Mechanisms of Protein Degradation

Biotherapeutics Pharm Sci: PhRDMechanisms of Protein DegradationKevin King14 November 2010

Biotherapeutics Pharm Sci: PhRDContent• Overview of protein structure• Chemical degradation• Physical degradation2

Biotherapeutics Pharm Sci: PhRDContent• Overview of protein structure• Chemical degradation• Physical degradation3

Biotherapeutics Pharm Sci: PhRDBiotherapeutics…..the Nature of the Beast

Biotherapeutics Pharm Sci: PhRDQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSComplexQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSBiotherapeuticQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESS1000+ amino acidsQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSQMVESSCVASGQMVESSCVASGQMVESSCVASGQMVESSCVASGSSSSSS

Biotherapeutics Pharm Sci: PhRDProtein StructurePrimary Secondary Tertiary Quaternary-helixmonomerdimertrimertetramer…amino acidsequenceb-sheets=> Correct primary and higher order structures required for function

Biotherapeutics Pharm Sci: PhRDImportance of Understanding Degradation ProfilesSmall moleculesBiotherapeuticsFormulate This…bioavailability…shelf life!

Biotherapeutics Pharm Sci: PhRDPotential Routes of DegradationDeamidation (Asn, Gln)Oxidation (Met, Trp, Cys)V LV HCDR1CDR2CDR3CDR3CDR2CDR1V HV LDR1CDR2CDR3C 1 HSC 1 HCDR2CDR3Glycation(Lys)CLS H SS HSSC 2HC 2HSSCLIsomerization (Asp)C 3HC 3HPeptide bondhydrolysis/truncations(Asp-Pro, Asp-Xxx)Pyroglutamate Formation(N-terminal Gln, N-terminal Glu)Denaturation, Aggregation,precipitation

Biotherapeutics Pharm Sci: PhRDWhat Factors Affect ChemicalDegradation?• pH• Buffer species• Other excipients• Light• Oxygen• Metal ions• TemperatureHydrolysis, deamidationDeamidationMaillard reactionPhoto decompositionOxidationHydrolysis, oxidationMost routes

Biotherapeutics Pharm Sci: PhRDContent• Overview of protein structure• Chemical degradation• Physical degradation11

Biotherapeutics Pharm Sci: PhRDDeamidationDeamidation of two key asparagines (shown in red)in Bcl-xL prevents this protein from carryingout its normal function, to block cell suicideYarnell, C&EN, 81, 47, Feb17, 2003

Biotherapeutics Pharm Sci: PhRDDeamidation of Proteins• Non-Enzymatic reaction which occurs at Asn and Gln• Hydrolysis reaction requiring water• Pathways depend on pH• Cyclic imide pathway under slightly acidic to basicconditions• Direct hydrolysis under acidic conditions• Minimum rate in pH range ~ 3 – 4• Buffer catalytic effect in pH range ~ 7 - 12• Rate is 5-10x faster for Asn than for Gln• Generates both normal and beta peptide bonds• Rate is affected by the N+1 residue•Interconvertibility isoAsp-Y to Asp-Y

BiotherapeuticsDeamidationPharm Sci: PhRDof AsparagineAsnz = 0-carbonylOO. . .NHNHNH. . .OOHOONHOOONH 2NORNH3- 17 Da -carbonyl. . .ROOH 2 O+18 DaNH. . .. . .NHNHNH. . . . . .NHNH O NH. . .O ROH ROpH range (~ 5 – 12)Succinimide(cyclic imide)OOProducts ratioIso-Asp : AspBasic ~2 – 3 : 1Asp, +1 Da, z = -1by hydolysis at -carbonyliso-Asp, +1 Da, z = -1by hydrolysis at -carbonylAdapted from JCarroll

Biotherapeutics Pharm Sci: PhRDDeamidation of AsparaginepH range (~ 1 - 2)Not observedAcidicDirect HydrolysispathwayPatel and Borchardt, PharmRes, 7, 703, 1990

