#1Biophysical characterization of excipient combinations for mAb formulation development Subhashchandra Naik, PhD PEPTALK 23 January 18, 2023 .)< Comera LIFE SCIENCES#2Content ● ● ● ● Need for and problems with Subcutaneous biologic administration Comera SQore TM platform technology Identifying and characterizing excipients Computational screening ● New stabilizing excipients Experimental validation. Case studies on formulation development of mAb A Formulation Buffer screening ● ● • Viscosity and stability optimization ● DOE for formulation optimization ● Conclusions Comera LIFE SCIENCES 2#3Conversion of IV to SQ Administration ● IV Pain and discomfort Time commitment Risk of infection Reduced compliance Space and nursing time requirements Increased costs ● ● ● MIN (<) Comera LIFE SCIENCES SQ Less pain, less time Reduced risk of infection Potential self- administration Higher patient satisfaction. Improved quality of life. Reduced cost 3#4Developing SubQ mAb formulations is challenging Viscosity (CP) High Low Monomeric Concentration (mg/ml) TH Network tein-protein interactions (P High Increase in Viscosity Decrease in Stability PPI Comera LIFE SCIENCES 4#5SQore platform helps in IV to SQ Conversion ● >200 EXCIPIENT COMPOUNDS ● • SQore excipients SQore ● + Viscosity reducers • Stabilizers ● PROPRIETARY EXCIPIENT ENGINEERING They are known chemical structures They have well established toxicology profiles PPI High Concentration and high PPI (High viscosity) H3C SQore™ CH3 CH3 Caffeine (1,3,7-trimethylxanthine) CAS # [58-08-2] MW 194.19 g/mol Comera LIFE SCIENCES High Concentration and Low PPI (Low viscosity) 5#6Developing subQ mAb formulations using SQore platform DSF ELISA BLI Octet Light scattering Biophysical studies (CD, UV, FL) LC SQore™ In silico studies Experimental work Validation Final Formulation Comera LIFE SCIENCES 6#7Computational studies to identify viscosity hotspots. Protein-protein and protein-excipient blind docking helps identify interaction hotspots and the residues involved. Results overlaid with the protein ligand docking to identify excipient binding sites, interfering with mAb self association Infliximab fab fragment (pdb ID:5VH3) In silico protein protein interaction Comera LIFE SCIENCES In silico protein ligand interaction Excipient interfering with residues involved in mAb self association 7#8Identifying screening sites and desired pharmacophores. Surface Hydrophobicity Surface charge Computational studies allows 1.Identifying self interaction hotspots in mAbs 2.Identifying excipients that decrease self association 3.Identify excipients that can stabilize the mAb Identifying sites involved with Self interaction Identify single interaction site and Screen SQore library within the pocket Pharmacophore Results Name RMSD Hass Rinds 0 2 2 4 4 374 MalPort-002-964-400 0.450 Mofort-839-455-121 0.611 265 MolFort-039-455-121 9013 265 Moort-001-771-995 0.766 274 MolFort-001-771-998 0.756 276 MoPort-001-771-999 0.760 Mort-001-771-998 0.766 Port 002-771-908 0.706 MaPort-001-771-998 0.766 HaPort-001-771-998 0.756 MolFort-001-771-998 0.766 MalPort-020-138-470 0.720 MoPort-046-304-15 0.710 MuPort-002-657-242 0.739 MyPort-002-857-242 0.739 MoPort-002-857-242 0.739 342 MaPort-002-657-242 MaPort-030-230-707 342 343 MoPort-039-330-797 0.605 343 MoPort-029-230-707 9.609 343 224 114 376 342 Showing 1 to 20 of 6 h Comera LIFE SCIENCES 1 2 3 4 Next 4 4 *M 4 4 4 5 5 5 5 5 5 3 3 3 Ranked screening results 8#9MD simulation data of protein excipient interaction 10 ns time scale Excipient Protein Ligand RMSD (nm) 0.8 06 04 02 RMSD (nm) RMSD Hackbone after hq fit to Backbone 4 Time (m) RMSD LIG after sq fit to Protein уни ду мучилист 4 6 Time (ts) 10 10 Number 340 300- 310 300 280 05- 10 9 20000 2000 Hydrogen Bonds 4x8 4000 Time (4) Time (ps) 10X00 10000 8000 Simulation data allows to identify regions 1. 