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Non-Native Aggregate Assembly and Structure: A Multi-Scale Problem

Christopher J. Roberts, Ph.D.Associate Professor of Chemical Engineering

University of Delaware

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Students & Post-DocsPrateek Shah (Ph.D. `08) Jennifer (Andrews) Woods (Ph.D. `08)Yi Li (Ph.D. `09) Michelle Spatara (Ph.D. `09)Teresa Young (Ph.D. `09) William F. Weiss IV (Ph.D. `09)Rebecca Brummitt(Ph.D. `10) ErincSahin(Post-doc Res. Assoc.)Charles Woods Marco BlancoRon Maurer Andrew Kroetsch(BChE `10)Nayoung Kim

Current / Recent CollaboratorsAnne Robinson (UD) BramieLenhoff(UD)Erik Fernandez (UVA) Aming Zhang (UVA)TundeOgunnaike(UD) AdeolaGrillo (HGSI)Melissa D. Perkins (HGSI) Doug Nesta(GSK)Lucy Chang (GSK / PFZ) Alan Hunter (MedImmune)MagdaIvanova(UCLA) Tom Laue, Sue Chase (UNH)Richard Remmele(Amgen) Dean Liu (Amgen)Tapan Das (Pfizer) Kevin King (Pfizer)

Funding (current + previous)National Institutes of HealthMerck Research LaboratoriesNational Science Foundation Human Genome SciencesGlaxoSmithKline PfizerMedImmune AmgenBoehringer Ingelheim

Acknowledgements

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Dobson CM “Protein Folding and Misfolding” Nature (2003)

“Aggregation” occurs via competing contributions

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Unfolding-mediated Aggregation is Multi-Step:Product(s) Range from Oligomers to Visible

Particles

200 nm 200 nm50 nm

Weiss, Hodgdon, et al. Biophys. J. (2007)

Li et al., J. Pharm. Sci.(2010)

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Non-Native Agg’s are Typically Net “Irreversible”w/out Extreme T, p, [denaturant]

200 nm 200 nm50 nm

Weiss, Hodgdon, et al. Biophys. J. (2007)

High T (~ 100 C)High p (~ 103 bar)High [denaturant] (~ 4+ molar)

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Aggregation rates are very sensitive to temperature need (at least local) unfolding

-3

-2

-1

0

1

2

3

4

3 3.2 3.4 3.6

bG-CSF shelf life (t90) vs. T, pH>4.5

Erroneous(Arrhenius)Prediction

1000 / T

log 1

0(ti

me

for 1

0% lo

ss)

pH 7.5

pH 4.8

1 year

1 mo.

1 day

1 hour

5 min.

LENPPrediction

5°C20°C30°C40°C

Roberts et al JPhSci (2003)

Hun ~ 102 kcal/mol at T ~ Tm

Hun ~ 0 kcal/mol at T ~ 10 - 20 oC

PBD 1BGC

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Small length scales: Inter-protein contacts can be only small segments of the entire protein

Zhang, Ivanova, Weiss, Roberts, Fernandez, Biochemistry (2010)

Akin to modified “domain swapping” model (e.g., Sambashivan et al., Nature (2005))

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Small length scales: Difficult to reliably predict (exposed) “hot spot” sequences in foldable proteins

Adapted from Roberts, Das, & Sahin Int. J. Pharm. (2011)

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Small length scales: single point mutations can greatly change kobs via “hot spots” a/o Gun

Roberts, Das, & Sahin Int. J. Pharm. (2011)Sahin, Jordan, Spatara, Naranjo, Robinson, Fernandez, Roberts Biochemistry (2011)

M69 S130

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Roberts, Das, & Sahin Int. J. Pharm. (2011)Sahin, Jordan, Spatara, Naranjo, Robinson, Fernandez, Roberts Biochemistry (2011)

M69 S130

Small length scales: single point mutations can greatly change kobs via “hot spots” a/o Gun

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Intermediate length scales: colloidal interactions & (near) neighbors

lim [protein] 0G22 = -2B22

Blanco, Das, and Roberts (in preparation)

S(q 0) [light / neutron scattering]

