PDA: A Global Association New Control Strategies for Monitoring of Endotoxin Masking in Biologics Basel, 24.09.2014 Johannes Reich Outline 1) Background 2) Endotoxin masking 3) Endotoxin demasking 4) Take home message 2 Background LER = Low Endotoxin Recovery (Endotoxin Masking) Ref.: R. Mello, 2014 3 Background NOE = Naturally Occurring Endotoxin Ref.: R. Mello, 2014 4 Background Ref.: R. Mello, 2014 5 Background Clinically relevant? - Safety database: Too many “cofounding variables” at this time - LAL vs. RPT: Contrary results Ref.: R. Mello, 2014 6 Background Requests from FDA for biologics: … provide evidence that the bulk drug substance and drug product formulation (containing phosphate, citrate, polysorbate, etc.) does not interfere with endotoxin recovery of the LAL test … … conduct spiking studies on the undiluted drug substance and drug product with known amount of endotoxin and simulate the worstcase hold conditions to evaluate endotoxin masking over time … … based on the results of the studies, modify the endotoxin release test and/or determine the suitability of alternative endotoxin test methods … 7 Background Endotoxin •Endotoxins are breakdown products of Gram-negative bacteria •Chemically, endotoxins are Lipopolysaccharides (LPS) •Lipid A is the highly toxic unit •Biological activity and detectability varies with aggregation state Structure: • • Conserved Lipid A and core Heterogeneous O-antigen Physical properties: • • • • Ionic Amphiphilic Surface active Self assembling +X +X A c t i v i t y 8 Background Loss of endotoxin detectability (Low Endotoxin Recovery) Endotoxin masking monitoring 100*[Endotoxin]H2O,t0/[Endotoxin]Sample,tx Undiluted sample spiking 2. Incubation (0.5h, 1d, 3d,…) at RT 3. Sample preparation (dilution) 4. Detection Recovery [%] 1. Recovery [%] = 100 90 80 70 60 50 40 30 20 10 0 0 4 40 Time 9 Masking Common formulation components of MABs Commercial Drug: Active Ingredient: Formulation: Remicade Infliximab Phosphate, Polysorbate, Sucrose Avastin Bevacizumab Phosphate, Polysorbate, Trehalose Benlysta Belimumab Citrate, Polysorbate, Sucrose Humira Adalimumab Phosphate, Citrate, Polysorbate, Mannitol, Sodiumchloride Soliris Eculizumab Phosphate, Polysorbate, Sodiumchloride Tysabri Natalizumab Phosphate, Polysorbate, Sodiumchloride Mabthera Rituximab Citrate, Polysorbate, Sodiumchloride Actemra Tocilizumab Phosphate, Polysorbate, Sucrose Single components Formulation Multiple components Recovery [%] Formulation Recovery [%] Water 100 Citrate buffer + Polysorbate 80 <1 Citrate buffer, 10 mM 100 Citrate buffer + Polysorbate 20 <1 Phosphate buffer, 10 mM 75 Phosphate buffer + Polysorbate 80 <1 Polysorbate 80, 0,05 % (w/v) 88 Phosphate buffer + Polysorbate 20 <1 Polysorbate 20, 0,05 % (w/v) 84 10 Masking Substantial doses of endotoxin “disappear” 50000 13.900 Recovery [EU/mL] 5000 500 65,0 50 5 1,25 0,5 0,05 <0,005 <0,005 <0,005 250 25 2,5 0,005 250000 25000 2500 Endotoxin Spike [EU/mL] Material & Methods: • Endotoxin: X EU/mL E.coli O55:B5 • Buffer: 10 mM Sodium citrate (pH 7,5) • • • Surfactant: 0,05 (w/v) % Polysorbate 20 Masking: 7 days, room temperature Detection: EndoZyme® 11 Masking Endotoxin recovery depends on formulation condition Temperature (citrate + polysorbate) 100,00 4°C 80 RT 60 Recovery [%] Recovery [%] 100 Surfactant (+citrate) 37°C 40 20 80,00 