Non-Native Aggregate Assembly and Structure: A Multi-Scale ... · (Arrhenius) Prediction 1000 / T...

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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

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

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

“Aggregation” occurs via competing contributions

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)

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)

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

3 3.2 3.4 3.6

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

Erroneous(Arrhenius)Prediction

1000 / T

pH 7.5

pH 4.8

1 year

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

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))

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

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

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

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

Intermediate length scales: colloidal interactions & (near) neighbors

lim [protein] 0G22 = -2B22

Blanco, Das, and Roberts (in preparation)

S(q 0) [light / neutron scattering]

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)

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

40 50 60 70 80

Excess Specific Heat C

apacity

(cal g

‐1K‐

omer Fraction, m

Temperature / deg. C

CH2CH3

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

CH2CH3

FabTSML

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.

Brummitt et al JPhSci (2011b)

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

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)

200 nm 200 nm50 nm 50 nm

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).

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)

Nucleation (Monomers)&Mon-Agg Interactions

Nucleation / Seeding&Agg-Agg Interactions

Small L (“local” structure)

Small & Large L