf(cid:147) u
`
`Ex. 1017 - Page 4 of 60
`
`

`

`W. Wang :International Journal of Pharmaceutics 185 (1999) 129–188
`
`133
`
`for proteins has been reported in the following
`ranges: 21–63 kJ:mol (Kristja´nsson and Kinsella,
`(Volkin and Klibanov,
`1991); 5–20 kcal:mol
`1989); or 5–10 kcal:mol (Jaenicke, 1990; Pace et
`al., 1996).
`The low DGf(cid:147) u values indicate that conforma-
`tional stability of a protein in aqueous solution is
`equivalent to a few H-bonds or ion pairs. A single
`hydrogen bond can lower the protein’s free energy
`by 0.5–2 kcal:mol and an ion pair by 0.4–1.0
`kcal:mol (Vogt and Argos, 1997). After analyzing
`more than a dozen mutant proteins, Pace et al.
`(1996) found that average free energy gain is
`1.1–1.6 kcal:mol per hydrogen bond and 1.18
`kcal:mol per -CH2- for hydrophobic effect. These
`values suggest significant contribution of hydro-
`gen bonding to DGf(cid:147) u relative to that from hy-
`drophobic effect, however,
`if the polar groups
`must be buried to form intramolecular hydrogen
`bonds, the net gain is only about 0.6 kcal:mol per
`hydrogen bond. In comparison, the strength of a
`hydrogen bond in water is about 5 kcal:mol, and
`the energy of van der Waals interaction amounts
`to about 1 kcal:mol at 25°C (Darnell et al., 1986).
`
`2.2. Protein unfolding:denaturation
`
`In solution, the folded state of any protein is
`not infinitely stable (Shortle, 1996). It may un-
`fold:denature into an inactive form—a process of
`protein conformational changes. These conforma-
`tional changes are due to predominant
`inter-
`molecular
`protein–solvent
`interactions
`over
`intramolecular
`interactions, which keeps
`the
`folded state (Jaenicke, 1990). On the other hand,
`solvent-induced inactivation of proteins may oc-
`cur without disruption of protein conformation
`(tertiary structure) (Cowan, 1997).
`
`2.2.1. Protein unfolding process
`Protein unfolding can be described generally by
`a single transition step between the completely
`folded and unfolded states since any intermediate
`state is highly unstable and only exists in negligi-
`ble amounts (Chan and Dill, 1991; Jaenicke,
`1991). This is true at least for small globular
`proteins (Kristja´nsson and Kinsella, 1991). Others
`may have more than one unfolding process such
`
`as human placental alkaline phosphatase (Hung
`and Chang, 1998).
`The following equation describes the two-state
`model:
`N (native)v U (unfolded:denatured)
`[ A (aggregated)
`
`or
`Pfoldedv Punfolded[ A.
`For most proteins, the unfolded state (U) is insol-
`uble and favors aggregation. Under certain condi-
`tions, a particular U state exists, which is highly
`compact and has significant amounts of residual
`secondary structures such as thermally-unfolded
`RNase T1 (Pancoska et al., 1996) and acid-un-
`folded apomyoglobin (Staniforth et al., 1998).
`This particular U state is termed molten globule
`(Goto and Fink, 1989; Shortle, 1996).
`The N state may unfold reversibly or, depend-
`ing on the condition, irreversibly to the U state.
`For example, unfolding of wild-type barnase and
`some of its mutants are reversible based on the
`repeatability of DSC endotherms or CD transi-
`tions on rescanning (Johnson et al., 1997). Ther-
`mal treatment of IFN-b-1a (Runkel et al., 1998)
`and b-galactosidase (Yoshioka et al., 1994a)
`causes irreversible denaturation of both proteins
`and eventually leads to formation of aggregates
`(A).
`Proteins unfold locally and globally. Local and
`global unfolding occur concurrently and indepen-
`dently. Increasing denaturant concentration or
`temperature can selectively promote global un-
`folding because global unfolding exposes more
`surface and gives rise to higher chain entropy and
`enthalpy (at high temperatures) than local unfold-
`ing (Bai et al., 1994).
`An important
`thermodynamic parameter in
`two-state transition is the change in heat capacity,
`DCp. The large and positive DCp observed in
`protein denaturation is due primarily to exposure
`of nonpolar groups (Pace et al., 1996; Johnson et
`al., 1997). The molar enthalpy of protein denatu-
`ration, DH, at low temperatures may be either
`positive or negative but increases markedly with
`temperature. Since the driving force for protein
`unfolding is the increased conformational entropy
`
`Ex. 1017 - Page 5 of 60
`
`