Biotherapeutics Pharm Sci: PhRDDeamidation of Proteins (Asn)Effect of the (n+1) th ResidueFirst-order deamidation half times of pentapeptidesGlyXxxAsnYyyGly in days at pH 7.4, 37.0°C, 0.15 M Tris·HCl• Degree of side-chain branchingcritical in (n+1) th residue• Charge of residue not critical(exceptions: His, Ser, Thr)• Little effect of (n-1) th residueFrom analogous Gln pentapeptides:• Gln-Gly half-time~ 400- 600 days• Other Gln- sequences even longerRobinson and Robinson, PNAS, 98, 944, 2001

Biotherapeutics Pharm Sci: PhRDDeamidation of ProteinsImpact of pH / Buffer / Ionic Strength• Ionic Strength: Little to no effect• pH: Increased rate w pH• Buffer: Catalysis by Phosphate, CarbonateBrennan and Clarke, Ch 5. in Deamidation and Isoaspartate Formation in Proteins andPeptides, DWAswad Ed. CRC Press, 1995

Biotherapeutics Pharm Sci: PhRDDeamidation of ProteinsImpact of Structure• Asn is a “weak helix breaker”, somewhatinfrequent in -helices• 2 0 structures (-turns, -helices) tend tostabilize Asn residues against deamidation– Effect weak in -helices; may even destabilize– Stabilization probably due to conformational restrictions(critical distance of approach)– Stabilization also probably due to reduced nucleophilicactivity of the backbone NH centers, which are H-bonded in -helices and in all positions except one of the-turns– Expect stabilization in -sheets alsoXie and Schowen, JPS, 88, 8, 1999

Biotherapeutics Pharm Sci: PhRDDeamidation of ProteinsImpact of Structure• Probability of deamidation increases when alabile sequence falls in a flexible domain of theprotein.• Computational method for prediction of rate ofdeamidation of Asn residues gives mostaccurate results when both primary sequenceand 3D structure data is used (96.5% vs 79.8%with primary sequence alone and vs 64.3% with3D structure alone) (Robinson, PNAS, 99, 5283, 2002)(www.deamidation.org)

Biotherapeutics Pharm Sci: PhRDDeamidation of ProteinsImpact on Activity• Asn30 to Asp on LC: Potency ~70%. Asp102 to isoAsp on HC: Potency < 30%(Harris et al, JChromB, 752, 233, 2001)• Homodimers of SCF with IsoAsp in place of Asn10 was 50X less potent, while Asphomodimers had 150% of normal activity (Hsu et al., Biochemistry, 37, 2251, 1998)• Calbindin D 28K : Asn203 to Asp conversion has no impact on Ca 2+ binding affinity and(the high) cooperativity. Asn203 to isoAsp reduces affinity at multiple sites leading toreduced affinity and sequential binding (Vanbelle et al., ProtSci., 14, 968, 2005)• Deamidation of N146 and not N78 had a significant impact on chaperone activity andstructural properties of B-crystallins (Gupta and Srivastava, IOVS, 45, 206, 2005)• Presence of < 5% deamidated Amylin20-29 leads to aggregation and amyloidformation (Nilsson, ProtSci, 11, 342, 2002)• Enhanced bioactivity of mammalian somatotropin through selective deamidation(Asn to isoAsp in region 96-101 of bSt) (WO 1987/01708)• Activity of a protein may or may not be impacted by deamidationdependent upon– Position of deamidation and impact on structure/conformation– Extent of deamidation in pool

Biotherapeutics Pharm Sci: PhRDDeamidation of ProteinsBiological Relevance• Intestinal T-cell response to -gliadin in adult celiac diseaseis focused on a single (enzymatically) deamidated Gln(targeted by tissue transglutaminase) (JEM, 191, 603, 2000)• Deamidation of N146 in B-crystallins has been implicatedin the formation of cataracts (IOVS, 45, 206, 2004).• Two of (9) Asn in Bcl-x L undergo selective deamidation thatblocks its pro-survival function (blocking pro-apoptopicproteins) allowing cell death (Cell, 111, 51, 2002; PLoS Biology, 5, 0039, 2007).• In vivo deamidation half-life of Asn55 in an IGg1 (located inCDR2 of HC) is around 140 hrs irrespective of route ofadministration (IV or SC) (AnalChem, 77, 1432, 2005)• Ageing / Molecular clocks (Asn half-lives of 1 d to 50 yrs)

