Analysis of Subvisible Particles. Linda O. Narhi Formulation and Analytical Resources, R&D Amgen, Inc.

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1 Analysis of Subvisible Particles Linda O. Narhi Formulation and Analytical Resources, R&D Amgen, Inc.

2 Outline Introduction: Protein aggregates Techniques for analyzing sub micron and micron particles Generation and characterization of protein aggregates for immunogenicity assessments Future directions and conclusions

There are multiple pathways by which proteins can lose their native structure Heterogeneous nucleation dependant

g due to air-water interface) /chemical Silicone oil or Nano-particles Structural Changes Adsorption/ Assembly

2002, Biochemistry, 41: 6422-31 Chi EY et al 2003, Protein Science, 12: 903-13 Chi EY et al 2003, Pharm Research.

3 There are multiple pathways by which proteins can lose their native structure Heterogeneous nucleation dependant aggregation Conformational or colloidal stability dependant aggregation Assembly Structural Changes (e.g due to air-water interface) /chemical Silicone oil or Nano-particles Structural Changes Adsorption/ Assembly /chemical Thirumangalathu R et al 2009, J Pharm Sci (online) Bee J et al 2009, J Pharm Sci (online) Krishnan S et al 2002, Biochemistry, 41: Chi EY et al 2003, Protein Science, 12: Chi EY et al 2003, Pharm Research. 20: Particles can have different morphology based on the mechanism of formation Thanks to Eva Chi (Univ of New Mexico) and Krishnan Sampath

4 Protein aggregates can have widely varying properties, depending on exposure during process and storage Distribution of particle sizes Shape Reversibility Stability Total amount of aggregate Native vs non-native conformation Chemical Modification Density Exposed T and B-cell epitopes Covalent vs. non-covalent bonds Morphology (Crystalline structure, amorphous, etc.) Protein aggregates represent an extremely low fraction by mass of the total protein, are typically dynamic and can be influenced by many factors, including transportation and the drug product container/closure. Potential effect on safety (immunogenicity) and efficacy.

5 Protein aggregates range in size (1 million fold) Definitions by size (all are aggregates): Oligomers:10 nm 0.1 µm Particles: - Submicron: 0.1 to 1 µm - Micron: 1 to 125 µm - Visible: 125 µm 0.001µm 0.01µm 0.1 µm 1 µm 10µm 100µm 1000 µm 10,000 µm Currently there are no generally accepted definitions for particulates; these can be defined based on either size or mechanism of formation

6 Analysis of protein aggregates in solution

7 Light Obscuration is currently the common compendial method used for measuring subvisible particles in therapeutics. Applicable size range: μm

8 Modifications to USP<788> enable more accurate testing of subvisible particles but challenges still exist Reduced sample volume required for testing (single units 1 ml), allows measurement of unit to unit variability Extended size range for particle measurements: 2, 5, 10, 15, 20, 25, 50 μm Modified sample handling and include degassing step to minimize bubbles (false reading) and improve method performance Enabled (product specific) method development, qualification and validation Lack of a protein particle standard still poses challenge to accurate measurements 10 & 25 μm: required by USP 2 & 5 μm: recommended to USP Others (e.g. 15, 20, 50 μm): optional

9 Vacuum degassing of protein solution improved method performance 10 μm particles/ml Allow sample to equilibrate prior to testing Vacuum (75 Torr) degas Particles/m L Particles/m L Degas Duration (hour) Degas Duration (hour) Sample handling is critical to avoid both false positives and false negatives

10 Subvisible particle levels vary and are dependent on the container closure and formulation µm 5 µm 10 µm µm 5 µm 10 µm Type I Glass Vials Pre-filled Syringe Particles/mL 100 Particles/mL Sample 1_T1 Sample 1_T2 Sample 1_T3 Sample 2_T1 Sample 2_T2 Sample 2_T3 1 Sample 3_T1 Sample 3_T2 Sample 3_T3 Sample 4_T1 Sample 4_T2 Sample 4_T3 Samples Samples µm 5 µm 10 µm Particles measured in the absence of protein 1000 Particles/mL In vials Placebo PFS Samples Light obscuration can only provide information on number and size of particles

11 Camera Based Technologies MFI Flow cam A third system, the FPIA, from Malvern, is also available For all systems the output is heavily dependent on the analytical algorithms employed by the software.

