Validating and Implementing Subvisible Particle (SbVP) Testing for Biotechnology Products throughout Development

Transcription

1 Validating and Implementing Subvisible article (SbV) Testing for Biotechnology roducts throughout Development Linda Narhi and Yijia Jiang Amgen Inc, Thousand Oaks, CA, USA

2 Outline Background rotein aggregates and particles US<788>: challenges Drivers for a SbV analysis method suitable for protein therapeutics Qualifying and validating the subvisible particle analysis method Implementing SbV testing: application of the subvisible particle analysis method Summary and future directions 2

3 Multiple athways Can Lead to rotein Aggregation N I1 I2 U aggregate(a1) A2 A3 Ig rotein Aggregates by FIA Imaging N=Native state, I=intermediate state, U=Unfolded state and A=aggregate state Reversible and irreversible reactions can occur Solvent conditions, temperature, protein concentration, etc. Determinein which state the majority of the protein will exist Roberts et al., Biotechnol Bioeng, 98 (2007) (Images provided by Nancy Jiao, Amgen) Manufacturing rocess Steps and roduct Fermentation urification Formulation Storage Shipping Administration? Stress Conditions Heat Freeze-thaw Cross-linking rotein concentration Formulation change ph, salt Chemical modification Mechanical Stress Surface effects Nano-particles rotein aggregates can be induced under forced conditions and can occur simultaneously during biopharmaceutical manufacturing 3

4 Not all Aggregates or articles are the Same. Dissociable Dimer Non Dissociable Dimer Non Dissociable Aggregate Subvisible articles Visible rotein articles 4 Visible Extraneous articles

5 Aggregates and articles Cover a Size Range of > 1 million Fold Aggregates: 10 nm 0.1 µm (> dimers = oligomers, soluble) articles: - Submicron: 0.1 to 1 µm (sub-micron, often soluble aggregates) - Subvisible: 1 to 150 µm (sub-visible, often insoluble aggregates) - Visible: 150 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 5

6 Analytical Tools for Measuring rotein Aggregates and articles Dynamic Light Scattering AUC FFF-MALS Static Light Scattering SEC nm m mm cm (10 7 nm) monomers & oligomers Aggregates Mass% Coulter rinciple (Electrozone) Flow through microscope (MFI/FIA/FlowCam) submicron particles articles Microscope Automated Inspection Light Obscuration subvisible particlesvisible particles articles/ml Visual Inspection US <788> articulate Matter in Injections = Subvisible articles 10 µm, LO and microscopic 6

7 Reasons for Subvisible article Analysis for rotein Solutions atient safety Immunogenicity is largest concern: Large amounts of small protein particles (e.g. 2 m) may be more immunogenic than small amounts of large protein particle (e.g. 10 m) roduct consistency and clinical experience are key Occlusion concern roduct and process development: Consistency and relative ranking are key Quantifying the amounts of small protein particles (e.g. 2 m) may be more informative articles/ml Sample roduct Lot articles/ml at 2 µm articles/ml at 10 µm Control (unfilt., Lot 1) 339 ± 78 Below LOQ 1x filtration Lot 1 29 ± 6 Below LOQ 2x filtration Lot 1 19 ± 9 Below LOQ Control (unfilt., Lot 2) 680 ± 51 Below LOQ 1x filtration Lot 2 20 ± 3 Below LOQ

8 Challenges in Using US <788> Method for rotein Solutions rotein particles are often not captured by the filtration step in the microscope method Importance of appropriate sample handling Does not monitor SbV below 10 microns, perhaps the most importance size for protein particles 25 ml / test sample requirement Optical properties of protein particles are not that different from that of the solution, particularly at high protein concentration Undercounting at high concentration due to changes in optical properties (RI, OD) and increased background Different techniques give different counts results are relative Lack of protein particle standards. ertinence of the concentration limits (6000/600) to protein therapeutics Differentiating between protein and other particles

