Compatibility and Physical Stability of Monoclonal Antibodies After Dilution into Different IV Administration Bags

Compatibility and Physical Stability of Monoclonal Antibodies After Dilution into Different IV Administration Bags David Volkin Macromolecule and Vaccine Stabilization Center, Department of Pharmaceutical Chemistry Colorado Workshop on Protein Aggregation and Immunogenicity July 2012

Outline of Presentation Introduction Monoclonal antibodies and routes of administration Pharmaceutical challenges with IV delivery and proteins Regulatory guidance Limited examples in scientific literature Case Study with IgG4 mab Future Directions and Considerations

Monoclonal Antibody Structure, Post-Translational Modifications and Stability Cellular Folding and Disulfide-bond Formation Glycosylation Proteolytic Cleavage C-terminal Lysine Oxidation, Deamidation, Isomerization Aggregation due to misfolding http://www.path.cam.ac.uk/~mrc7/igs/img09.jpeg http://www.abcam.com/ps/cms/images/abstructure.jpg Chemical Pathways: Oxidation Deamidation Isomerization Disulfide exchange Peptide fragmentation N-terminal pyroglutamic acid Glycation Environmental Physical Pathways: Aggregation Particle Formation Adsorption Precipitation Conformational Changes Native Molten globule Incompletely disordered Random coil

Number of antibodies approved each year FDA-Approved Monoclonal Antibody Drugs (1986-2011) 5 4 3 2 1 0 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 35 30 25 20 15 10 5 0 Cumulative number of approved antibodies Reichert, mabs 1,387 (2009); Nelson et al., Nat. Rev. Drug Discov. 9, 767 (2010); Mullard, Nat. Rev. Drug Discov. 10, 82 (2011); Mullard, Nat. Rev. Drug Discov. 11, 91 (2012)

Route of Administration for FDA-Approved mabs Intravenous (IV): 21 Subcutaneous (Sub Q): 8 Other (IM, eye): 2 ~ 68% (~21 of 31) mab-based treatments administered to patients by IV injection Of these IV-delivered mabs, ~67% (~14 of 21) for oncology indications Zhang et al, pgs. 711-763 in: Therapeutic Monoclonal Antibodies: from bench to clinic, Ed Z. An, Wiley (2009) ; Online Product Inserts from Individual Drugs

IV Delivery and Protein Stability IV Bags: Different Diluents Different Types of Plastic Adapted from: http://www.ebme.co.uk/arts/vis/index.htm IV Setup Varies: Volume and Flow Rate Bag Size Transport Storage IV Administration Kits: Different Types of Plastic Different Filters https://shop.life-assist.com http://www.shopmedvet.com

Examples of Variables To Consider During IV Administration of Protein Therapeutic Drugs Protein and Formulation Protein: Formulation: IV Bags and Kits Diluents: Plastics: Filters: IV Setup Headspace: Environment: Storage: Sensitivity to different environmental stresses Types and amount of excipients Isotonic NaCl, Dextrose solutions Polyvinylchloride (PVC), Polyolefin (PO) 0.22 to 1 micron filters Diluent volume, size of bag Agitation, ambient light and temperature Hold times during day / freeze-thaw

Regulatory Guidelines ICH-M4Q Common Technical Document Quality Section 3.2.P.2.6, Compatibility The compatibility of the drug product with reconstitution diluents or dosage devices (e.g., precipitation of drug substance in solution, sorption on injection vessels, stability) should be addressed to provide appropriate and supportive information for the labeling. USP <797> Pharmaceutical Compounding, Sterile Preparations ensuring that compounded sterile preparations are accurately identified, measured, diluted and mixed, and are correctly purified, sterilized, packaged, sealed, labeled, stored, dispensed, and distributed.

Previous Studies Examining Stability of mabs During IV Administration Although compatibility data generated for regulatory filings, Published literature on stability of mabs in IV bags very limited. Especially compared to large number of papers on mab stability data in primary containers (vials, syringes). Examples of mab stability under simulated IV conditions: 2008: Chemical stability, 2 mabs in dextrose IV bag (LC-MS) 2009: Physical stability, mab in saline, dextrose solution (FFF, Fluor, TEM) 2010: Dose of 2 mabs, PVC IV bags in saline (ELISA, visual) 2012: Formulation variables, 3 mabs in saline IV bag (SEC, HIAC, OD) 2012: Case study from today s talk (2008) Fisher et al, Eur J Pharm Biopharm 70, 42.; (2009) Demeule et al, mabs 1, 142; (2010) Ikesue et al, Am J Health Syst Pharm 67, 223; (2012) Sreedhara et al, J Pharm Sci 101, 21.

