Excipient effects on the stuctural and colloidal stability of proteins A rational approach to the formulation of protein

1. 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 (pwesth@ruc.dk)

2. 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.

3. Counteracting the physical instability • Some main strategies: • Solvent based stabilization (excipients) • Modifications (PEG, Glycans etc.) • Mutations (site directed mutagenesis)

4. Stabilization of Pharmaceutical Proteins • Stabilization against which transition? • Reversible unfolding or irreversible transitions • Lumry-Eyring model (Lumry and Eyring 1954) NDI 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

5. 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 Tm=80°C Tm<80°C Tm<80°C

6. 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) Fundamentals: Constant P: qp=DH=CpDT

7. 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.

8. The use of Kirchkoff´s equation on DSC (and other) data lead to counterintuitive results! Proteins denature both upon heating and cooling

9. Effects of solute additives (or excipients) • on the ND equilibrium

10. 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

11. 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

12. Preferential Interactions • Preferential Binding: Excipient-protein interactions are stronger than water-protein interactions. • Preferential exclusion: Water-protein interactions are stronger than excipient-protein interactions. • FAVORABLE INTERACTION=LOWER FREE ENERGY! m°Prot Preferentially excl. excipient No Excipient Preferentially bound excipient D D DG° N N DG° D N DG° Stabilization Destabilization

13. Hofmeister effects and the Lyotropic Series Kosmotropes Chaotropes sucrose sorbitol betaine Glycine Urea 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

14. Bound and excluded additives Timasheff et al.

15. Effects of additives: alcohols Small alcohols do not, however, universally destabilize the protein Td vs. [alcohol] Conclusions 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

16. 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)

17. 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

18. 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

19. An interfacial view Gibbs adsorption equation (The surface excess) • Osmotic gradient • Smoother interface • Tighter molecular packing

20. 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.

21. Effects of solute additives on the irreversible UI transition

22. 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 NDI

23. 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

24. Aggregates, amyloids, inclusions, fibrils Jahn & Radford 2004 Separate mechanisms or a universal propensity of polypeptides to form intermolecular b-structure?

25. 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.

26. Electrostatics: the double layer The simplest case: a plane surface (negatively charged) Interactions between two particles For a particle (e.g. a protein)

27. Ionic strength and the double layer Salts may strongly modulate the stability of liquid protein formulations – but not always in a bad way !

28. 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-

29. Reversibel precipitation Urea and the solubility of apo-myoglobin • DeYoung et al. 1993 N(aq) D  Ag  Low urea  High urea

30. 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

31. Irreversible denaturation of a-Amylase Thermal stability of amylase and apo-amylase Bacillusa-Amylase B-aA Ca3 B-aA Ca Nielsen et al 2003a

32. o 60 C 100 J/sec) 50 m Denaturation Heat flow ( Injection 0 Calcium removal 0 30 60 Time (min) D q [ ] - = × D × × × kt Heat flow : k H V P e 0 den cell D t 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 Ca-stripping.

33. 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”

34. Dual effect: Urea-aAmylase N  D  Ag urea D form accumulates in Urea solutions Urea promotes accumulation of the D-form in accordance with its lyotropic properties Buffer, glycerol or betaine [D]~0 in buffer or solutions of stabilizers

35. 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 !!!

36. 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 ([N]=[D]=16mM) 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-). buffer Non-elec-trolytes salts Bagger et al, 2007

37. MALLS RI MALLS RI SEC-MALLS-RI analysis of quenched samples MALLS-Signal proportional to CmMw 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

38. Aggregate properties after ½hr at Tm SEC-MALLS-RI analysis of BSA thermal aggregation Fraction aggregated Size BSA/particle Particle concentration EFFECTS OF ADDITIVES ON BSA AGGREGATION Non-electrolytes: Many small aggregate particles – no net effect on ”life-time”. Electrolytes: Few and small particles – promotes kinetic stability. No ”Hofmeister-effects” in either case. Sucrose Sorbitol Urea NaSCN Na2SO4

39. 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

40. 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.

41. 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 5mM HEPES Aggregate size HEPES+ 10mM  NaCl □ KCl  NaCH3COO HEPES+ 5mM HEPES Aggregate Concentration (nM) HEPES HEPES+ 5mM  NaCl □ KCl  NaCH3COO HEPES+ 10mM CONSTANT: adsorption to existing particles.

42. 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

43. 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

44. 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

45. Does salt change the path or the product? 2nd derivative FTIR spectrum of native and aggregated BHA with and without added salt Low salt D1 N D2 High salt Intermolecular b-sheet a helix

46. Salt induced change in kinetic order 2nd order (UU complex) 1st order (N is rate limiting)

47. 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

48. 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

49. 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.

50. 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). ”MLVO-behavior” Strongly bipolar proteins: Coulombic protein-protein interactions drives proteins together – salts weaken this initial step of aggregation. ”ANTI-MLVO” E.g. a-amylase Phytase E.g. Serum albumin