Biotherapeutics Pharm Sci: PhRDDeamidation and ApoptosisRobinson et al., PNAS, 66, 753, 1970

Biotherapeutics Pharm Sci: PhRDIsomerizationRacemization

Biotherapeutics Pharm Sci: PhRDIsomerization in Proteins• Asn, Asp and Gln• Succinimide intermediate• Asp much slower than Asn via thismechanism• Primary route is the deamidation whichyields iso-Asp and Asp in a roughly 3:1ratio.• Rate and propensity determined bylocation and mobility.

Biotherapeutics Pharm Sci: PhRDIsomerization and Racemization in ProteinsRates:N isoDD isoDDaniel et al., BiochemJ., 317, 1, 1996

Biotherapeutics Pharm Sci: PhRDDeamidation, Isomerization andRacemization (Asx)Yoshioka and Stella, Ch. 5, 2000

Biotherapeutics Pharm Sci: PhRDHydrolysis of Peptide Bonds• -x-Asp-y sequence is considered labile• Hydrolysis in dilute acid at least 100X faster than otherpeptide bonds• Mechanisms similar to deamidation succinimideformation, followed by racemization and isomerization• Asp-Pro (Pro-Asp?) susceptible (8-20X faster compared toother Asp-x or x-Asp) under acidic conditions• Asp-Gly susceptible under highly acidic conditions (pH 0.3 –3)• -x-Ser-, -x-Thr-: Cleavage at N-terminal side faster than otherpeptide bondsMarcus, IntJPeptProtRes., 25, 542, 1985; Han et al., IntJBiochem, 15, 875, 1983;Oliyai and Borchardt, PharmRes., 10, 95, 1993

Biotherapeutics Pharm Sci: PhRDHydrolysis of Peptide BondsDaniel et al., BiochemJ., 317, 1, 1996Geiger and Clarke, JBC, 262, 785, 1987

Biotherapeutics Pharm Sci: PhRDDisulfideBreakage and Scramblinghttp://www.ehscenter.org/dbd/

Biotherapeutics Pharm Sci: PhRDDisulfides: Cleavage and ScramblingcysteinesCleavage: Reducing conditions e.g. in cytosolcystineThiol-disulfide exchange(neutral or alkaline environments; pH > ~8;protonated thiols are not reactive, pKa ~ 8.3)Disulfide reshuffling faster than formation/reduction of disulfides

Biotherapeutics Pharm Sci: PhRDGlycation• Reducing sugars reacting through classic Maillard reaction toamino groups• ε-amino group of Lys, N-terminal -amino susceptible• Implicated in complications of diabetes and ageing (stiffeningof arteries, vascular narrowing, cataract formation –• AGE: Advanced Glycation Endproducts• Accumulates in slow-turnover proteins (eg collagen, -crystallins)• Impacts structure and function• Acidification [loss of positive charge(s)]• Insolubility• Cross-linking• Resistance to heat denaturation• Generation of chromophores (brown, yellow)• Impacted by pH, pka of aminoacid group, adjacent AA,Temperature, Concentration of reducing sugar, ConcProtein

Biotherapeutics Pharm Sci: PhRDGlycation (Maillard Reaction)glycatedglycated--Luthra and Balasubramanian, JBC, 268, 18119, 1993

Biotherapeutics Pharm Sci: PhRDDiketopiperazine

Biotherapeutics Pharm Sci: PhRDDiketopiperazine (DKP)• Often seen with dipeptide esters and amides• Nucleophilic attack of N-terminal nitrogen on theamide carbonyl between 2 nd and 3 rd residues• Peptides containing Gly as the 3 rd residue, or Pro in2 nd residue from N-terminal particularly susceptible• Favored in neutral or alkaline medium• DKP from x-Pro is impacted by cis-trans equilibriumof Pro, and dependent on charge distribution aroundpeptide bond. Cis facilitates ring closure.Powell, ACS567, Ch.7, 1994; Goolcharran and Borchardt, JPS, 87, 283, 1998