Examining Physical Stability of mabs: Newer Analytical Methods for Aggregates and Particles Aggregates and Particles of Varying Size Soluble aggregates 0.1µm or less Submicron Particles 0.1-1 µm Subvisible Particles 1-100 µm Visible particles 100 µm or more Size-range, Number, Morphology, Composition Effect of Environmental Stresses Focus of this talk: application to IV administration of mabs

Outline of Presentation Introduction Case Study with IgG4 mab Future Directions and Considerations

Case Study with IgG4 Monoclonal Antibody Recently Published Case Study Kumru O, Liu J, Ji J, Cheng W, Wang J, Wang T, Joshi S, Middaugh R, Volkin D Compatibility, Physical Stability and Characterization of an IgG4 mab after Dilution into Different IV Administration Bags J Pharm Sci (2012) Online Early View Background Continuation of work of Sreedhara et al (2012) with different mab and additional analytical methods Physical stability of IgG4 mab after dilution in IV bags with 0.9% NaCl diluent. Various formulation conditions Detailed characterization of particles formed

Initial Experiment Dilute IgG4 mab in IV bags containing 0.9% NaCl and varying levels of PS20 and protein Incubate in IV bag (PO) at 30 C, 100 rpm in a shaking incubator; 0-6 hrs. Remove mab-containing solutions from IV bag Analysis by: Protein concentration PS20 levels Optical Density SE-HPLC HIAC

Effect of PS20 level on mab Physical Stability: Dilute IgG4 into IV Bag (0.9% NaCl) Agitation Time (hours, 30 C) PS 20 in IV bag (w/v) Protein in IV bag (mg/ml) Turbidity (Ave OD from 340-360 nm) SE-HPLC (% Monomer) HIAC (Particles >2 µm/ml) 0 3.6 0.01 98.0 950 2 3.6 0.01 97.3 3549 0.004% 4 3.6 0.01 96.6 7808 6 3.6 0.01 96.0 4133 0 3.6 0.01 97.9 379 2 3.6 0.01 98.0 598 0.012% 4 3.6 0.01 98.0 408 6 3.7 0.01 98.0 1054 See Kumru et al, J Pharm Sci (2012) online for additional time-points and concentrations

SE-HPLC of IgG4 mab After Dilution into IV Bag (0.9% NaCl) with varying PS20 levels

Experimental Flow Chart for Further Characterization Dilute IgG4 mab to 1 mg/ml PS20 in IV bags containing 0.9% NaCl Incubate in different IV bags at 30 C, 100 rpm in a shaking incubator; 0-6 hrs. Remove mab-containing solutions from IV bag Amount and size range: SE-HPLC NTA MFI Turbidity Characterization: TEM and MFI SDS-PAGE Free thiol content FTIR Microscopy Extrinsic Fluorescence (ANS)

Counts, # Counts, # Counts, # MFI Analysis of Effect of PS20 and Headspace: Dilute IgG4 mab into IV Bag (0.9% NaCl) 1000000 100000 PVC IV bag PVC IV Bag 0hr 2hr 4hr 6hr 10000 Add 0.015% PS 20 (in IV bag) 1000 100 Remove Headspace 1000000 100000 10000 PVC IV bag w/ 0.06% PS20 10 >2 <5 >5 <10 >10 <25 >25 <50 >50 <70 >70 <100 0hr 2hr 4hr 6hr ECD ( m) 1000000 100000 10000 PVC IV bag, No headspace 0hr 2hr 4hr 6hr 1000 1000 100 100 10 >2 <5 >5 <10 >10 <25 >25 <50 >50 <70 >70 <100 ECD ( m) 10 >2 <5 >5 <10 >10 <25 >25 <50 >50 <70 >70 <100 ECD ( m)