60,00 40,00 20,00 0,00 0,00001 0 0 4 time [hours] Buffer (+ polysorbate) 10 mM Citrate RT 60% 10 mM Phosphate RT 20% 0% 0 100 200 300 0,01 0,1 1 Endotoxin: 100 EU/mL E.coli O55:B5; Buffer: 10 mM Sodium citrate (pH 7,5), Surfactant: X (w/v) % Polysorbate 20; Masking: 7 days, room temperature; Detection: EndoZyme® pH (buffer + polysorbate) 400 time [min] Endotoxin: 100 EU/mL E.coli O55:B5; Buffer: 10 mM Sodium citrate, 10 mM Sodium phosphate, 5 mM EDTA, Surfactant: 0,05 (w/v) % Polysorbate 20; Masking: X min, room temperature; Detection: EndoZyme® Recovery [%] Recovery Active Pharmaceutical Ingredient 5mM EDTA RT 80% 40% 0,001 Polysorbate (v/v) [%]] Endotoxin: 100 EU/mL E.coli O55:B5; Buffer: 10 mM Sodium citrate (pH 7,5), Surfactant: 0.05 (w/v) % Polysorbate 20; Masking: X hours, 4°C, room temperature, 37°C; Detection: EndoZyme® 100% 0,0001 40 200 180 160 140 120 100 80 60 40 20 0 Phosphate Citrate 0 2 4 6 pH 8 10 12 14 Endotoxin: 100 EU/mL E.coli O55:B5; Buffer: 10 mM Sodium citrate, 10 mM Sodium phosphate, Surfactant: 0,05 (w/v) % Polysorbate 20; Masking: 7 days, room temperature; Detection: EndoZyme® 12 Masking Heterogeneity of endotoxin preparations Recovery [%] 100 10 1 NOE E.coli O55:B5 NOE E.coli O113 NOE E.cloacae NOE E.cloacae (EDTA) NOE B.capeci LPS E.coli O55:B5 0 0 1 2 Materials & Methods: • Endotoxin: 100 EU/mL Endotoxin • Buffer system: 10 mM Sodium citrate (pH 7.5) 3 4 Incubation time [days] • • • 5 6 7 Surfactant: 0.05 (w/v) % Polysorbate 20 Masking: 7 days, room temperature Detection: EndoZyme® 13 Masking Masking principle Endotoxin masking is a two-step mechanism: – Destabilization of LPS-aggregates (1) – Change of LPS-aggregation state (2) Factor C can not be activated •Cooperative binding mechanism (Tan et al., 2000) •Endotoxin-aggregates are the active state (Müller et al., 2004) 14 Masking Summary • Masking is depending on: - time - temperature - pH - buffer - surfactant - protein • Different endotoxins show different masking kinetics • High masking capacities in common formulations • Endotoxin masking ≠ Test interference 15 Demasking Challenge: Endotoxin re-arrangement into Limulus lysate active state + + Energy + “de-masking” Active LPS Active LPS masked LPS Reaction pathway 16 Demasking Polysorbate - Citrate 100 EU/mL 6 EU/mL 100% Recovery [%] Recovery [%] 100% 10% 10% 1% 1% Positive Control 1 2 Masked 3 7 time [days] Positive Control 1 Masked De-Masked 2 3 time [days] 7 De-Masked 25 EU/mL Recovery [%] 100% Materials & Methods • Endotoxin: E.coli O55:B5 • Buffer system: 10 mM Sodium citrate (pH 7.5) • Surfactant: 0.05 (w/v) % Polysorbate 20 • Masking: 1 – 7 days, at 4 °C • De-Masking: after 1- 7 days, 1 hour • Detection: EndoLISA® 10% 1% Positive Control 1 Masked 2 3 time [days] 7 De-Masked 17 Demasking Polyclonal Antibody Phosphate + NaCl + Polysorbate 20 (e.g. formulation similar to Avastin ®) 2. Endotoxin in sample 3. Demasking 120 120 100 100 100 80 80 80 60 40 Recovery [%] 120 Recovery [%] Recovery [%] 1. Endotoxin in H2O 60 40 60 40 20 20 20 0 0 H2O Materials & Methods • Endotoxin: 50 EU/mL E.coli O55:B5 • Buffer : 10 mM Sodium Phosphate(pH 7.5), 50 mM NaCl • Surfactant: 0,05 (w/v) % Polysorbate 20 Buffer + PAK + Polysorbate 20 • • • • 0 De-masking Protein: 10 mg/mL bovine polyclonal IgG antibody (PAK) Masking: 3 days at room temperature De-Masking: 1 hour