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Excipient effects on the stuctural and colloidal stability of proteins A rational approach to the formulation of protein pharmaceutics?. Agenda: Protein stability, reversible and irreversible transitions Preferential interactions and reversible stability Aggregation and colloidal stability

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Excipient effects on the stuctural and colloidal stability of proteinsA rational approach to the formulation of protein pharmaceutics?

  • Agenda:
  • Protein stability, reversible and irreversible transitions
  • Preferential interactions and reversible stability
  • Aggregation and colloidal stability
  • Electrostatic and ”Hofmeister” effects on protein aggregation

Bratislava, May 20th 2008

Peter Westh (

protein conformations are physically unstable
Protein Conformations are physically Unstable !

Typical stability for a small globular protein at “normal” conditions

DG° ~ 20-50 kJ/mol

Hen lysozyme has ~200 intramolecular hydrogen bonds – the bond energy for each of these is ~25kJ/mol

Evolutionary aspects –

Is it so hard to design a stable protein conformation?

Functional aspects –

An enzyme is a nanomaschine often doing 100-100,000 cycles per sec

Technological aspects –

Product stability may be a limiting factor

Hen lysozyme with 185 bound water molecules

Wilson et al 1992.

counteracting the physical instability
Counteracting the physical instability
  • Some main strategies:
  • Solvent based stabilization (excipients)
  • Modifications (PEG, Glycans etc.)
  • Mutations (site directed mutagenesis)
stabilization of pharmaceutical proteins
Stabilization of Pharmaceutical Proteins
  • Stabilization against which transition?
  • Reversible unfolding or irreversible transitions
  • Lumry-Eyring model (Lumry and Eyring 1954)


Equilibrium transition

K=[D] / [D] and hence DG°=-RTlnK

Far from Equil. mI << mD

Kinetically controlled

The irreversible transition is directly relevant to formulation protocols. The reversible transition is experimentally and theoretically accessible and empirically related to the kinetic stability

cooperativity of n d
Cooperativity of ND
  • The reversible denaturation of small proteins is often highly cooperative – This implies non-additivity of domain stability.
  • NIn1In2In3...InND (Ini:intermediates)
  • Ini are sufficiently sparsely populated to be neglected, hence
  • ND

Does not happen since

DG°blue+red > DG°blue + DG°red

The difference is the red-blue domain interaction free energy




equilibrium denaturation n d
Equilibrium denaturation ND
  • Differential Scanning Calorimetrymeasures the heat flow (in Watt=J/sec) required to heat the sample at a constant rate

Calorimetry is a simple experimental principle (Lavoisier had nice calorimeters in 1780) which has been developed to extreme sensitivity (10-100nW or ~ 10-7°C)


Constant P: qp=DH=CpDT

historic sweep
Historic sweep
  • One of the oldest analytical principles still in use – Lavoisier had rather precise calorimeters by 1780.
  • Readily measured thermodynamic function.
  • Heat cannot be measured – temperature can.
  • Heat is NOT at state function – enthalpy and internal energy are.
the use of kirchkoff s equation on dsc and other data lead to counterintuitive results
The use of Kirchkoff´s equation on DSC (and other) data lead to counterintuitive results!

Proteins denature both upon heating and cooling

stability and specific binding
Stability and specific binding

The binding of a ligand to the native state brings abour stabilization – The dicplacement of the peak along with the change in transition enthalpy quantifies the binding strength

protein stability solute excipient effects
Preferential exclusion : inhibits the formation of interface

Stabilize Precipitate

Native protein

Denaturered protein

Præferential binding : promotes larger interfaces

Destabilize Solvate

Protein stability – solute (excipient) effects
  • Solutes effect stability, solubility and oligomerization according to their adsorption or repulsion form the protein interface
preferential interactions
Preferential Interactions
  • Preferential Binding: Excipient-protein interactions are stronger than water-protein interactions.
  • Preferential exclusion: Water-protein interactions are stronger than excipient-protein interactions.