2. 3. 4. 5. Comera LIFE SCIENCES Where the excipient binds How the excipient binds Strength of binding Interaction time Residues it can interact with MD Simulation gives a better predictive power and more reliable analysis of protein-ligand dynamics and improve screening results 9#10Screening and MD simulation with Mab A DSF ELISA BLI Octet Light scattering Biophysical studies (CD, UV, FL) Excipients CS1 CS2 CS3 CS4 CS5 LC FDA Inactive Ingredient Yes No No Yes Yes SQore USP / GMP Yes Yes Yes Yes Yes Prior injectable use Yes No No No Yes Computational GRAS Yes Yes Yes Yes Yes Comera LIFE SCIENCES 10#11Effect of stabilizers on mAb thermal stability Ellipticity change 2 Ellipticity change 1 0 -1 35 7 6 5 0 -1 Nivolimumab Thermal Melt -Ctrl -CS1 CS4 CS3 -Sucrose <-CS1 <-Cs3 -CS4 <-Suc 45 control 20 Temp (C) 75 pembrolizumab Thermal Melt Temp (C) 80 85 100 Ellipticity change 8 Normalized Ellipticity 7 6 NHO5 -1 20 15 10 UT 5 -10 - Ctri <-CS4 -CS1 20 CS3 -Sucrose ctrl CS1 -CS3 -CS4 30 Trastuzumab Thermal Melt 45 55 65 Temp (C) 40 Ustekinumab CD thermal melt 50 60 Temp (C) ^ 75 70 85 80 95 90 ● ● Stabilizers validated with CD thermal melt Comera LIFE SCIENCES mAb conc: 0.2 mg/ml mAb Stabilizer conc: 20 mM Temp ramp 1C/ min Novel stabilizers improve the thermal stability by shifting melting temp Thus they appear to be similar or better than sucrose in stabilizing mAbs against thermal stress 11#12Effect of stabilizers on Isothermal stability of mAb ● ● ● ● % monomer remaining 100 95 90 85 BO 75 70 65 60 allill CS1 No stabilizer CS3 Pembrolizumab 4 week monomer loss study 5 mg/ml mAb C54 Stabilizers saccharide conc: 0.2M Sucrose Treh CS2 MT4 % monomer remaining 100 95 90 85 80 75 70 65 60 Control CS3 Nivolimumab 4 week Monomer loss study MI Sucrose CS1 mAbs were formulated with the stabilizers and isothermally incubated at 40C for 4 wk CS4 Stabilizers All stabilizers were effective in improving thermal stability as compared to no stabilizer control Treh CS2 T4 Comera LIFE SCIENCES 12#13Effect of stabilizers on Freeze thaw stability of mAbs 1000000 100000 10000 1000 100 10 1 Ctrl Nivolimumab FT with stabilizers CS3 Treh 100000 10000 1000 100 10 1 Suc Ctrl particles/ml ■2-10 nm 10-20 nm ■20-50 nm CS3 1000000 CS1 100000 10000 1000 100 10 1 Pembrolizimumab FT with stabilizers CTRL Treh Suc Treh HGG FT with stabilizers CS1 Stabilizer particles/mil ■2-10 nm 10-20 nm CS4 ■ 20-50mm CS3 Sucr total 2-10um >10um >25um ● Comera LIFE SCIENCES mAb conc: 10 mg/ml saccharide conc: 0.2M 10 FT cycles Samples analyzed by flowcam Stabilizers showed effectiveness in preventing particulate formation due to freeze thaw stress 13#14● ● ● Binding kinetics and mechanism of stabilizers Interaction of stabilizers with mAbs was investigated using BLI octet Infliximab was biotinylated and loaded onto SSA tips. Binding kinetics determined by direct interaction with up to 1M stabilizers Kd calculated as 5.7 m M for CS1 and 6.9 for CS4 (44) Supay #16 At 412 KO ** 11 Se 400 mM CS1 200 mM CS1 100 mM CS1 800 mM CS4 400 mM CS4 200 mM CS4 100 mM CS4 50 mM CS4 Some CS1 binding kinetics Firmwa CS4 binding kinetics Se 1 S Comera LIFE SCIENCES 14#15)< Comera LIFE SCIENCES Case Study Developing an optimal formulation for mAb A using combination of excipients 15#16mAb liquid formulation development: Buffer screening Rh (nm) 50 45 40 35 30 25 20 15 10 5 ● ● M DLS screen Makan Varferz hiff rettent Many 5 10 time (h) 15 20 -pH 6 his -pH 6 MES pH 5.5 