`

`134
`
`W. Wang :International Journal of Pharmaceutics 185 (1999) 129–188
`
`in aqueous solution, DSf(cid:147) u is expected to be
`positive.
`
`2.2.2. Protein unfolding:melting temperature
`Proteins unfold above certain temperatures.
`During a thermal unfolding process, the tempera-
`ture at which 50% of protein molecules are un-
`folded (DG(cid:30)0 at this time) is defined as the
`unfolding
`(or melting:denaturation:transition)
`temperature (Tm). Thermal unfolding of a protein
`is usually endothermic. The Tm of many proteins
`have been determined (Table 1) and appear
`mostly in the range of 40–80°C. When the level of
`protein hydration decreases, Tm may increase
`sharply due to destabilization of the unfolded
`state (Rupley and Careri, 1991).
`The higher the Tm, the greater the thermal
`resistance of a protein. For example, glyceralde-
`hyde-3-phosphate dehydrogenase (GAPDH) from
`hyperthermophilic bacterium Thermotoga mar-
`itima has a melting temperature of 109°C
`(Jaenicke, 1996). However, there is no particular
`relationship between Tm and protein stability as
`measured by DGf(cid:147) u (Dill et al., 1989). For exam-
`ple,
`the Tm of serum albumin from different
`sources
`has
`the
`following
`order:
`human
`(59.7°C)\dog (59.5°C)\rabbit
`(57.8°C)\rat
`(57.6°C)\bovine (56.8°C), yet their temperatures
`of maximum stability (DG) are all similar at about
`20°C (Kosa et al., 1998). The DGs of three SH3
`domains of the Tec family of tyrosine kinases are
`below 12–16 kJ:mol, but their melting tempera-
`tures are relatively high between 69 and 80°C
`(Knapp et al., 1998).
`A protein may have two or more melting tem-
`peratures, depending on the experimental condi-
`tions and analytical techniques used (Table 1).
`For example, Kosa et al. (1998) demonstrated
`that dog and rabbit albumin have one transition,
`but human, bovine, and rat albumin have two
`transitions, suggesting the presence of a stable
`intermediate. The apical domain (residues 191–
`376) of GroEL protein shows two reversible melt-
`ing temperatures at 35 and 67°C by far UV CD,
`which are attributed to unfolding of the C-termi-
`nal helices and the domain core, respectively.
`Protein huPrP(90–231) has one melting tempera-
`ture in the presence of GdnHCl at pH 5.0 and 7.2,
`
`but shows two transition temperatures at pH 3.6
`and 4.0 with a stable unfolding intermediate
`(Swietnicki et al., 1997). By DSC, interleukin-1
`receptor (IL-1R, 50 kD) shows two unfolding
`transitions near 48°C and 65°C, but after decon-
`volution, three melting transition peaks are iden-
`tified, representing three different domains in the
`protein (Remmele et al., 1998).
`Multimeric, chimeric, or modular proteins often
`have more than one melting temperature. For
`example, native recombinant human placental
`factor XIII (rFXIII) is a non-covalent dimer and
`each subunit consists of 3 thermolabile domains
`(56 kD, N-terminal) and two thermostable do-
`mains (24 kD, C-terminal). The intact protein
`melts in two distinct temperature regions by DSC:
`69°C at pH 8.6, representing three thermolabile
`domains; and 90°C in 2 M GdnHCl, representing
`two thermostable domains
`(Kurochkin et al.
`1995). The chimeric protein toxin sCD4(178)-
`PE40 (sCD4-PE40), consisting of HIV binding
`domains of the T-cell membrane protein (CD4)
`and the cytotoxic domains of Pseudomonas exo-
`toxin A (PE-40), has two transition temperatures
`originating from both components. In addition,
`unfolding of the less stable PE-40 induces unfold-
`ing of the more stable CD4 component because
`the free form of CD4 denatures at a higher tem-
`perature (56°C) than that in the protein complex
`(46°C) at pH 6.5 (Davio et al., 1995).
`
`2.3. Mesophilic 6ersus thermophilic proteins
`
`In describing basic properties of proteins, it is
`necessary to mention thermophilic proteins, a dif-
`ferent class of proteins having much higher ther-
`mostability than that of mesophilic counterparts
`like those in humans. Mesophilic proteins usually
`retain their native structures over a narrow range
`of temperatures from about 5 to 50°C. For ther-
`mophilic proteins, the upper limit of thermostabil-
`ity is usually 20 to 30°C higher, corresponding to
`an increase in protein stability by 5–7 kcal:mol. A
`DG of this order may be achieved by 1 or 2
`additional salt bridges inside the protein globule,
`several additional hydrogen bonds, or 7–10 addi-
`tional CH3- groups in the hydrophobic nucleus of
`the protein (Mozhaev and Martinek, 1984).
`
`Ex. 1017 - Page 6 of 60
`
`