12 Images of MFI versus FPIA Protein particles by MFI Protein particles by FPIA Particle standards Silicone oil droplet Protein particles Size can be expressed in different manners: ferret diameter based on largest diameter ECD or equivalent circular diameter, etc.

13 FlowCam vs MFI: Placebo from PFS MFI Placebo PFS_MFI high mag (>10 um feret) µm Placebo PFS_MFI low mag (>15 um feret) µm FlowCam

14 The majority of particles measured from product stored in a prefilled syringe are silicone oil Images obtained with MFI Product in PFS_ Product in vial Imaging systems provide information about shape and morphology that can help differentiate between protein and other particles

15 Overview of Coulter Theory How the Technology Works Particles suspended in a weak electrolyte solution are drawn through a small aperture. Across the aperture a voltage is applied. This creates the sensing zone. A particle passing through the aperture displaces a volume of electrolyte solution equal to its own volume. Displaced electrolyte causes change in resistance across aperture resulting in voltage pulse. Pulse intensity is proportional to the particle volume (converted to Equivalent Spherical Diameter).

16 Overview of Coulter Theory What do the pulses depend on? Particle characteristics Volume Shape Conductivity Porosity Aperture Current Size Electrolyte solution Aperture diameter: Nominal diameter μm (Dynamic range 2-60%: μm) 100 μm aperture: dynamic sizing range: 2 60 μm Aperture sizes available (µm) Isoton II Electrolyte: 1% salts solution (NaCl, KCl, Phosphate) (1% NaCl = 171 mm) Formulation buffers: 100 mm NaOAc: with excipients 10 mm NaOAc: with excipients

17 Duke PS Standard Linearity on 3 Instruments: Low conductivity buffer 5 µm standard in a formulation buffer Coulter: 100um aperture, current -200A, gain 16 ( um) 5 µm standard in a formualtion buffer Coulter MFI HIAC % conc Total P/mL Total P/mL Total P/mL Too P/mL R 2 = 1 R 2 = R 2 = % Conc Coulter MFI HIAC Linear (Coulter) Linear (MFI) Linear (HIAC) Linearity: PS standard is linear over the dilution range on all 3 instruments. Particle Counts: All 3 instruments have comparable particle counts.

18 IgG a Linearity on 3 Instruments IgG a is at 20 mg/ml (100%) in 50mM NaAc and 100 mm NaCl buffer IgG a in formulation buffer Coulter MFI P/mL R 2 = R 2 = R 2 = % Conc HIAC Linear (Coulter) Linear (MFI) Linear (HIAC) The results for this IgG are linear over the dilution range on all 3 instrument. Coulter counter is in agreement with MFI; HIAC gives much lower counts.

19 IgG b Linearity on 3 Instruments: 100 mm NaAcetate buffer IgG b is at 150 mg/ml (100%) in 100mM NaAc buffer IgG b in 100 mm NaAc R 2 = Coulter MFI P/mL R 2 = R 2 = HIAC Linear (Coulter) Linear (MFI) Linear (HIAC) % Conc This IgG is linear over the dilution range on all 3 instruments. The counts of SbVPs: Coulter > MFI > HIAC

20 The absolute quantity of particles varies between techniques HIAC MFI Particles/mL ECD (µm )

21 Conclusions: Electrazone, LO, Imaging systems Coulter/Elzone HIAC Flow Cam MFI Results Concentration, Size, Mass Concentration, Size Concentration, Size, Shape, Transparency Concentration, Size, Shape, Transparency Principle Electrical sensing zone Light obscuration Flow microscopy Flow microscopy Size 0.4 µm to 1.2 mm µm 1 µm to 3 mm µm Shape No No Yes Yes Transparency No No Yes Yes Volume required 10 ml 2 ml 1 ml 1 ml Sampling Efficiency 10% (1/10) 60% (1.5/2.0) 30% (0.3/1.0) 50% (0.5/1.0) Calibration yes yes no no Interference by shape no yes yes yes Interference by RI no yes yes yes Electrolyte required yes No no no Sampling efficiency = 100 x volume analyzed/total volume required

22 Mab Monomer/Oligomer Separation by AF4: Satisfactory UV 220 nm (relative scale) 1.0 SEC Monomer: 70.2% HMWS: 29.8% FFF Monomer: 74.0% 0.5 HMWS: 26.0% time (min)

23 Sub-μm Particle Quantification by FFF-MALS is Possible in Some Cases (enriched in SbVP) relative radius (nm) scale 50.0 T = 15 min r = 23 nm x x10 Angular Dependence R(theta) x x sin²(theta/2) time (min)

24 FFF-MALS detection is not sensitive enough to detect levels of SbVP present in product samples 1.0 FFF-MALS-UV 0.8 relative scale sub-μm particles would be here time (min) Detection sensitivity limits FFF, AUC, etc.