Preferentially excl. excipient

No Excipient

Preferentially bound excipient












hofmeister effects and the lyotropic series
Hofmeister effects and the Lyotropic Series








Michael Chaplin

Relies on surface charge density and polarizability

Anionic kosmotropes (e.g. F- and SO42-) bind 10-20 water molecules strongly but leaves the bulk rather unperturbed.

Anionic chaotropes affect bulk properties

Koga et al. 2004, Westh et al. 2006

Naturally occurring inorganic osmolytes are kosmotropes

effects of additives alcohols
Effects of additives: alcohols

Small alcohols do not, however, universally destabilize the protein

Td vs. [alcohol]


At 10C ethanol stabilizes lysozyme up to ~4M

At 40C ethanol is neutral up to ~1M

Propanol strongly reduces Td for Lysozyme

Velichelebi and Sturtevant 1981

cutinase stabilizers and destabilizers
Cutinase: Stabilizers and destabilizers

Calorimetric and neutron scattering studies

provide a ”state diagram” for cutinase in SDS

Effetcs of a ”traditional” stabilizer: Trehalose

Babtista et al Biopolymers 89 538 (2008)

Nielsen et al 2006, Nielsen J. Phys Chem B 111 2941 (2007)

preferential interactions rigorous approach
Preferential interactions: Rigorous approach

Preferential Interaction parameter3  (m3/m2)T,P,3

Gm3 is the number of excipient molecules, which has to be added to re-establish its chemical potential (activity) upon the addition of one protein molecule to the system.

3 > 0 : preferential binding

3 < 0 : preferential exclusion

linkage equations
Linkage equations

For non-specific interactions:

The effect of changing the logarithm of the activity (or roughly, the logarithm of the concentration) of any excipient on the standard free energy of protein unfolding is proportional to the adsorption of the excipient on the protein’s surface (the preferential interaction parameter) times the surface area change caused by unfolding.

Wyman & Gill, Binding and Linkage, 1990

an interfacial view
An interfacial view

Gibbs adsorption equation (The surface excess)

  • Osmotic gradient
  • Smoother interface
  • Tighter molecular packing
excipients and the n d transition closing remarks
Excipients and the ND transition: closing remarks
  • Any additive stabilizes the conformation with which it has the strongest interaction (LeChatelier’s principle)
  • This may be rationalized and quantified through the theory of preferential interactions and linkage theory
  • We do not know any reliable procedure to predict the preferential interaction of a protein-solute pair on the basis of their chemical structures
  • We do, however, have many empirical guide lines for ”semi-rational” formuation – for example the Hofmeister series or naturally occurring osmolytes such as sorbitol or trehalose.
irreversible transitions
Irreversible transitions
  • Definition: Transition far from equilibrium – in some cases it may in fact be reversed by changing the conditions.
  • In most cases, avoiding irreversible transitions is the real challenge in protein formulation.
  • In the simplest (Lumry-Eyring) picture
  • The kinetic stability is strongly liked to the equilibrium stability.
  • Irreversible transitions are both experimentally and theoretically less stringently described


conformational kinetic and colloidal stability
Conformational, kinetic and colloidal stability

N  D  I

  • Kinetic stability.
  • Common decay routes
    • Aggregate (Amorphous, Fibrils, amyloid plaques, inclusion bodies etc)
    • Adsorbed (to solid surface or inpurity)
    • Scrambled structures (stable intermediates)
    • Covalent variants (S-S rearrangements, deamidation, backbone hydrolysis)
    • Apoproteins (diffusive loss of prostetic groups)

Thermodynamic stability

Colloidal stability

As seen by DSC

aggregates amyloids inclusions fibrils
Aggregates, amyloids, inclusions, fibrils

Jahn & Radford 2004

Separate mechanisms or a

universal propensity of polypeptides to

form intermolecular b-structure?

stability of colloids
Stability of Colloids
  • Two main stabilizing effects counteracts the drive towards lower surface area:
  • Steric 2. Electrostatic

Kinetically stable dispersion of two phase system – emulsion, foam, suspension, aerosol etc.

electrostatics the double layer
Electrostatics: the double layer

The simplest case: a plane surface (negatively charged)

Interactions between two particles

For a particle (e.g. a protein)

ionic strength and the double layer
Ionic strength and the double layer

Salts may strongly modulate the stability of liquid protein formulations – but not always in a bad way !

in addition there are preferential solute interactions
In addition, there are preferential solute interactions

For ”non-specific” interactions, G scales with the surface area.

