Using sequence analysis and design to mitigate formulation concerns during pre-clinical development

Transcription

1 Using sequence analysis and design to mitigate formulation concerns during pre-clinical development Margaret Ricci, Riki Stevenson, Holly Huang, Francis Kinderman, Heather Franey, Stephen Brych, Xichdao Nguyen, Jie Wen, Cynthia Li, Randal R. Ketchem, Guna Kannan, Nicolas Angell, Yijia Jiang, Linda Narhi Process and Product Development, Amgen Inc. CaSSS July Forum NIH, Bethesda July 16, 12

2 Outline Overview of molecule screening & formulation design Case studies Solubility Viscosity Post-translational effects Formulation approaches Take-home messages 2

3 Molecule attributes that can impact process development & product formulation In vivo stability Structural stability Transport stability Chemical stability Molecule assessment Photostability Process compatibility Viscosity

4 Case study #1: Solubility Leptin formulation delivery challenges High doses required Low solubility at neutral ph 80 Injection site reactions 70 Formulations mg/ml 50 ph Solubility at neutral ph 30 Human leptin: 2-3 mg/ml 10 Murine leptin: 43 mg/ml Solubility (mg/ml) ph Reversibility

5 Murine leptin provided blueprint for sequence-dependent solubility HUMAN: MVPIQKVQDD TKTLIKTIVT RINDISHTQS VSSKQKVTGL DFIPGLHPIL MURINE: MVPIQKVQDD TKTLIKTIVT RINDISHTQS VSAKQRVTGL DFIPGLHPIL HUMAN: TLSKMDQTLA VYQQILTSMP SRNVIQISND LENLRDLLHV LAFSKSCHLP MURINE: SLSKMDQTLA VYQQVLTSLP SQNVLQIAND LENLRDLLHL LAFSKSCSLP HUMAN: WASGLETLDS LGGVLEASGY STEVVALSRL QGSLQDMLWQ LDLSPGC MURINE: QTSGLQKPES LDGVLEASLY STEVVALSRL QGSLQDILQQ LDVSPEC % Precipitation Final Concentration (mg/ml)

6 SOLUBILITY (mg/ml) Solubility analogs of leptin Murine leptin Over 100 analogs tested (in PBS, ph7 room temperature) Analogs 3X more soluble than murine leptin Analogs >50X more soluble than wild-type human leptin Human leptin 0

7 Tryptophan residues were critical to improving solubility Solubility (mg/ml) W101Q alone +W139Q alone +W101Q +W139Q -W101Q -W139Q Number of mutations Limited solubility at neutral ph is not due simply to piprecipitation or net hydrophobicity, but rather results from irreversible self-association involving specific residues

8 Case study #2: viscosity Sequence-dependent viscosity for mabs above 70 mg/ml 8

9 Molecule approaches Screening and selecting molecules with lower viscosity Exhibits extreme solubility and viscosity issues Not soluble in buffer above ~5 mg/ml (right) Highly viscous (observed, not measurable) A molecular model of the variable domain was generated using multiple structures from the PDB, structural CDR grafting and energy minimization 9

10 Applied molecular modeling to select Screening and selecting molecules with lower viscosity in MA Majority of the surface charge is predicted to be clustered in one region in the three dimensional structure. Two mutants were designed 1) Removed the patch of surface charge & replaced with germline residues 2) Lower pi 10

11 Mutations specific to charge patch area were effective in improving solubility and reducing viscosity 160 Maximum Solubility 35 Viscosity at ~150 mg/ml parental mab pi mutant surface charge mutant low viscosity mab surface charge mutant high viscosity mab A non-specific pi increasing mutant only slightly increased solution solubility (to ~10 mg/ml), whereas surface charge mutant improved solubility significantly (~150 mg/ml). Removal of the surface charge patch significantly reduced the viscosity, although on the high end compared to low/high viscosity mab controls Reducing surface charge within charge patch region enabling high concentration formulation but with reduced bioactivity 11

12 Viscosity (cp) Electronegative charge patch can contribute to high viscosity Electronegative Hydrophobic Parent parental Mutant mutant Parental mab with electronegative patch exhibited high viscosity. A point mutation was suggested by an in-house focused germlining method (basic to neutral amino acid substitution). A significant viscosity reduction was unexpectedly achieved and the thermal stability was maintained. 12