25 Studies with artificially generated subvisible particles

26 Known treatments and techniques can induce aggregates of various properties/populations for further evaluation Treatment Targeted Properties Heat (80C) Crosslink Hydrogen peroxide oxidation Metal catalyzed oxidation ph change Mechanical manipulation Protein-coated nanosphere Separate by Size (2 µm to > 10 µm) Non-native like conformation Native-like conformation; Irreversible repeatability Chemical modification Chemical modification; Covalently bound molecules Non-native like conformation; Native disulfide bonds Native and non-native conformation Regular array; Higher order structure Distribution of sizes

27 Classification of Aggregates from 2 IgG2 Molecules Class Number Treatment # Particles (+ = 10 1 ) HIAC Particle Size (µm) MFI Amorphous (A) Structured (S) MFI/ FlowCAM Reversible (R) Partially Reversible (PR) Irreversible (I) Secondary Structure (CC) FTIR Sup pellet Surface Hydrophobicity % ANS Binding Oxidation of Amino Acids Peptide Mapping % Protein Collected by Spinning Absorbance Class 1 not aggregated Class 2 chemically modified 1 untreated 2 untreated 1 H2O2 1 CuSO < 10 <30 < 10 < 40 A A A A - - R R % 8% 11% 27% None None Met Trp,Met,His < 1% < 1% < 3% < 5% 2 CuSO4 + <5 A R Met,Trp,His < 1% Class 3 small, native, reversible 1 ph < 20 A R % - ~ 4% 1 syringe SO (+) ++++ < 50 A PR % Met < 10% 2 syringe SO (+) +++ <25 A % - ~17% 1 syringe SO (-) +++ < 40 A PR 92-31% Met < 10% Class 4 small, native, irreversible 1 x-link ++ < % - < 3% Class 5 - small, partially native, partially reversible 1 stir 1d < 20 A PR % None ~ 25% 2 stir 1d <25 A PR % None ~45% 1 agitate SO (+) +++ < 10 A % - ~75% Class 6 medium, partially native, partially reversible 1 stir 3d < 60 A PR % Met,Trp ~ 100% 2 stir 3d <80 A PR 55-52% Met,Trp,Cys ~89% 1 65C ph < 130 A PR % - ~ 85% Class 7 large, unfolded, irreversible 1 80C +++ < 220 A I % - ~ 90% 2 80C +++ < 100 A I % - ~86%

28 Particles generated under different forced conditions have different population distributions 141, , , , size (µm) Differential Particle Counts untreated untreated store 37C 1d store 37C 24hr store 37C 8d store 37C 8d ph 3.5 ph 3.5 ph 4.3 ph 4.3 ph 8.5 ph 8.5 ph 11 ph 11 pipette pipette agitate 4C 8d agitate 4C 8d agitate wso 4C 8d agitate wso 4C 8d agitate 22C 3d agitate 22C 3d agitate wso 22C 3d agitate wso 22C 3d syringe no SO syringe no SO syringe wso syringe wso stir 1d stir 1d stir 3d stir 3d Crosslink - EDC Crosslink - EDC H2O2 H2O2 CuSO4 CuSO4 80C 80C 65C and ph C and ph storage ph mechanical stress Aggregate Treatment x-link oxidation heat

untreated diameter 10µm Aggregate Sets have Distinct Morphological

H2O2 diameter 5µm syringe SO (-) diameter 40µm syringe SO (+)

29 untreated diameter 10µm Aggregate Sets have Distinct Morphological Features IgG2 1 images taken by MFI: 1mg/ml aggregates diluted X in A5, 0.5 ml analyzed. CuSO4 diameter 40µm stir 1d diameter 20µm stir 3d diameter 60µm H2O2 diameter 5µm syringe SO (-) diameter 40µm syringe SO (+) diameter 50µm 65C ph 8.5 diameter 130µm 80C diameter 220µm agitate 22C SO (+) diameter 5µm ph 11 diameter 20µm