Biotherapeutics Pharm Sci: PhRDDiketopiperazine (DKP)Phe-Pro N-terminus in rhGHBattersby et al., IntJPepProtRes, 44, 215, 1994

Biotherapeutics Pharm Sci: PhRDPyroglutamic Acid (PyroE)Q PyroEE PyroE• N-terminal Gln can spontaneously cyclize to form PyroE• Gln-Gly reacts much fasterE PyroEN-terminal Glutamine (Q): Common, quantitativeCharge change (-1, loss of basic residue)Mass Change (-17 Da)N-terminal Glutamic Acid (E): Less commonCharge neutralMass Change (-18 Da)Adapted from JCarrol; Powell, ACS567, Ch.7, 1994; Chelius et al., AnalChem, 76, 2370, 2006

Biotherapeutics Pharm Sci: PhRDImpact on Charge Heterogeneity inProteinsChange on Stability• Deamidation (more acidic) (z = -1)• Succinimide (more basic or neutral)• Glycation (more acidic)• Pyroglutamate formation (more acidic) (z = -1)• Peptide Bond Hydrolysis (either acidic or basic)JCarroll

Biotherapeutics Pharm Sci: PhRDOxidation ProductsResiduesMetCysOxidation productsMet-Sulfoxide-S-S-disulfide crosslinks, sulfenicacid/sulfinic acid/solfonic acid2-oxoimidasoline, aspartate/asparagineN-formylkynurinine, kynurenineHisTrpTyr Try-Try crosslink, 3-4-dhydroxyphenylalanineBorchardt R. T. et al., Biotechnology and Bioengineering, Vol. 48, 490-500, 1995Shen F. J. et al., Techniques in Protein chemistry, Vol. III, 275-284

Biotherapeutics Pharm Sci: PhRDProtein Oxidation‣ Covalent modification of a protein‣ Induced by reactive oxygen intermediates or other oxidants‣ Can be caused by• Chemical reagents (H 2 O 2 , ·O 2 , metal ions, excipients)• UV light‣ Commonly modifies the residues• Met, Cys, Trp, His, Tyr‣ May cause reduced potency of protein, depending on site‣ May cause conformational change in protein, leading toaggregation

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsAgents that can lead to Protein Oxidation• Chemical Reagents• (H 2 O 2 , Fe 2+ , Cu 1+ , Glutathione, HOCl, HOBr, 1 O 2 , ONOO - )• Activated phagocytes (oxidative burst activity)• -Irradiation in the presence of O 2• UV light, Ozone• Lipid peroxides (HNE, MDA, acrolein)• Mitochondria (electron transport chain leakage)• Oxidoreductase enzymes• (Xanthine oxidase, Myeloperoxidase, P-450 enzymes)• Drugs and their metabolitesShacter, Virtual Free Radical School http://www.healthcare.uiowa.edu/research/sfrbm/virtual.html

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsTypes and Mechanisms Site-Specific Oxidation• Catalyzed by transition metals e.g., Fe(II) or Cu(I)• Involves generation of ROS and oxidation at metal binding site• His, Pro, Arg, Lys, Thr, Tyr, Met, Cys most susceptible Non-Site Specific Oxidation• Caused by (contaminant) oxidants such as H 2 O 2 , HOCl, oroxidizing radiation (e.g., light)• All susceptible residues Primary Formulation development relevant residues• Met, Cys, His, Trp, TyrShacter, DrugMetabRev, 32, 307, 2000; Stadtman, AnnuRevBiochem, 62, 797, 1993

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsMCO is a Site-Specific ProcessFe(III)+Fe(II)Peroxideor O 2 etcOxidized &NonfunctionalReactiveSpeciesAdapted from Shacter (Virtual Free Radical School); Stadtman and Levine, Ann NY AcadSci., 899, 191, 2000

