PROTEIN-PROTEIN INTERACTIONS, VISCOSITY AND …

1 PROTEIN-PROTEIN interactions , VISCOSITY AND injectability IN MULTI protein CO-FORMULATIONS- bevacizumab AND rALBUMINSECOND GENERATION ALBUMIN FUSIONSJens T. Bukrinsky, PhDSenior formulation ScientistAt Novozymes Biopharma A/SCambridge October 20132 AGENDACase study on aggregation prevention Analytical challenges and preliminary data Mechanism SAXS DLS AF4 Viscosity3 HUMAN SERUM ALBUMIN - PROPERTIES Structure 585 amino acids, Single chain Three domains 17 disulfide bridges Cys34 is unpaired One tryptophan 66472 g/mol Hydrophobic patches/cavities IpH = Soluble up to >400 g/L Approximately 50 g/L in blood High physical stability Long plasma life 19-20 days Inert SafeLocation of long chain fatty acid binding sitesCurry et al.

2 (1999) BBA1441, 131-1404 How does rAlbumin stabilize protein formulationsHSAin bloodrAlbin formulationMultiple hydrophobic binding sitesCoat hydrophobic surfaces in primary packaging materialsIncreases the colloidal stability of bloodPrevent self-association of protein drugsNatural antioxidant in bloodPrevent oxidation of protein drugsANALYSIS OF AGGREGATES- A CHALLENGE FOR COMPLEX FORMULATIONS- PRELIMINARY DATAANALYSIS OF AGGREGATES- A CHALLENGE FOR COMPLEX FORMULATIONS- PRELIMINARY DATAP revention of protein aggregation in high concentration mAb formulationsMethod

3 Set-upAvastin ( bevacizumab )Recombumin Alpha (rAlb)orbufferTransfer to Glass vialAF4-UV-MALS-RI analysisFormulation29 days40oCAF4-UV-MALS-RI ANALYSIS AF4: Asymmetric Flow Field Flow Fractionation Separation takes place in the channel based on differences in diffusiontime (min) Albumin monomerA2. Albumin dimerM1. mAb monomerM2. mAb dimerA1(A2)M1 M2AF4 separation works well with 4 out of 4 tested mAbs:Omalizumab (blue)Rituximab (green)Tocilizumab (red) bevacizumab (next slide) AF4 separation:Short channelSpacer 350S10 kDa RC membrane50 mM NaNO3, pH flow 1 ml/minCross flow gradient 3-1 mL/min (15 min) bevacizumab :rAlb mixture 100:50 mg/mL (in red) Avastin 25 mg/mL(in blue)Aggregation prevention of 100 mg/mL bevacizumab by 50 mg/mL rAlbumintime (min) M2 Confirmed A1.

4 Albumin monomer ( kDa) A2. Albumin dimer ( kDa) M1. bevacizumab monomer (149 kDa) M2. bevacizumab dimer (298 kDa)Not yet confirmed but ration matches FAB HC-HC A1(A2)M1 M2 Monomer DimerAvastin93,06,4 bevacizumab +albumin92,32,2 Prevention of freeze-thaw induced formation of sub-visible particles in proteins/peptides?Method set-upRoActemra (Tocilizumab)Albucult (rAlb)orbufferTransfer to Eppendorff tubeAnalysis by Micro-flow Imaging (MFI)Sub-visible particles (1-100 m)20 C1 hX 3-20 C>23 hAlbucult prevents the formation of sub-visible particles in Tocilizumab (a monoclonal antibody)020000400006000080000100000 Particle counts/mL1-10 m particlesFT cycle 0FT cycle 1FT cycle 2FT cycle 3FT cycle 40100020003000400050006000 Particle counts/mL11-100 m particlesFT cycle 1FT cycle 2FT cycle 3FT cycle 4 Reference formulations m ( mg/mL) Tocilizumab (diluted w milliQ)

5 137 m (20 mg/mL) Tocilizumab (RoActemra , Roche) 15 m ( mg/mL )Albucult (dil. w milliQ) 150 m (10 mg/mL) Albucult (dil. w milliQ)Test samples mg/mL Tocilizumab formulated with Albucult in mAb:rAlb molar ratios 1:1 1:5 1:10 ALBUCULT PREVENTS FORMATION OF FREEZE THAW INDUCED SUB-VISIBLE PARTICLES IN MAB FORMULATIONSM echanism behind aggregation preventionMechanism behind aggregation preventionSmall Angle X-ray Scattering Determination of solution structureMaxLab Beamline I911-SAXS 164 164 78 78 44 44 Pair distribution functionrAlb is a monomer with a concentration dependent repulsive behaviour Repulsion evaluated from peak Repulsion evaluated from I(0)No concentration dependent self-association of bevacizumab (up to 30 mg/mL)