Counts, # Counts, # Counts, # MFI Analysis of Effect of PS20 and Headspace: Dilute IgG4 mab into IV Bag (0.9% NaCl) 1000000 100000 10000 PO IV bag PO IV Bag 0hr 2hr 4hr 6hr Add 0.015% PS 20 (in IV bag) 1000000 100000 PO IV bag w/0.06% PS20 1000 100 10 >2 <5 >5 <10 >10 <25 >25 <50 >50 <70 >70 <100 0hr 2hr 4hr 6hr ECD ( m) 1000000 100000 PO IV bag,no headspace Remove Headspace 0hr 2hr 4hr 6hr 10000 10000 1000 1000 100 100 10 >2 <5 >5 <10 >10 <25 >25 <50 >50 <70 >70 <100 ECD ( m) 10 >2<5 >5<10 >10<25 >25<50 >50<70 >70<100 ECD ( m)

Particle concentration >2mm (# particles/ml) Total particle concentration >2 m (# particles/ml) MFI Analysis of Effect of Saline Source: Dilute IgG4 mab into 0.9% NaCl (IV Bag vs. Glass Vials) A IV Bags Total Subvisible Particle Concentration: B Glass Vials 1E7 1000000 PVC PO PVC0.06PS20 PO0.06PS20 PVCSaline POSaline 1E7 1000000 2-5 PVC PO PVCSaline POSaline LabSaline 100000 100000 5-10 10000 10000 10-25 ECD (mm) 1000 1000 25-50 100 100 50-70 10 0 2 4 6 Time (hrs) 10 70-100 0 2 4 6 Time (hrs)

Particle concentration >2mm (# particles/ml) Characterization of Subvisible Particles by MFI Dilute IgG4 mab into IV Bag (0.9% NaCl) A Total Subvisible Particle Concentration: B Morphology: 1E7 1000000 PVC PO PVC0.06PS20 PO0.06PS20 PVCSaline POSaline 2-5 PVC PO 100000 10000 1000 ECD (mm) 5-10 10-25 25-50 100 50-70 10 0 2 4 6 Time (hrs) Key trends in MFI Data: 1. PVC > PO 2. No PS20 > 0.015% PS20 3. PO + 0.015% PS20 ~ Background level 4. No difference in morphology vs. IV bag type 70-100

Characterization of Submicron Particles: NTA and TEM Dilute IgG4 mab into Different IV Bags (0.9% NaCl) PVC IV Bag PO IV Bag Key trends: 1. Low, measurable levels in NTA: PVC > PO 2. High levels PS20 in IV bags had background in NTA (not shown) 3. No difference in morphology vs. IV bag type by TEM

Summary of Aggregation and Particulate Data: Dilute IgG4 mab into Different IV Bags (0.9% NaCl) Condition SE-HPLC NTA MFI Turbidity Time 0 (% Soluble Aggregate) (% Total Area) (Size, nm) (10 6 particles/ml) (total >2 m/ml x10 4 ) (NTU) PVC 1.3% 0.1 100% 118 11 0.5 0.3 4.7 0.8 1.5 0.7 PO 1.2% 0.2 100% 108 2 0.4 0.2 6.7 4.2 1.9 0.5 PVC + PS20 1.4% 0.1 100% ND 1.0 0.5 0.6 0.1 PO + PS20 1.5% 0.1 100% ND 0.2 0.1 0.5 0.1 PVC No Headspace 1.4% 0.1 100% < LOQ 5.8 3.1 0.9 0.2 PO No Headspace 1.4% 0.1 100% < LOQ 1.0 0.5 0.9 0.1 Time 6hr PVC 0.9% 0.1 74.9% 0.7 131 7 1.7 1.5 350 45.9 55 5.7 PO 1.4% 0.1 91.3% 5.4 125 12 0.7 0.4 31 7.9 14 6.3 PVC + PS20 1.6% 0.1 99.7% 0.8 ND 5.0 0.6 23 7.7 PO + PS20 1.5% 0.1 99.4% 0.4 ND 0.3 0.2 0.6 0.1 PVC No Headspace 1.4% 0.1 99.2% 1.5 < LOQ 43 13.1 3.0 0.7 PO No Headspace 1.4% 0.1 99.1% 2.9 < LOQ 24 10.1 3.1 1.4 ND- No data. Samples containing PS20 interfered with NTA measurements. < LOQ: Particle concentration was below the limit of quantitation (LOQ)