`

`W. Wang :International Journal of Pharmaceutics 185 (1999) 129–188
`
`135
`
`Table 1
`Melting temperatures (Tm) of proteins
`
`Proteins
`
`Protein solution compositions
`
`Methods
`
`Tm (°C)
`
`References
`
`AcP (98 aa)
`HSA (66 kD)
`
`0.4 mg:ml in 50 mM acetate, pH 5.5
`20 mM in 100 mM phosphate, pH 7.4
`
`88 mM in 100 mM phosphate, pH 7.4
`0.1 mM in 67 mM phosphate, pH 7.4
`
`BSF (48 kD)
`
`2.5 mg:ml in 50 mM phosphate, pH 6
`
`2.5 mg:ml in 50 mM phosphate, pH 7
`
`2.5 mg:ml in 50 mM phosphate, pH 8
`
`em(cid:30)
`
`CD (222 nm)
`Fluorescence (l
`345 nm)
`DSC
`DSC
`
`DSC
`
`DSC
`
`DSC
`
`a-Chymotrypsin
`
`0.3 mg:ml in 10 mM phosphate, pH 6.0
`
`UV (281 nm)
`
`0.3 mg:ml in 10 mM phosphate, pH 7.0
`
`UV (281 nm)
`
`0.3 mg:ml in 10 mM phosphate, pH 8.0
`
`UV (281 nm)
`
`Cytochrome c
`
`2 mg:ml in 600 mM NaCl
`
`rhDNase
`
`Elastase
`
`aFGF
`
`bFGF
`
`10 mg:ml in water, pH 6.8
`
`20 mg:ml in 10 mM acetate, pH 5.0
`
`100 mg:ml in PBS, pH 7.2
`
`CD (228 nm)
`
`1 mg:ml in phosphate-citrate-borate, pH 4
`
`1 mg:ml in phosphate-citrate-borate, pH 9
`
`GA (82 kD)
`
`1.8 mg:ml in 50 mM phosphate, pH 6
`
`1.8 mg:ml in 50 mM phosphate, pH 7
`
`1.8 mg:ml in 50 mM phosphate, pH 8
`
`0.18 mg:ml in 10 mM citrate, pH 6.0
`1 mg:ml in 10 mM citrate, pH 6.0
`3 mg:ml in 10 mM citrate, pH 6.0
`3.3 mg:ml in 5 mM phosphate, pH 6.0
`
`rhGH (22 kD)
`
`IgG (mouse)
`
`Recombinant IFN-b-1a
`
`Recombinant IFN-b-1a,
`deglycosylated
`Lysozyme
`
`100 mg:ml in 100 mM Na2HPO4, 200 mM
`NaCl, pH 7.2
`100 mg:ml in 100 mM Na2HPO4, 200 mM
`NaCl, pH 7.2
`In 0.1 M NaCl, 0.1 M acetate, pH 5.4
`
`In 50 mM citrate, pH 4.0
`
`In 50 mM citrate and 1 M sucrose, pH 4.0
`
`DSC
`
`57
`62
`
`63
`60
`
`60
`
`54
`
`48
`
`47
`
`44
`
`42
`
`77
`
`67
`
`66
`
`45
`
`50
`
`64
`
`64
`
`62
`
`58
`
`89
`81
`79
`74
`
`67
`
`63
`
`66
`
`74
`
`80
`
`Chiti et al., 1998
`Farruggia et al.,
`1997
`Pico´, 1995
`Kosa et al.,
`1998
`Wang et al.,
`1996a
`Wang et al.,
`1996a
`Wang et al.,
`1996a
`Lozano et al.,
`1997
`Lozano et al.,
`1997
`Lozano et al.,
`1997
`Lo and Rah-
`man, 1998
`Chan et al.,
`1996
`Chang et al.,
`1993
`Volkin et al.,
`1993
`Wang et al.,
`1996b
`Wang et al.,
`1996b
`Wang et al.,
`1996a
`Wang et al.,
`1996a
`Wang et al.,
`1996a
`Bam et al., 1998
`Bam et al., 1998
`Bam et al., 1998
`Vermeer et al.,
`1998
`Runkel et al.
`1998
`Runkel et al.
`1998
`Shoichet et al.,
`1995
`Liu and Sturte-
`vant, 1996
`Liu and Sturte-
`vant, 1996
`
`DSC
`
`DSC
`
`DSC
`
`DSC
`
`DSC
`
`DSC
`
`DSC
`
`DSC
`
`CD (222 nm)
`CD (222 nm)
`CD (222 nm)
`DSC
`
`UV (280 nm)
`
`UV (280 nm)
`
`CD
`
`DSC
`
`Ex. 1017 - Page 7 of 60
`
`