30 Conclusions and Next Steps

31 Results and correlations will likely be specific to the product and the aggregate involved, and need to be evaluated on a case by case basis Conclusions Limitations with current technology include: Lack of specificity to protein particles Lack of protein aggregate standards Differences in absolute quantitation between analytical methods Lack of technology to quantitate and characterize below 2µm Lack of validated predictive models to assess immunogenicity Limitations of the current technology and inherent variability of aggregate population confound establishing meaningful acceptance criteria for release testing and comparability Additional testing and characterization of subvisible particles is necessary to better understand the nature and utility of monitoring subvisible particles Additional testing is necessary to understand limits and utility of assays for immunogenicity, and ability to predict clinical outcome

32 Next Steps Continue to explore the relationship between amounts of protein particles of different sizes (what species are on pathway to SbVP?) Monitor 2 and 5 µm sizes during process and formulation development Work with vendors to develop technology to distinguish between silicone oil droplets, foreign particles and proteins Develop methods to characterize small amounts of particles with minimal sample manipulation Develop and verify in vivo and/or in vitro models to evaluate the significance of subvisible particles and immunogenicity.

33 Acknowledgements Shawn Cao Nancy Jiao Joey Pollastrini Yijia Jiang Marisa Joubert John Gabrielson Monica Pallitto

34 Back-up slides

SE-HPLC Slight increase in HMWC due to dissociable aggregates FT-IR Spectra show presence of Amide I and

35 Analysis of isolated particles can be used to compare biochemical and biophysical properties rce-sds Subvisible particles isolated from a product sample retain full potency and similar quality profile SE-HPLC Slight increase in HMWC due to dissociable aggregates FT-IR Spectra show presence of Amide I and II bands Reference Standard Particle C B A Second Derivatives of FT-IR Spectra indicate presence of β-sheet structures

36 The absolute number of particles varies between techniques although relative number relationships are consistent 1.E+07 Particles/mL 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 FFF PMS HIAC 1.E+01 1.E+00 1.E Diameter (um) Note: For FFF there is dilution during the analysis; Error bars represent STD for triplicate measurements; PMS = Particle Measuring System (based on static light scattering)

37 Applications of analytical methods

38 Information on subvisible particles below 10µm can be useful for process characterization Sample Particles/mL at 2 µm Particles/mL at 5 µm Particles/mL at 10 µm Particles/mL at 25 µm Control (unfilt., Lot 1) 339 ± ± 2 Below LOQ Below LOQ 1x filtration Lot 1 29 ± 6 11 ± 5 Below LOQ Below LOQ 2x filtration Lot 1 19 ± 9 14 ± 9 Below LOQ Below LOQ 5x filtration Lot 1 50 ± ± 9 Below LOQ Below LOQ Control (unfilt., Lot 2) 680 ± ± 4 Below LOQ Below LOQ 1x filtration Lot 2 20 ± 3 5 ± 2 Below LOQ Below LOQ Filtration by 0.2 µm filter has removed particles at 2 μm 1x filtration is effective in removing the particles Particle concentrations are below LOQ at 10 and 25 μm

39 A relationship exists between particles of different sizes, with the slope being product-dependent Native Protein Light & Heat Stressed Protein 1000 Particles/mL Particle Size (um) Visible particles observed in 80% units Particles/mL 10 No visible particles observed Sizes (µm)

40 FTIR Spectra of IgG2 1 Aggregate Groups Correlation Coefficient Treatment CC Total CC Sup CC Pellet Absorbance Units Pellet untreated freeze-thaw LN ph 11 x-link EDC agitate SO (+) syringe SO (-) syringe SO(+) stir 1d stir 3d H2O Wavenumber cm-1 CuSO ph C ph C = not tested

41 Particles can also be generated by Crosslinking in Solution Mechanism of EDC Mechanism of Glutaraldehyde Differential Particle Counts EDC Crosslinking of IgG Time (minutes) More EDC added Differential Particle Counts HIAC Particle Counts 0 Glutaraldehyde Crosslinking of IgG low ph Time (minutes) 2 µm ~ 163,000 5 µm ~ 28,000 high ph size (µm)

Analytical methods to characterize aggregates and subvisible particles

Analytical methods to characterize aggregates and subvisible particles Andrea Hawe 9 th P4EU Meeting 30 th November 2015 * MPI * Martinsried 1 Background of company privately held, independent service