DG~0 DG<0

N  D  Ag

Do stabilizers promote aggregation ?

Do denaturants promote shelf-life?

Effect of typical stabilizer such as Trehalose or SO42-

Are these (equilibrium) considerations relevant to a kinetically controlled process ?

Typical destabilizer such as Urea or ClO4-

reversibel precipitation
Reversibel precipitation

Urea and the solubility of apo-myoglobin

  • DeYoung et al. 1993

N(aq) D  Ag

Low urea

High urea

transition state picture
Transition state picture

Conceptual scheme (if D is reasonably populated):

N  D  D Ag

Eyring 1935

Bagger 2007

Rate governed by the concentration [D]

Equlibrium constant: K= [D]/[D]

Solute effects on aggregation kinetics depends on relative size of DG and DG

TS theory and protein aggregation

Chi et al. 2003

Baines & Trout 2004

irreversible denaturation of a amylase
Irreversible denaturation of a-Amylase

Thermal stability of amylase and apo-amylase


B-aA Ca3

B-aA Ca

Nielsen et al 2003a

titration calorimetry and ion stripping








Heat flow (



Calcium removal




Time (min)


























Titration calorimetry and ion-stripping

Isothermal titration calorimetry detects the heat of reaction when a small amount of titrand is added to a calorimetric (stirred) cell

Addition of EDTA to amylase at 60°C


a dual effect
A ”dual effect” ?

Aggregation of a-amylase at 60C

  • Eronina et al. 2005

”Additives which stabilize the N-state of glucagon phosphorylase also promotes the protein’s aggregation”

dual effect urea a amylase
Dual effect: Urea-aAmylase

N  D  Ag


D form accumulates in Urea solutions

Urea promotes accumulation of the D-form in accordance with its lyotropic properties

Buffer, glycerol or


[D]~0 in buffer or solutions of stabilizers

how important are hofmeister effects on the irreversible step
How important are Hofmeister effects on the irreversible step?
  • “Dual effects” have be detected
  • But
  • many studies have reported:
  • “kosmotrope ~ kinetic stability”
  • and
  • “chaotrope ~ kinetic instability”
  • Are Hofmeister effects important compared to electrostatic interactions between proteins?

Almost negligible !!!

thermal aggregation of bovine serum albumin
Thermal aggregation of bovine serum albumin

BSA forms “-aggregates” in which a moderate part of the native helices are converted into intermolecular -sheets.Militello et al. 2004

Time course of aggregation at Tm


Real-time SLS

  • Lag phase.
  • “1st order kinetics”
  • All solutes appear to retard aggregation. Salts are stronger inhibitors.
  • No correlation to “Hofmeister effects” (Urea~sorbitol and SCN-~SO42-).




Bagger et al, 2007

sec malls ri analysis of quenched samples


SEC-MALLS-RI analysis of quenched samples

MALLS-Signal proportional to


RI and UV Signals proportional to Cm

30 min at Tm (buffer and 0.5 M sucrose)

Reference (unheated)

Ratio of MALLS:RI signals is a measure of Mw

aggregate properties after hr at t m
Aggregate properties after ½hr at Tm

SEC-MALLS-RI analysis of BSA thermal aggregation

Fraction aggregated

Size BSA/particle

Particle concentration



Many small aggregate particles – no net effect on ”life-time”.


Few and small particles – promotes kinetic stability.

No ”Hofmeister-effects” in either case.






summary bsa aggregation
Summary – BSA aggregation:

Hofmeister effect – preferential interactions – are not important for the rate of aggregation

Salts promote stability ????

Electrolyte efffects generally relate to lower concentrations !