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsNon-Site Specific Oxidation: Initiation• H 2 O 2 , HOCl, Organic peroxides, etc.• Directly• Through free radical mechanisms (initiated bylight / transition metals /H 2 O 2 2HO •H 2 O 2 + O 2 2O•-2 + 2H +H 2 O 2 + O 2 HO 2• + O•-2 + H +H 2 O 2 + O 2 2HO•2pH dependent• Main Source: Impurities in excipients,Contamination

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsMethionine ResiduesRSCH3 RS(O)CH3 RS(OO)CH3Methionine Methionine MethionineSulfoxide Sulfone• Methionine very susceptible to oxidation• Multiple pathways possible (free radical, singlet oxygen,nucleophilic substitution)• Reactivity varies with conformation• pH sensitivity (acid catalyzed: slight increase in rate from pH 5 to 1)• Key component in the system for regulation of cellular metabolism

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsMethionine Residues as Endogeneous AntioxidantsMethionines are key components in the system for regulation of cellularmetabolism‣ Readily oxidized by a variety of “abuses” (generally to MetSulfoxide)‣ Repair mechanism: Methionine Sulfoxide Reductases (MSR) widelypresent and capable of reducing free or protein-bound MetSulfoxidesback to Met‣ Reversible covalent modification functions as a regulator of biologicalactivity though alteration of catalytic efficiency and through modulationof surface hydrophobicitye,g• In HDL, apolipoprotein-I reduces Cholesteryl ester hydroperoxides toalcohols at the cost of oxidation of two Mets to MetSulfoxides. (Garner et al.,JBC, 273, 6088, 1998)• Oxidized apolipoprotein-I is reduced by MSR allowing catalytic activityof the protein. (Sigalov and Stern, FEBS Lett, 433, 196, 1998)

Biotherapeutics Pharm Sci: PhRDOxidation of Proteins: Cysteine ResiduesRS - + M n + RS• + M(n-1)+R’SH + RS • + O 2 R’S-SR + H + + O•-2 (cystine disulfide)RS • + O 2 RSOO •RSOO • + RSH RSO • + RSOH (sulfenic acid)RSOH + R’SH RS-SR’ + H2ORSH + O•-2 RS• + HO2-RS • + R’S • RS-SR’RSH + R’S - + H 2 O 2 RS-SR’ + OH - + HO •RSH RSOH RSO 2 H RSO 3 Hthiol sulfenic sulfinic sulfonic• Cysteine also quite susceptible to oxidation• Cysteine oxidation forms inter- or intra-molecular disulfide bonds• Further oxidation can yield sulfenic acid• Oxidation mechanism strongly catalyzed by transition metals• Rate accelerated with higher pH, optimum around 6 (-SH pKa ~ 8.5)• Cystine disulfide less sensitive to mild oxidants such as H2O2compared to Met or Cys• In absence of a nearby thiol for disulfide formation, oxidation canyield sulfenic, sulfinic, and sulfonic acids.

Biotherapeutics Pharm Sci: PhRDOxidation of Proteins: Histidine Residues• Histidine also quite susceptible to oxidation (MCO or Photocatalyzed)• Histidine often part of metal-binding sites• MCO leads mostly to 2-Oxo-Histidine. Chain scission rarely seen.• Photoxidation in presence of photosensitizers via singlet oxygen 1 O 2• By-products proposed include asparagine / aspartic acid• Oxidation propensity and rate impacted by residues in proximity andconformation• Colored Solution?Li et al., BioBio, 48, 490, 1995

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsTryptophan Residues• Tryptophan susceptible to oxidation by ROS as well as Photo• Low abundance but highest molar absorption coefficient (280 – 305 nm)• Impacted by sequence position, exposure to solvent, access to oxygen• Colored Solution ?Li et al., BioBio, 48, 490, 1995; Bummer and Koppenol, 2000; Kerwin and Remmele, JPS, 96, 1468, 2007