Detection: EndoLISA® 18 Demasking Polyclonal Antibody Citrate + NaCl + Polysorbate 20 2. Endotoxin in sample 3. Demasking 120 120 100 100 100 80 60 40 Recovery [%] 120 Recovery [%] Recovery [%] 1. Endotoxin in H2O 80 60 40 80 60 40 20 20 20 0 0 H2O Materials & Methods • Endotoxin: 50 EU/mL E.coli O55:B5 • Buffer : 10 mM Sodium Citrate (pH 7.5), 150 mM NaCl • Surfactant: 0,05 (w/v) % Polysorbate 20 Buffer + PAK + Polysorbate 20 • • • • 0 De-masking Protein: 10 mg/mL bovine polyclonal IgG antibody (PAK) Masking: 3 days at room temperature De-Masking: 1 hour Detection: EndoLISA® 19 Demasking Contaminated Monoclonal Antibody (e.g.: formulation similar to Mabthera®) 10 Recovery [%] Endotoxin [EU/mg] 12 8 6 4 2 2. Addition of Polysorbate 3. Demasking 200 200 150 Recovery [%] 1. Solvation of MAK33 in Citrate/NaCl 100 50 150 100 50 0 0 MAK33 Endotoxin contamination source unknown (“NOE”) Materials & Methods • Endotoxin: 11 EU/mg (naturally occurring endotoxin) • Buffer : 25 mM Sodium Citrate(pH 7.5), 150 mM NaCl • Surfactant: 0,07 (w/v) % Polysorbate 80 Buffer + MAK + Polysorbate 80 0 De-masking Masked endotoxin Demasked endotoxin contamination contamination • • • • Protein: 10 mg/mL mouse monoclonal antibody (MAK 33) Masking: 3 days at room temperature Demasking: 1 hour Detection: EndoLISA® 20 Demasking Concept A. B. C. D. + A.: Destabilization of masking complex B.: Adsorption of surfactants C.: Modulation of adsorber D.: Disruption and reconfiguration of endotoxin aggregates LPS: Polysorbate: A: B: C: D: 21 Demasking Masking conditions need their solution Endotoxin masking Formulation API Component: Example: Properties: Chelator: Citrate vs. EDTA pH, denticity, etc. Surfactant: Polysorbate vs. Triton HLB, CMC, etc. API: Antibody vs. Lysozyme Charge, pI, hydrophobic interfaces, etc. Endotoxin demasking Concept for demasking Destabilization of masking complex Formulation API Adsorption of surfactants Modulation of adsorber Disruption and reconfiguration of endotoxin aggregates 22 Take home message • Simulate worst-case conditions for hold time studies (FDA) • Endotoxin masking is depending on sample composition • Masking capacity of common formulations is high (10000 EU/mL) • Demasking is a sample preparation step • Specific demasking protocol for each product recommended • Masking is a reversible process! • Endotoxin masking is not irreversible, thus the endotoxin is not detoxified 23 Acknowledgement Universität Regensburg Prof. Dr. H. Motschmann Dr. H. Grallert Dr. W. Mutter M. Vogl K. Heinzelmann 24 References T. Nakamura, F. Tokunaga, T. Morita, S. Iwanaga, Interaction between Lipopolysaccharide and Intracellular Serine Protease Zymogen, Factor C, from Horseshoe Crab (Tachypleus tridentatus) Hemocytes, The Journal of Biochemistry, 103, 370-374, 1988 M. Mueller, B. Lindner, S. Kusumoto, K. Fukase, A. Schromm, U. Seydel, Aggregates Are the Biologically Active Units of Endotoxin, The Journal of Biological Chemistry 279, 25, 26307-26313, 2004 N. Tan, M. NG, Y. Yau, P. Chong, B. Ho, J. Ding, Definition of endotoxin binding sites in horseshoe crab Factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides, The FASEB Journal, 14, 12,1801-1813, 2000 R. Mello, LER: An FDA Reviewer’s Perspective, PMF Bacterial Endotoxin Summit, Philadelphia, 2014 25
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