Preferential interactions ~0.2-2 M

Electric double layer ~ 0.01 – 100 mM

sec malls a amylase
SEC-MALLS : a-Amylase

SEC-MALS measurements of a-Amylase aggregation in 5mM

HEPES buffer, pH 8, 60C. LS signal (@90) is shown in panel A,

UV signal panel B.

electrolyte additives and a amylase
Electrolyte additives and a-amylase

Colloidal stability (pH 8.0, 60C) of a-amylase (net charge -10)

5mM HEPES + 10 mM NaCl

5mM HEPES + 5 mM NaCl


Aggregate size


 NaCl

□ KCl




Aggregate Concentration (nM)



 NaCl

□ KCl



CONSTANT: adsorption to existing particles.

electrolyte additives and a amylase1
Electrolyte additives and a-amylase

Colloidal stability (pH 9.0, 60C) of a-amylase (net charge -16)

Monovalent counterion (Na+)

5 mM buffer (Na-Borate)

buffer + 10 mM NaCl

buffer + 20 mM NaCl and buffer + 40 mM

Trivalent counterion (Co3+)

buffer + 10 mM Co[(NH3)6]Cl3

20 and 30 mM Co[(NH3)6]Cl3

60 and 120 mM Co[(NH3)6]Cl3

1st or 2nd order kinetics ?

Buffer alone

40 mM NaCl

120 mM Co[(NH3)6]Cl3

critical electrolyte concentration
Critical Electrolyte Concentration

Evans & Wennerstrøm, The Colloidal Domain 1999

At CCC the potential barrier is negligible – further addition of salt will not accelerate the aggregation.

Note counter-ion valens to the 6th !!

Hence 700 x increase from +1 to +3 – We see ca 400 X

1st or 2nd order kinetics
1st or 2nd order kinetics?

The time course suggested 1st order when salt was added and 2nd order in pure buffer.

This is confirmed in rate vs. protein concentration measurements

does salt change the path or the product
Does salt change the path or the product?

2nd derivative FTIR spectrum of native and aggregated BHA with and without added salt

Low salt




High salt

Intermolecular b-sheet

a helix

salt induced change in kinetic order
Salt induced change in kinetic order

2nd order (UU complex)

1st order (N is rate limiting)

mlvo theory and a amylase aggregation
MLVO theory and a-amylase aggregation

High salt: 1st order

Production of U from N is rate limiting

Experimentallyobservedlevel in 5 mMBorate, pH 9.0

Low salt : 2nd order

electrolytes and aggregation a hypothesis
Electrolytes and aggregation: A hypothesis
  • N  U
  • Low charge density (weak coupling)
  • hydration of ions – ”Hofmeister effects”
  • Type of ion is central – type of protein is unimportant
  • U  Ag
  • High charge density (strong coupling)
  • Columbic forces –”polyelectrolyte effects§”
  • Type of protein is central – type of ion is unimportant (except valance)

§Record et al. 1991, 1998

a comment
A comment
  • Kosmotropes such as sugar alcohols are often useful stabilizing excipients.
  • This is primarily due to their stabilization of the N state and hence reduction of the ”reactant” concentration for the second irreversible step.
different effects of electrolyte additives a hypothesis
Different effects of electrolyte additives:A hypothesis

Weakly bipolar proteins (even charge distribution):

Coulombic protein-protein interactions promotes colloidal stability – conventional colloid stability (salts promote aggregation).


Strongly bipolar proteins:

Coulombic protein-protein interactions drives proteins together – salts weaken this initial step of aggregation.






Serum albumin

  • Lars Øgendal, Life Sci. Univ. Of Copenhagen
  • Yoshikata Koga, UBC Vancouver Canada
  • Lise Arhlet, Life Sci. Univ. Of Copenhagen
  • John Carpenter, Denver, USA
  • Kim Andersen, Novozymes A/S, Denmark
  • Kim Borch, Novozymes A/S, Denmark
  • Phd students
  • Søren Olsen
  • Heidi Bagger
  • Anders Nielsen