Biotherapeutics Pharm Sci: PhRDOxidation of Proteins: Tyrosine Residues• Tyrosine subject to Photooxidation• Products include DOPA or Di-tyrosine (cross-linking reaction)• Dityrosine has unique fluorescence signature (410-415 nm emission, 315 nm excitation)• During photooxidation, Tyr can transfer energy to disulfides and Trpresulting in Tyr radical cations (Tyr •+ ), disulfide-based radical (RSSR •+ ),or triplet-state Trp ( 3 Trp)Kerwin and Remmele, JPS, 96, 1468, 2007; Bummer and Koppenol, 2000

Biotherapeutics Pharm Sci: PhRDOxidation of Proteins: Phenylalanine Residues• Phenylalanine subject to Photooxidation• Products include ortho- and meta-tyrosine derivatives

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsGeneralizations‣ Cys, Met and His are highly susceptible to MCO‣ Trp and His are main targets of Photo-induced oxidation‣ Rates of photooxidation follow the orderHis > Trp > Met > Tyr‣ Specific oxidation mechanisms influenced by• Intrinsic factor: Structure• Buried residues less easily oxidized by non-sitespecific mechanisms (eg H2O2, Light)• Site-Specific MCO less impacted by steric effects andmore by proximity of residue to metal binding site• Exogeneous factors: pH, Temperature, Buffer, ExcipientsLi et al., BioBio, 48, 490, 1995

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsGeneralizations (cont’d)Exogeneous factors: pH• Oxidation potential of system increases with pH• Metal binding will improve due to ionization of functionalgroups (but solubility of transition metals may decrease) withincreasing pH• Ionization of side chains (Cys, His) enhances lability withincreasing pH• At pH < 4, only Met and Trp can be readily photooxidizedLi et al., BioBio, 48, 490, 1995

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsGeneralizations (cont’d)Exogeneous factors: Temperature• Effect both through impact on 2 0 and 3 0 structure as well aspotential pH shifts• Reduction of rate with temperature often not significant dueto increased aq. solubility of oxygen at lower temperatures(1.29e-3 mmol/mL at 25ºC; 2.18e-3 mmol/mL at 0ºC)• Change in temperature may change rate controlling step inmechanism.Li et al., BioBio, 48, 490, 1995

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsGeneralizations (cont’d)Exogeneous factors: Buffer species• Major effect of buffer species actually comes throughcontaminants, metal chelating ability, and possible interactionwith ROS• Tris is a Fe and Cu chelator but also a hydroxyl radicalscavenger• Citrate is a chelator; phosphoric acid (weak), tartaric acid• Fenton chemistry requires bicarbonate ion• No clear generalizationsLi et al., BioBio, 48, 490, 1995; Stadtman, AnnRevBiochem, 62, 797, 1993

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsGeneralizations (cont’d)Exogeneous factors: Excipients• Major effect of common Excipient species actually comesthrough contaminants• Peroxides (e.g. in Polysorbates)• Trace Metals, esp Fe, Cu• High concetrations of sugars and polyols seems to inhibitMCO (though a metal complexation mechanism?)Li et al., JPS, 85, 868, 1996; Li et al., BioBio, 48, 490, 1995

Biotherapeutics Pharm Sci: PhRDOxidation of ProteinsChelators: Use with caution• Strong 1:1 Chelators (e.g., EDTA, desferrioxamine,nitrilotriacetic acid) can impact oxidation rates based uponstoichiometry (chelator/Fe ratio)• Rate increases until all Fe is sequestered, max at ratio 1• Almost complete inhibition at ratio 1.1 – 1.2• Weak chelators (e.g., o-phenanthroline) require largeexcess (~5 - 20) to inhibit• Site-specific oxidation is impaired by Chelate-Fe(III)complexes, but non-site specific oxidation is enhanced• Chelates extract iron from potential binding sites• Chelate-Fe(III) complex promote formation of ROSspecies different than those at metal-binding sitesStadtman, AnnRevBiochem, 62, 797, 1993; Zhao et al., PharmRes, 13, 931, 1996