`

`136
`
`W. Wang :International Journal of Pharmaceutics 185 (1999) 129–188
`
`Table 1 (continued)
`
`Proteins
`
`Protein solution compositions
`
`Methods
`
`Tm (°C)
`
`References
`
`M-CSF
`wt-hPAH (452 aa)
`
`RNase A
`
`1.2 mg:ml in 20 mM PolyB buffer
`14-18 mg:ml in 20 mM HEPES buffer,
`pH 7.4
`In 0.04 M glycine, pH 2.8
`
`DSC
`IR, peak ratio of 1619
`to 1650 cm(cid:28)1
`UV (287 nm)
`
`In 0.03 M MES, pH 5.8
`
`UV (287 nm)
`
`In 0.03 potassium phosphate, pH 6.7
`
`UV (287 nm)
`
`2 mg:ml in 600 mM NaCl
`
`DSC
`
`0.1 mg:ml in water
`0.2 mg:ml in 20 mM citric acid, pH 2.3
`0.2 mg:ml in 20 mM citric acid, pH 3.1
`0.2 mg:ml in 20 mM citric acid, pH 3.1
`About 2 mg:ml in 50 mM glycine, pH 2.8
`
`CD (222 nm)
`CE
`CE
`CD (222 nm)
`DSC
`
`About 2 mg:ml in 50 mM citrate, pH 6.0
`
`About 2 mg:ml in 50 mM citrate and 1 M
`sucrose, pH 6.0
`About 2 mg:ml in 50 mM citrate and 1 M
`glycine, pH 6.0
`25 mg:ml in 5 mM HEPES and 0.8 M
`GdnHCl, pH 8
`
`25 mg:ml in 5 mM HEPES, 0.8 M GdnHCl,
`and 1 mM MnCl2, pH 8
`
`DSC
`
`DSC
`
`DSC
`
`CD (222 nm)
`
`CD (222 nm)
`
`RNsae H
`
`87
`57
`
`41
`
`60
`
`64
`
`62
`
`65
`36
`49
`50
`48
`
`65
`
`70
`
`68
`
`30
`
`41
`
`wt-RNase T1
`
`About 1 mg:ml in 30 mM MOPS, pH 7
`
`Optical rotation (295 nm)
`
`48
`
`About 0.2 mg:ml in 30 mM MOPS, pH 7
`
`UV (286 nm)
`
`About 0.2 mg:ml in 30 mM MOPS, pH 7
`
`CD (238 nm)
`
`About 0.2 mg:ml in 30 mM MOPS, pH 7
`
`CD (284 nm)
`
`1 mg:ml in 25 mM phosphate and 400 mM
`NaCl, pH 6.5
`1 mg:ml in 25 mM phosphate and 400 mM
`NaCl, pH 7.9
`1 mg:ml in 25 mM phosphate and 400 mM
`NaCl, pH 9.9
`0.1mg:ml, pH 7.2
`0.1mg:ml, pH 7.2
`1 mg:ml, pH 7.2
`1.5 mg:ml in HCl solution, pH 2.5
`
`DSC
`
`DSC
`
`DSC
`
`Fluorescence
`CD
`DSC
`DSC
`
`2 mg:ml in 50 mM phosphate, pH 6.0
`2 mg:ml in 50 mM phosphate, pH 7.0
`2 mg:ml in 50 mM phosphate, pH 8.0
`
`DSC
`DSC
`DSC
`
`48
`
`47
`
`48
`
`57
`
`54
`
`48
`
`42
`55
`48
`52
`
`63
`52
`48
`
`Thrombin
`
`TP40 (40kD)
`
`Trypsin
`
`YEI (240 kD)
`
`Schrier et al., 1993
`Chehin et al. 1998
`
`Lin and Timasheff,
`1996
`Lin and Timasheff,
`1996
`Lin and Timasheff,
`1996
`Lo and Rahman,
`1998
`Tsai et al., 1998a
`McIntosh et al., 1998
`McIntosh et al., 1998
`McIntosh et al., 1998
`Liu and Sturtevant,
`1996
`Liu and Sturtevant,
`1996
`Liu and Sturtevant,
`1996
`Liu and Sturtevant,
`1996
`Goedken and
`Marqusee,
`1998
`Goedken and
`Marqusee,
`1998
`Shirley et al.,
`1989
`Thomson et al.
`1989
`Thomson et al.,
`1989
`Thomson et al.,
`1989
`Boctor and Mehta,
`1992
`Boctor and Mehta,
`1992
`Boctor and Mehta,
`1992
`Sanyal et al., 1996
`Sanyal et al., 1996
`Sanyal et al., 1996
`Boctor and Mehta,
`1992
`Wang et al., 1996a
`Wang et al., 1996a
`Wang et al., 1996a
`
`Ex. 1017 - Page 8 of 60
`
`