9 Current ractice An Example Modify the light obscuration (LO) compendial method Apply appropriate sample handling procedures to minimize false positives (e.g. air bubbles) and false negatives (e.g. reversible) Reduce sample amount to 5 ml or less / test enerate data for SbV 2 & 5 µm in addition to 10 & 25 µm Use the modified LO method as work horse Verify method performance Key parameters hase appropriate Use orthogonal methods to complement when necessary Focus on consistency and trending articles/ml Test 1 Test 2 Test Sample Volume (ml)

10 Qualifying and Validating the SbV Analysis Method 10

11 Modifications to US <788> Reduced sample volume required for testing (single units 1 ml); allows evaluation of unit to unit variability Single technique can be used from product development to commercialization Extended size range for particle measurements: 2, 5, 10, 15, 20, 25, 50 m Modified sample handling and use a degassing step to minimize bubbles (false reading) and improve method performance Enabled (product specific) method development, verification, qualification and validation 10 & 25 m: required by US 2 & 5 m: recommended to US 11

12 SbV Method Qualification uiding rinciples Qualify as a quantitative method Qualify 2 m, 5 m, 10 m, and 25 m measurements Use protein product solution / particle as much as possible Demonstrate equivalence to US <788> method for LO methods 12

13 Method Qualification and Validation arameters Specificity rotein particles Accuracy Measurement of article Counting Standard (CS - polystyrene spheres) at certified concentrations recision Repeatability Intermediate precision rotein & CS Linearity and range rotein & CS Limit of detection (LOD) / limit of quantification (LOQ) Robustness Simulation & experimental confirmation 13

14 Qualification Results with CS Shawn Cao, Yijia Jiang and Linda Narhi, US harmacoepial 14 Forum, , Vol. 36(3), 2010

15 Qualification Results with a rotein Solution The SbV method performs accurately and reproducibly and gives results that are comparable to those of the compendial method It can be used to determine the particle numbers at 10 µm, as well as measure particles in the 2 to 10 µm range. The variability increases as particle concentration decreases. The SbV method is capable of reproducibly counting particle concentrations between approximately 10 and 18,000 particles/ml. Shawn Cao, Yijia Jiang and Linda Narhi, US harmacoepial Forum, , Vol. 36(3),

16 Implementing SbV Testing: Application of the Subvisible article Analysis Method 16

17 FIH_No degas FIH_2hr Vacuum FIH_2hr RT CD D_No degas CD D_Vacuum 2hr CD D_RT 2hr /ml articles/ml articles/ml Sample Handling Matters - Degas Allow sample to stand at RT prior to testing Degas Duration (hour) rotein X Vacuum (75 Torr) degas Degas Duration (hour) rotein Y FIH vs CD_HIAC 2 µm , Samples Sample handling is critical to avoid false positives 17

18 Sample Handling Matters Dilution (I) Dilution of FS samples is needed due to higher SbV counts at 2 m sometimes going over sensor limit (18000 p/ml) rotein Z 2 µm 5 µm Linear (2 µm) articles/ml R 2 = R 2 = ercentage of Drug roduct Dilution was justified by the linear response of particle concentration with dilution 18

19 Sample Handling Matters Dilution (II) New findings with high concentration protein samples Nonlinearity observed at higher protein concentrations rotein Y, 120 mg/ml, 2 um by HIAC rotein Y, 120 mg/ml, 2 um by Coulter Consider dilution for SbV analysis in high protein concentration samples for this Mab 1:3 dilutions will be used for both DS and D Sample handling is critical to avoid false negatives 19

20 Sample Handling Matters Lyophilized roduct Reconstitution (I) SbV levels in WFI from different sources and containers vary significantly, some are quite high at 2 and 5 µm and above Hospira 10mL WFI_ DK_vial 1 in 15 ml bottle ECD >= 6 µm The shape of SbVs is round and typical of silicone oil droplets likely from syringes and vial stoppers. 20

21 Cumulative Counts per ml Sample Handling Matters Lyophilized roduct Reconstitution (II) Measured SbV in lyophilized samples can be affected by those in the WFI used for reconstitution 2 µm SbV Counts_rotein O Lyo D Recon with mq or WFI_HIAC 3000 WFI 2,556 2, ,247 1,606 1,215 2,001 1, Recon mq_v1 Recon mq_v2 195 Recon mq_v3 Recon WFI_V1 Recon WFI_V2 Recon WFI_V3 Samples The SbV levels are generally lower in the product samples reconstituted with WFI than in the corresponding WFI used. Therefore, WFI background subtraction can not be used to correct for the contribution of SbV in the reconstituted D from WFI Use Milli-Q water to reconstitute lyophilized products for SbV testing 21