Biotherapeutics Pharm Sci: PhRDOxidation – Case StudyOne lot of lyophilized drug candidate showed small number of vials had a high and erratic level of oxidized protein(up to 20%), which was randomly distributed among all the samples. It was suspected that the oxidation was dueto contamination of sanitizing agent used during the filling/lyophilization process through vapor transfer.• Sanitizing/oxidizing agents can oxidize protein product readily through vapor transfer not only in aclosed system but also in an open system.• The amount of oxidation is proportional to the time of sample exposure and the type andconcentration of the sanitizing/oxidizing agents.Wei Wang et al., PDA J. Pharm. Sci. and Tech. Vol. 58, 121-129, 2004

Biotherapeutics Pharm Sci: PhRDContent• Overview of protein structure• Chemical degradation• Physical degradation58

Biotherapeutics Pharm Sci: PhRDWhat Factors Affect Physical Degradation?HydrophobicsurfacesHeatingDenaturationLyophilisationReconstitutionAdsorptionOrganicsolventsAggregationShakingand shearPrecipitation

Biotherapeutics Pharm Sci: PhRDProposed Mechanisms of ProteinAggregationOrdered aggregates associate via mechanisms such asdomain swapping (ds), strand association (sa),edge-edge-association (ee), or -strand stacking (bs).BBA 2008: 469, 100-117

Biotherapeutics Pharm Sci: PhRDExperimental ApproachesN– Native; U– Unfolded; I– Intermediate; O– Oligomeric; A-- AmyloidFEBS J. 2005: 272, 5962-7

Biotherapeutics Pharm Sci: PhRDAggregation of Proteins‣ The term usually refers to multimers of proteins– e.g. dimers, trimers, tetramers…. to large polymers‣ Aggregates can be non-covalent or covalent (disulfide-linked)‣ Aggregates can be present as:– Fully soluble in a clear solution– Partially insoluble in a turbid solution (usually extremely large)– Mostly insoluble as a precipitate that collects in the bottom ofthe container‣ Formed by non-specific protein : protein association resultingfrom interactions among solvent exposed hydrophobic groupsNMozier

Biotherapeutics Pharm Sci: PhRDAggregation of Proteins‣ Assembly of initially native, folded proteins resulting in nonnativestructures.‣ Aggregates: Irreversible, may contain high levels of nonnativeintermolecular -sheet structures‣ Onset, rate, final morphology dependent on solutionconditions such as pH, salt species, salt concentration,cosolutes, excipients, surfactants‣ Function of relative intrinsic thermodynamic stability of thenative stateChi et al., PharmRes, 20, 1325, 2003

Biotherapeutics Pharm Sci: PhRDModels of Aggregation (1)N (Native)U (Unfolded)A (Aggregate)• Denatured / unfolded molecules aggregate due to hydrophobicinteractions• Aggregate formation driven by hydrophobic effect• Normally buried hydrophobic regions are exposed• Rate increases with temperature (U increase with temp.)• First order kinetics

Biotherapeutics Pharm Sci: PhRDModels of Aggregation (2)N (Native) I (Intermediate) U (Unfolded)A (Aggregate)• Protein aggregates are generally formed from intermediates inthe folding/unfolding process• Misfolded intermediates (often thermodynamically stable)can also aggregate• Intermediates can be part of the native state ensemble• Hence, aggregation is not an “unnatural” state for a protein• Aggregation can occur even under conditions favoring native state

Biotherapeutics Pharm Sci: PhRDAggregation as aNucleation and Growth ProcessGazit, AngewChemIntEd, 41, 257, 2002

Biotherapeutics Pharm Sci: PhRDClassification of Aggregates Non-Covalent– Weak association (easily reversible by dilution, milddetergent)– Strong association (not easily reversible) Covalent‣ Soluble‣ Insoluble Visible Invisible (subvisible)

Biotherapeutics Pharm Sci: PhRDTypes of Aggregates• Weakly associated Non-Covalent– Reversible by dilution– Dimers to multimers• Strongly associated Non-Covalent– Not reversible by dilution– Dimers to multimers– Precipitates possible• Covalent– Not reversibleMonomerMultimerSmallAggregateLargeAggregatePrecipitate