`

`W. Wang :International Journal of Pharmaceutics 185 (1999) 129–188
`
`137
`
`There are seemingly two basic mechanisms re-
`sponsible for enhanced thermal stability of ther-
`mophilic proteins. First,
`thermophilic proteins
`may possess structural characteristics different
`from those of mesophilic proteins. These struc-
`tural characteristics include (1) increased hydro-
`phobic
`interactions;
`(2)
`formation of
`extra
`hydrogen bonds and:or salt bridges; (3) more
`compact protein structures (may have crystal-like
`density); and (4) presence of few Cys, high con-
`tent of Arg, and low content of Lys (Mozhaev
`and Martinek, 1984; Vieille and Zeikus, 1996;
`Vogt and Argos, 1997). The second mechanism of
`stabilization is favorable interactions of proteins
`with other cellular components or accumulated
`low-molecular-weight
`compounds:osmolytes as
`thermoprotectants. The interaction reduces the
`contact area of nonpolar fragments in proteins
`with water, leading to a decrease in free energy of
`the system and stabilization of proteins. These
`osmolytes include potassium salt, sugars, a-gluta-
`mate, cyclic 2, 3-diphosphoglycerate (DPG), 2-O-
`b-mannosylglycerate, di-glycerol-phosphate, and
`di-myo-inositol-1,1%(3,3%)-phosphate
`(Mozhaev
`and Martinek, 1984; Huber et al., 1989; Rupley
`and Careri, 1991; Martins and Santos, 1995; Mar-
`tins et al., 1997).
`Is there a major force that contributes the most
`to stability of thermophilic proteins? Many ther-
`mophilic proteins show good correlation between
`stability and high hydrophobicity as measured by
`the hydrophobic index, a ratio of the volumes of
`polar to nonpolar amino acids (Mozhaev and
`Martinek, 1984). Detailed analysis indicates that
`protein stability depends not on total content of
`hydrophobic amino acid residues but on those
`inside the protein. Recent findings suggest that
`hydrogen bonding may play a major role in stabi-
`lizing thermophilic proteins. By comparing a
`group of thermostable proteins, Vogt and Argos
`(1997) recently concluded that increasing hydro-
`gen bonding density at the protein surface is a
`major factor for increased thermal stability. After
`analyzing 16 protein families containing a total of
`56 proteins from thermophilic, mesophilic, and
`thermophobic sources, Vogt et al. (1997) found
`that hydrogen bonding can provide the most gen-
`eral explanation for therma