13 Possible dipole moment effect Mutation The point mutation altered the dipole moment and zeta potential Electronegative patch was likely not the only contributor to viscosity for this molecule 13

14 Viscosity (cp) Framework sequence can contribute to high viscosity parental mutant Mutations in framework residues reduced the viscosity 5 0 parental mutant 14

15 Case study 3: Post-translational effects Effect of glycosylation on solubility & stability Mab candidate with extra N-linked glycosylation Unknown effect of extra glycosylation on formulation stability and manufacturability Engineer mab analog missing 2 nd N-linked glycosylation for comparison Assess conformational & stability attributes Biophysical properties Conformation Particle size Thermal stability Acid ph-induced unfolding Self-association Analytics CD FTIR Fluorescence Raman spectroscopy DSC AUC Dynamic light scatte 15

16 Cp(cal/ o C) Effect on thermal stability monitored by DSC The Tm of the protein at neutral ph (citrate) is 70ºC, as is expected for a native, folded mab Analog Red: AMG 811 Analog (C7N) Parental Black: AMG 811 (C7N) However, the Tm of the analog is about 1 C lower & irreversible, and the protein precipitates immediately after melting Temperature ( o C) 16

17 Rh, nm Polydispersity (%) Effect on self-association monitored by dynamic light scattering 11 Hydrodynamic radius 70 Polydispersity 10 AMG Parental Parental AMG 811a 9 AMG Analog 811a 50 Analog AMG ph ph Removal of the extra N-glycosylation site resulted in a less soluble protein with greater tendency to self-associate 17

18 Cp(cal/ o C) Effect of redox refolding and mutation on stability V L C L V H C H 2 N 298 glycan C H 3 unpaired Cys C H 1 C 22 C 23 C 96 C 104 C 89 C 145 C 1 C 134 C 221 C 194 C 227 C 214 C 262 C 230 C 368 N 298 glycan AMG control refolded 76.4 o C CHO 70.6 o C Black: FPB Red: 16-hour refolding 66 o C 82.9 o C Blue: 40-hour refolding Redox refolding and mutation cause thermal transition shift from 70.6 C to 76.4 C, indicative of significantly increased stability of refolded material Narrow and symmetric DSC transitions of mutant suggest refolding improves the homogeneity Redox refolding and mutation are equally effective CHO mutation AMG 714 C104S o C 67 o C 82.9 o C Temperature ( o C)

19 Viscosity (cp) Viscosity (cp) Case study 4: Formulation approaches Reducing solution viscosity through excipients Example 1: calcium acetate Example 2: proline no calcium acetate 10mM calcium acetate No proline 3% Proline Concentration (mg/ml) Concentration (mg/ml) Before After Mitigate charge patch mediated viscosity by excipients No 'one size fits all' Other formulation parameters include ph control 19

20 Viscosity (cp) Feed Pressure (psi) Viscosity modifiers and temperature can be used to achieve high concentrations mM Ca 10mM Ca 15mM Ca mm Ca 23mM Ca AMG Protein 785 Concentration (mg/ml) Temperature 23C 40C Retentate Concentration (mg/ml) Excipient addition, high temperature operation, and lower cross-flow rate were required to achieve 1.5X overconcentration

21 Mitigation of challenges in delivering stable formulations MOLECULE FORMULATION Select/Design molecules with improved properties Optimize formulation to mitigate protein interactions & instabilities PROCESS DEVICE Utilize new technologies to manufacture high concentration formulations Implement device technologies to deliver dose in acceptable form Effective strategy requires coordination across molecule, formulation, process and device work streams 21

22 Take-home messages Found clues in nature to sequence-dependent solubility Implemented screening assays to select low viscosity molecules Applied molecular modeling to select favorable sequence attributes & eliminated localized charge patches Reengineered molecules for improved in vitro and in vivo stability Demonstrated that engineering the molecule for desired solution properties is an effective approach to mitigate formulation concerns in pre-clinical development Applied molecule selection principles in conjunction with formulation, process, and delivery device strategies 22

23 Acknowledgements Margaret Karow Taruna Arora Rutilio Clark Rick Jacobsen Twinkle Christian Chris Clogston David Le Elaine Smith Tim Osslund Amgen for funding these studies 23