Biotherapeutics Pharm Sci: PhRDCauses of Aggregation• Process– Fermentation/Expression Inclusion bodies– Purification Shear, pH, ionic strength– Filtration Surfaces, Shear– Fill/Finish Surfaces, Shear,Contamination (eg silicone oil)– Freeze/Thaw Cryoconcentration, pHchanges, Ice-solution interfaces– Shipping Agitation, Temperature cycling– Lyophilization Cryoconcentration, pHchanges, Ice-solution interfaces,dehydration– Administration Diluents, Component materialsand surfaces, Leachables

Biotherapeutics Pharm Sci: PhRDAll Aggregates Are Not Made EqualStorage studyIncreasingprotein concIncreasingprotein concAgitation studyTreuheit et al, PharmRes., 19, 511, 2002

Biotherapeutics Pharm Sci: PhRDAggregation and TurbidityCaution: Turbidity is not necessarily an indicator ofAggregationClear / Opalescentboundary~3 NTU = Ref 1Sukumar et al, PharmRes., 21, 1087, 2004

Biotherapeutics Pharm Sci: PhRDAggregation Induced by OxidationLi et al., Biochem, 34, 5762, 1995

Biotherapeutics Pharm Sci: PhRDHeterogeneous Nuclei: Silicone OilNo impact seen by CD, DSCPan et al., NBC2006, Boston, #M1050

Biotherapeutics Pharm Sci: PhRDAggregation: Why Important• Potential impact on bioactivity– Potency– PK/PD• Potential impact on immunogenicity

Biotherapeutics Pharm Sci: PhRDMisfolding and ImmunogenicityMaas et al., JBC, 282, 2229, 2007

PeptideBiotherapeuticsSequencePharm Sci: PhRDHot-Spots1 02 01 0Powell, ACS567, Ch. 7, 1994

Biotherapeutics Pharm Sci: PhRDProtein Sequence Hot-SpotsPathwaySiteDeamidationAsn, Gln, -Asn-X-, -Asn-Gly-, Asn-His-, -Asn-Ser-, -Asn-Ala-,-Asn-Asp-, -Asn-Thr-HydrolysisOxidationAsp, -Asp-Pro-, -Asp-Gln-, -Pro-Asp, -Asp-Tyr-, -X-Ser-, -X-Thr-Met, Cys, His, Trp, TyrIsomerizationDiketopiperazineGlycationBeta-EliminationPyroglutamic acidAsn, Asp, -Asn-Gly-, -Asp-Ser-, -Asn-Ser-, -Asp-Ser-X-X-Gly, X-Pro-LysSer, ThrH2N-Gln-, H2N-Gln-Gly-, H2N-Glu-

Biotherapeutics Pharm Sci: PhRDSomavert ®20 3010F D N A M L R A D R L N Q L A F D T Y Q E F E EAYS R L IPKLE P V Q F L R S V F A N S L V Y G A S D S N V110 YOxidation “Hot Spots”D90LNorleucine misincorporationPEG - 39%LT R P130EPLQGSGK40DQIWIELDRLKSFGETPEG - 78%M L T Q I K EY140180PQ KS R120 Not susceptible SFI QV C Q V T R L Fto oxidationN-SuccDisulfideT170FL TES V RSDes-PheMLNH2 L YG S C G F COOHDTrisulfideQL SAPEG - 100% 80190(S-S-S)PEG - 80%NS K Deamidation “Hot Spots”NPIF FRD PEG - 79%TQInternal ClipLN STLH NSED D A L L K N YG L L Y C 50150LL160DisulfideNFCSSKQ Q T E E R N S P T P I SEPEG - 26%7010060PEG - 81%Adapted from: Olson et al, Polyethylene Glycol: Chemistry & Biological Applications, ACS, 1997, p170

Biotherapeutics Pharm Sci: PhRDContent• Overview of protein structure• Chemical degradation• Physical degradation79

Biotherapeutics Pharm Sci: PhRDAcknowledgementsJames CarrollNing LiBilikallahalli MuralidharaSatish SinghWei Wang