Characterization of Particles by SDS-PAGE Dilute IgG4 mab into IV Bag (0.9% NaCl) S, supernatant P, pellet Disulfide cross-linked species observed in pelleted mab % Free thiol content of mab: 0.16% in native IgG4; 0.38% in GuHCl unfolded IgG4

Characterization of Particles by FTIR Microscope Second-derivative amide I bands of IgG4 particle from IV Bags,0.9% NaCl mab- Native control mab- PVC bag mab- PO bag mab- Heat-treated control Native control: Minima at 1636 cm -1, ~1690 cm -1 Heat aggregate control: IV bags aggregates: Minimum at 1624 cm -1, loss of two native minima Mostly native protein secondary structure (intramolecular -sheet)

Characterization of Particles by ANS Fluorescence Spectroscopy Fluorescence Intensity 480nm (cps) Dilute IgG4 mab into IV Bags (0.9% NaCl) 700000 650000 600000 550000 500000 450000 400000 350000 300000 250000 200000 150000 100000 50000 0 mab1 Stock PVC Sup PO Sup PVC Pellet PO Pellet mab1 Heat Native control: Heat-aggregate control: IV bags aggregates: Lower ANS fluorescence intensity Higher ANS fluorescence intensity Intermediate ANS fluorescence intensity (loss of some protein tertiary structure)

Lessons Learned from Case Study Properly designed formulation studies critical to support IV administration: Optimize analytical methods for testing samples Diluent only studies not sufficient Optimize formulation excipients not only for storage in primary container, but subsequent IV administration Effect of type of IV Bag and headspace Physical degradation can be minimized and controlled Nature of mab physical instability in IV bags with 0.9% NaCl diluent: Aggregates and particles of varying size and number can form Particles formed had partially altered tertiary structure with some nonnative S-S bonds, but still retained significant native secondary structure

Outline of Presentation Introduction Case Study with IgG4 mab Future Directions and Considerations Additional formulation design comments Challenges of compatibility assessments during development

Additional Formulation Design Considerations Different proteins have different stability properties Different formulations result in different stability profiles Different diluents result in different stability profiles Different IV bags and sets have different properties Polymer type Surface morphologies Leachable and impurity profile Manufacturing process/ vendors PO- Polyolefin PVC- polyvinyl chloride http://www.graylineinc.com/tubing-materials/

Our Case Study Covered Only Part of a Larger IV Compatibility Design Space IV Bags: Different Diluents Different Types of Plastic Adapted from: http://www.ebme.co.uk/arts/vis/index.htm IV Setup Varies: Volume and Flow Rate Bag size Transport (Agitation and Light) Storage (Time and Temperature) IV Administration Kits: Different Types of Plastic Different Filters https://shop.life-assist.com http://www.shopmedvet.com

Challenges of Compatibility Assessments During Development of Protein Therapeutics Early Development Preparing IV pharmacy manual for a new protein candidate Limited stability information Analytical methods in development Wide dose range in early clinical trials At the same time, typically limited number of clinical sites Later Development Preparing IV pharmacy manuals for larger trials with more (worldwide) clinical sites Supporting a larger IV compatibility design space with many variables that potentially could affect protein stability At the same time, more knowledge about protein stability, methods and dose.

Designing and Developing Protein Formulations During Clinical Development Compatibility and Delivery in Clinical Trials DS/DP Process Changes and Comparability Formulation Design and Development Analytical Method Changes and Stability Profiles

Summary of Presentation Introduction Case Study with IgG4 mab Future Directions and Considerations Encourage more scientific publications in this important area!!!

Acknowledgements Authors listed in published case study presented in talk Financial support from Genentech Special Acknowledgments: Genentech: KU: John Wang, Jun Liu Ozan Kumru, Sangeeta Joshi, Russ Middaugh

KU Macromolecule and Vaccine Stabilization Center Unique and innovative academic center specializing in the characterization, formulation and stabilization of vaccines as well as protein and DNA based pharmaceuticals. http://web.ku.edu/~mvslab/index.shtml

Thank you for your attention! Questions?