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Often, Gun (short L) wins out over B22(intermed. L) for agg’n of “monomers”

Tagg = temp. for t50 ~ 1 hourTm from DSCb2*= (B22-B22

HS) / B22HS

Roberts, Das, & Sahin Int. J. Pharm. (2011)Sahin, Grillo, Perkins, Roberts J. Pharm. Sci (2010)

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Brummitt et al JPhSci 100:2104 (2011)

Intermediate L: antibody “chain polymerization” (small oligomers)

Lumry-Eyring Nucleated-Polymerization (LENP)

mass action model

pH 4.5

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0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

40 50 60 70 80

Excess Specific Heat C

apacity

(cal g

‐1K‐

1 )or  

Mon

omer Fraction, m

(T)

Temperature  / deg. C

Fab

CH2CH3

Local unfolding is (particuarly?) important for aggregation of large proteins

CH2CH3

FabTSML

DSC

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Initiation via irreversible dimerization of partially unfolded monomers

Nucleation: n ~ C01-x

Chain Growth: g ~ C0-1

Andrews & Roberts J Phys Chem B (2007); Weiss et al Biophys J (2007)Li & Roberts J Phys Chem B (2009); Li et al J PharmSci (2010)

Lumry-Eyring Nucleated-Polymerization (LENP)

mass action model

Brummitt et al JPhSci 100:2104 (2011)

Dimer…LargeSoluble /Insoluble Agg.

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Brummitt et al JPhSci (2011b)

Large L: further aggregate growth via both monomer add’n & aggregate coalescence

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Aggregate growth mechanism ispH-dependent

pH 3.5Slow growthOnly soluble

aggregates

pH 4.5Rapid growthSoluble & “insoluble” aggregates

pH 5.5Rapid growthPrimarily “insoluble” aggregates

(macroscopic “particles”)

Brummitt et al JPhSci (2011b)

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200 nm 200 nm50 nm 50 nm

100nm

Aggregate growth mechanisms depend strongly on charge & salt effects

Weiss et al. (2007)

Globular, HMW,Soluble Agg.

Insoluble Agg. / Precip. / Particles

Linear, HMW,Soluble Agg.

R. Hepler

Li et al., J. Pharm. Sci.p. 645-662 (2010).

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B22 Surrogate for agg – agg interactions & particle formation

200 nm 50 nm

Li et al., J. Pharm. Sci. 99:645 (2010)

All T < ca.Tm

NOTE: similar result for some IgGs (Sahin et al. 2010)

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[sal

t] (a

ppro

x. Io

nic

Str.

)

pH

Nucleation (Monomers)&Mon-Agg Interactions

Nucleation / Seeding&Agg-Agg Interactions

[sal

t] (a

ppro

x. Io

nic

Str.

)pH

Small L (“local” structure)

Small & Large L

200 nm 200 nm50 nm 50 nm

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Students & Post-DocsPrateek Shah (Ph.D. `08) Jennifer (Andrews) Woods (Ph.D. `08)Yi Li (Ph.D. `09) Michelle Spatara (Ph.D. `09)Teresa Young (Ph.D. `09) William F. Weiss IV (Ph.D. `09)Rebecca Brummitt(Ph.D. `10) ErincSahin(Post-doc Res. Assoc.)Charles Woods Marco BlancoRon Maurer Andrew Kroetsch(BChE `10)Nayoung Kim

Current / Recent CollaboratorsAnne Robinson (UD) BramieLenhoff(UD)Erik Fernandez (UVA) Aming Zhang (UVA)TundeOgunnaike(UD) AdeolaGrillo (HGSI)Melissa D. Perkins (HGSI) Doug Nesta(GSK)Lucy Chang (GSK / PFZ) Alan Hunter (MedImmune)MagdaIvanova(UCLA) Tom Laue, Sue Chase (UNH)Richard Remmele(Amgen) Dean Liu (Amgen)Tapan Das (Pfizer) Kevin King (Pfizer)

Funding (current + previous)National Institutes of HealthMerck Research LaboratoriesNational Science Foundation Human Genome SciencesGlaxoSmithKline PfizerMedImmune AmgenBoehringer Ingelheim

Acknowledgements

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