22 SbV Levels of Multiple Buffers in FS before and after Transportation 100,000 >= 2µm >= 5µm >= 10µm Cumulative Counts per ml Cumulative Counts per ml 10,000 1, ,000 10,000 1, ,303 4,989 21,398 2,051 2,144 6, ,180 42,810 14,987 3, mq placebo_static 11 >= 2µm >= 5µm >= 10µm 56,032 15,593 1,530 13, mq placebo_trnsp 5,450 2, Samples 1, , ,429 3,335 3,084 1,486 1,467 1, ,390 34,890 38,373 20,672 12,095 13,715 3,657 2,358 2, Static Transport Samples The amount of SbVs in FS varies depending on the buffer composition. Transportation increases SbVs in FS, which are mainly due to silicone oil droplets 22

23 SbVs below 10 µm Can be Useful for rocess Characterization Sample articles/ml at 2 µm articles/ml at 5 µm articles/ml at 10 µm articles/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 m 1x filtration is effective in removing the particles article concentrations are below LOQ at 10 and 25 m 23

24 Cumulative counts/container Cumulateive counts/container SbVs below 10 µm Can be Useful for Stability (I) Cumulative counts/container D_HIAC at 2 µm D Vial T0 40C 1d 40C 3d 40C 1wk 40C 2wk 40C 1M 25C 1wk 25C 2wk 25C 1M D_HIAC at 10 µm D Vial T0 40C 1d 40C 3d 40C 1wk 40C 2wk 40C 1M 25C 1wk 25C 2wk 25C 1M D_HIAC at 5 µm D Vial T0 40C 1d 40C 3d 40C 1wk 40C 2wk 40C 1M 25C 1wk 25C 2wk 25C 1M rotein Y D in vials SbV level increased at 2 µm for protein incubated at 40 C over time The trend was not clear at larger size ranges due to low particle concentration and high variability 24

25 Cumulateive counts/container Cumulateive counts/container SbVs below 10 µm Can be Useful for Stability (II) Cumulative counts/container D_HIAC at 2 µm D_HIAC at 10 µm D FS D Vial T0 40C 1d 40C 3d 40C 1wk 40C 2wk 40C 1M 25C 1wk 25C 2wk 25C 1M D FS D Vial T0 40C 1d 40C 3d 40C 1wk 40C 2wk 40C 1M 25C 1wk 25C 2wk 25C 1M D_HIAC at 5 µm D FS D Vial T0 40C 1d 40C 3d 40C 1wk 40C 2wk 40C 1M 25C 1wk 25C 2wk 25C 1M rotein Y D in FS SbV levels are much higher than those in vials There is no clear trend with incubation time and temperature 25

26 Summary and Future Directions The Light Obscuration method (with appropriate modifications) has been demonstrated to be a reliable quantitative method for particles 2 µm SbV testing should be carried out for both DS and D for release testing and stability SbV testing should also be implemented during formulation and process development and characterization Ultimate oal: New / revised US subvisible particle chapter for protein therapeutics The performance of the method needs to be verified to ensure proper sample handling 26

27 Summary and Future Directions (contd.) Begin to establish safe subvisible particle level in protein therapeutics (history) and be prepared to set appropriate product specific specifications Will need multiple D and DS lots before doing this Will be device dependent (FS might not be meaningful) Evaluate the correlation of SbVs with visible and semivisible particles Evaluate emerging technologies Quantify particles Orthogonal methods rovide ID, shape/morphology information 27

28 Acknowledgement Tony Mire-Sluis Nancy Jiao Shawn Cao Joey ollastrini Li Zhu David Brems 28

29 BACKU SLIDES 29

30 Accuracy and comparison of SV vs. compendial method 30

31 Repeatability 31

32 Intermediate precision 32

33 Linearity 33