Ac buffer -pH 7.5 PB -pH 6 his pH 5 citrate -pH 7.0 PB -pH 6.5 his -pH 6.5 MES -pH 5.0 Ac buffer -pH 6,5 5 PB 100 80 60 40 20 0 11 ctrl Accelerated stress screen DLS and BLI octet screening to identify pH and buffer Short Accelerated isothermal hold stress screening for 1-2 week Phosphate buffer was observed to improve stability Phos pH 7 Acet pH 5.5 Comera LIFE SCIENCES Phos-Ac pH 6 ■0 wk ■2wk 16#17mAb liquid formulation development: viscosity screen Binding (nm) 2.5 2.0 1.6 10 0.5 -0.5 -10 Self Assn 550 600 Sensor A6 Self Assn MES buffer pH 6 Phos buffer pH 6 660 BLI octet screen 700 750 800 Sensor C6 His buffer pH 6 Acetate buffer pH 6 850 Time (sec) His buffer pH 6 VR His buffer pH 6 Phos buffer pH 6 VR +Phos buffer pH 6 900 950 Sensor E6 1000 1050 Dissn HE 1100 Sensor G6 Dissn 11 Viscosity (CPs) 25C 100 90 80 70 60 50 40 30 20 10 0 50 100 150 Concentration (mg/ml) 200 Comera LIFE SCIENCES 250 DLS, BLI octet and microvisc help to identify and validate viscosity reducing excipients 17#18mAb liquid formulation development Rh (nm) 100 90 80 70 60 50 40 30 20 10 0 0 ● ● 5 DLS screening to identify stabilizing excipients Isothermal Accelerated stress screening at 45C DLS isothermal screening @40C www 10 15 20 25 Time (h) 30 35 40 45 Selected excipients % monomer 100 90 80 70 60 50 40 30 20 10 0 No stabilizer 4week isothermal Monomer loss 400 mM stabilizers CS1+ CSS CS1+ CS3 CS1 CS1+CS4 CS3 Sucrose CS3+Cs4 Trehelose Stabilizers Comera LIFE SCIENCES Mixtures were observed to improve stability better than single excipients 18#19mAb liquid formulation development using SQore excipients Viscosity (cps) CS1+ CS2 CS1+ CS3 CS1+CS5 100.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 50 Contral C51-CSS No stab control A CS1+ CS2 ●CS1.CS3 70 Excipient combination No stabilizer No viscosity reducer 90 110 130 150 conc (mg/ml) 170 190 Monomer stability 40C 52.19 58.63 85.21 20 87.3 210 230 250 Viscosity at 150 mg/ml 32.05 38.21 20.25 13.5 54.38 % monomer remaining 100 80 g 60 40 20 0 Viscosity(Cp) 60 50 40 30 20 10 O Compiled best stabilizer mixes 40C data no stab 0 ul CS1+ CS3 CS1+ CS2 Stabilizer mixtures 20 40 CS1+ CSS Stability 60 #4 wks Comera LIFE SCIENCES High stability + low viscosity 80 100 19#20● ● mAb A liquid formulation optimization by DOE Isothermal stability studies for optimizing m Ab A formulation Set up isothermal hold studies at 40C for 4 weeks mAb A: 150 mg/ml Sugars total conc: 200-500 m M Formulation A B ● CDEFGH ● с Н J pH 7 5 6 6 6.5 7 7 5 6.5 5 Recommended Formulation: mAb A conc (150 mg/ml) 5 mM phosphate buffer, pH 6.5 350 mM sugar mix Sugars (mm) 500 500 200 350 500 350 200 350 350 500 monomer 80 60 40 20 5 5.5 6 pH 100 90 80 70 60 50 40 30 20 10 0 6.5 A B C D E F G H I J DOE samples 7 DOE for formulating mAb A Lower pH detrimental for stability 500 450 350 300 Monomer 60 > 350 mM sugar mix helps stability 15 60 A Comera LIFE SCIENCES 20#21Summary and Conclusions ● ● ● ● ● (.)) Comera LIFE SCIENCES Comera SQore ™ platform provides excipient technology to address viscosity as well as stability issues for highly concentrated protein formulations enabling SQ administration of biopharmaceuticals. Comera can utilize computational as well as traditional screening to identify excipients Novel stabilizers show similar/improved profile as compared to sucrose or trehalose A mixture of stabilizers showed better stability profile as compared to single stabilizer at equivalent conc mAb can be easily optimized by DOE to obtain relatively stable low viscosity subQ formulation. 21#22Thank you! Comera Team Comera Life Sciences 12 Gill Street. Suite 4650 Woburn, MA 01801, USA comeralifesciences.com