6 Co-formulation of bevacizumab and rAlbuminBEV mg/mlHSA mg/mlMM (kDa) plot bevacizumab flexibility decreases with increasing rAlbumin concentrationrAlbumin stabilizes bevacizumab through molecular crowding curve fitting by linear combination Repulsive behavior of rAlbumin not perturbed by bevacizumab No interaction between rAlb and mAbHypothetical modelEvaluation of mAb-rAlbumin PROTEIN-PROTEIN interactions by DLSM ethod set-upAvastin ( bevacizumab )Recombumin Alpha (rAlb)Wyatt DynaPro Plate Reader (DLS)Formulation in different rAlb:mAb ratiosMixThe PROTEIN-PROTEIN interaction is too weak to detect.

7 Item Mass Ratio Radius (nm)ExpectedRadius (nm)Ratio7 B8 B9 B20 Avastin/Albucult 1 B21 Avastin/Albucult 1 B22 Avastin/Albucult 1 B23 Avastin/Albucult 1 C2 Avastin/Albucult 1 C3 Avastin/Albucult 1 B12 Avastin B13 Avastin ,533,544,555,5600,20,40,60,811,2 Radius / nmWeight RatioRadius vs. Ratio Observed radius of mixture comparable to expected radius of two non-interacting speciesVISCOSITY- A CHALLENGE FOR PROTEINS IN HIGH CONCENTRATIONSVISCOSITY- A CHALLENGE FOR PROTEINS IN HIGH CONCENTRATIONS01020304050600100200300400 5000 100 200 300 400 500 600 Average injection force (N) VISCOSITY ( = cSt)rAlb concentration (mg/mL)

8 ViscosityAverage injection forceCONCENTRATED rALB FORMULATIONS PRESENT AN EXCELLENT injectability *UP TO AT LEAST 300 MG/MLy = 0,1075e0,0168xR = 0,990211010010000 100 200 300 400 500 600 VISCOSITY ( = cSt)rAlb concentration (mg/mL) VISCOSITY and injectability are very well correlated properties for rAlb formulations The concentration of critical injectability is between 300 and 400 mg/mL where the VISCOSITY markedly levels up Between 280 and 500 mg/mL, the VISCOSITY of rAlb in solution increases exponentially with protein concentration*30G , 2 mL/min0102030405060010002000300040005000 600070000100 200 300 400 500 Average injection force (N) VISCOSITY ( = cSt)Beva.

9 Concentration (mg/mL)ViscosityAverage injection forcey = 1,083e0,0224xR = 0,99491101001000100000100 200 300 400 500 VISCOSITY ( = cSt)Beva. concentration (mg/mL)THE injectability *OF bevacizumab BECOMES CRITICAL BETWEEN 140 AND 180 MG/ML VISCOSITY and injectability are well correlated properties for bevacizumab formulations The concentration of critical injectability is between 140 and 180 mg/mL This is in agreement with the VISCOSITY increase between 150 and 230 mg/mL Between 100 and 400 mg/mL the VISCOSITY of bevacizumab in solution increases exponentially with protein concentration()( )*30G , 2 mL/min01020304050607080 Average injection force (N)

10 THE ADDITION OF rALB TO bevacizumab DOES NOT LEAD TO A SIGNIFICANT INCREASE OF THE INJECTION FORCE* The injection force of rAlb (50 mg/mL) is comparable to that of the buffer At a total protein concentration of 150 mg/mL, the addition of rAlb leads to a minor increase of the injection force At a total protein concentration of 250 mg/mL, it seems that rAlb eases the injection of bevacizumab to a measurable value*30G , 2 mL/min//MAJOR CONCLUSIONSM echanism behind aggregation prevention (one case study) Repulsive behavior and crowding effect as determinedby SAXS and confirmed by DLS, AF4 and Rheological studiesRheology and VISCOSITY The VISCOSITY of rAlb formulations is comparable to buffer solutions up to more than 100 mg/mL An exponential increase in VISCOSITY and injection force is observed for rAlb and two mAbs at high concentrations Adding rAlb to high concentration mAb formulations does not give an exponential increase in injection forceAnalytics Af4 (FFF)