Ten Lessons for the Formulation Development of Monoclonal Antibodies from Multimodal Thermal Unfolding Case Studies

Transcription

1 Ten Lessons for the Formulation Development of Monoclonal Antibodies from Multimodal Thermal Unfolding Case Studies Mark Brader Protein Pharmaceutical Development Biogen Idec PEGS Boston essential protein engineering summit May 4-5, 2015

2 Methodology Differential Scanning Calorimetry F ab C p (cal/ o C) C H 2 C H 3

3 Methodology Optim1000 simultaneous fluorescence and light scattering Thermal ramp Intrinsic fluorescence Static light scattering unfolding transition temperature (T m ) aggregation onset temperature (T agg )

4 Methodology DSC + Intrinsic Fluorescence (F350/330) As the first domain unfolds a transition is detected by the fluorescence change

5 Methodology DSC + Static Light Scattering As the second domain unfolds the protein aggregates

6 Methodology DSC SLS F 350/330 Superimposed mab7 screening buffer Fl 350/330 SLS DSC Initiation of fluorescence change coincides with unfolding of first domain Initiation of aggregation coincides with unfolding of second domain

7 In a simple world, where all proteins behave the same, and conventional wisdom applies well to mab formulation development

8 the everything is awesome scenario applies DSC F350/330 Screening buffer C P (cal/mole/ o C) F350/330 SLS SLS Formulated o

9 But unfortunately in the real world of protein product development it s a complicated world and everything is not always awesome

10 #1 A wide range of thermal unfolding profiles and conformational stabilities are observed for developable molecules

11 DSC profiles of 8 developable mabs in citrate buffer C P (cal/mole/ o C) T m (1) range T onset range Large Fab T m range

12 #2 Thermal shifts associated with the first unfolding transition may not correlate with improved pharmaceutical stability

13 DT m ( o C) DT m ( o C) DT m ( o C) Thermal shifts of T m (1) citrate buffer pharmaceutically optimized formulations 6 4 T m (1) DSC 10 8 T onset DSC mab1 mab2 mab3 mab4 mab5 mab6 mab7 mab8-8 mab1 mab2 mab3 mab4 mab5 mab6 mab7 mab8 7 T m (1) F 350/ mab1 mab2 mab3 mab4 mab5 mab6 mab7 mab8

14 #3 Improving thermal stability of the Fab domains may represent a better general formulation strategy than the more common approach of focusing on the first unfolding transition

15 DT m ( o C) Thermal shifts of T m (Fab) citrate buffer pharmaceutically optimized formulations (d) Shifts in Fab T m 6 of 8 mabs show clear positive shifts

16 C P (kcal/mole/ C) C P (kcal/mole/ C) mab2 formulated under conditions that greatly reduced Tm(1) but interestingly Fab Tm did not change importance of the Fab 66.5(±1.0) 73.5(±0.1) 82.3(±0.1) (±0.8) Temperature ( C) 73.5(±0.3) 82.7(±0.2) Temperature ( C) Cp(Y) CpBase(Y) CpFit(Y) CpPk1 CpPk2 CpPk3 Cp(Y) CpBase(Y) CpFit(Y) CpPk1 CpPk2 CpPk3

17 #4 Unusual or anomalous spectroscopic signatures do not necessarily translate into poor pharmaceutical stability

18 Intrinsic fluorescence profiles of 8 mabs in citrate buffer mab1 mab2 mab3 mab5 mab6 F350/330 F350/330 F350/330 F350/330 F350/330 F350/330 mab4 mab7 mab8 F350/330 F350/330

19 mab4 s unusual fluorescence transitions blue shift pre-unfolding transition red shift

20 % Bioactivity mab4 s unusual fluorescence transitions blue shift red shift pre-unfolding transition % Bioactivity after 6 months at 25 o C and after 3 months at 40 o C Excellent stability

21 mab4 s unusual fluorescence transitions blue shift red shift pre-unfolding transition % Aggregate by SE-HPLC 40 o C storage 25 % Bioactivity after 6 months at 25 o C mab1 and after 3 months at 40 o C % Aggregates by SE-HPLC mab2 mab3 mab4 mab5 mab6 mab7 mab8 5 Excellent stability Time (months)

22 #5 T agg does not appear to be a sensitive general indicator of improved formulation stability

23 DT m ( o C) Thermal shifts of T agg by SLS citrate buffer pharmaceutically optimized formulation mab1 mab2 mab3 mab4 mab5 mab6 mab7 mab8

24 #6 Formulation conditions can alter the cooperativity of domain unfolding

25 Formulation conditions can alter the cooperativity of domain unfolding C P (cal/mole/ o C) citrate buffer C P (cal/mole/ o C) pharmaceutically optimized formulation

26 A formulation strategy focused on the first Tm would miss the improved stabilization conferred by this formulation 63.3(±0.1) 70.6(±0.5) 82.6(±0.1) C P (cal/mole/ o C) citrate buffer 60.1(±0.2) 66.2(±0.1) 71.8(±0.1) 82.7(±0.1) C P (cal/mole/ o C) pharmaceutically optimized formulation

27 #7 & #8 Aggregation can be driven by different domains. The least conformationally stable domain is not necessarily the most aggregation-prone Formulation conditions can alter which domain drives aggregation

28 SLS reveals which domain mediates aggregation Formulation conditions can alter which domain drives aggregation mab7 mab4 citrate buffer Second domain aggregates First domain aggregates mab4 formulated formulation optimization Second domain aggregates

29 #9 Stability ranking at high temperature may not translate to pharmaceutically relevant temperatures

30 Poor correlation between Tm(1) and storage stability at 25 o C T m (1) by DSC versus aggregation rate by SE-HPLC at 25 o C for optimized formulations aggregation rate by SE-HPLC (%/month) T m (1) ( o C)

31 Pharmaceutical stability data increase in aggregation by SE-HPLC these developable mabs had <3.6%/month aggregation at 40 o C aggregation kinetics at 5 o C may be non-linear whereas aggregation kinetics at 25 and 40 o C are linear

32 #10 Prediction of low temperature stability from high temperature stability is unreliable

33 Simultaneous Multiple Sample Light Scattering monitoring of mab aggregation Wayne Reed, Tulane University Drenski et al. Analytical Biochemistry 437 (2013)

34 Conclusions Significant diversity in the thermal unfolding profiles mabs possessing a wide range of conformational stabilities and thermal unfolding profiles can be formulated into stable scalable products Formulation conditions shift thermal unfolding transitions Not all excipients confer pharmaceutical stability by increasing conformational stability Consideration of first transitions only can be misleading Stabilization of Fab appears to be more important than stabilization of the first transition

35 Acknowledgments Tia Estey Shujun Bai Roy Alston Karin Lucas Steve Lantz Kevin Maloney Pavel Landsman Reference Examination of Thermal Unfolding and Aggregation Profiles of a Series of Developable Therapeutic Monoclonal Antibodies Mark L. Brader,* Tia Estey, Shujun Bai, Roy W. Alston, Karin K. Lucas, Steven Lantz, Pavel Landsman, Kevin M. Maloney Molecular Pharmaceutics 2015, 12,