LER = Low Endotoxin Recovery LLR = Low Lipopolysaccharide Recovery and the LAL (Endotoxin Test) IVT Micro Week Meeting Cheryl Platco 1 14-16 Jun2017 Phila PA
What is LER? LLR • The observed masking of LPS standard (CSE,RSE) by the combination of Polysorbate containing Citrate and Phosphate platform buffer for monoclonal antibody preparations. • Enh/Inh issues are test interferences • LER/LLR is endotoxin related issues – Loss of Activity – Destruction of Activity – Masking of activity IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA 2
Why is LLR important? • The FDA questions sample validity for ability of activity assays to detect “endotoxin” or lipid A activity for inprocess and finished samples. • Theoretical Issue: – No missed pyrogenicity – No false negative LAL tests – (NEW concern) IIRMI • innate immune response modulating impurity IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA 3
What does this mean for firms? • The FDA is requiring masking studies for all mAb preparations • FDA requiring Rabbit Pyrogen test (also an activity test) if stability of assayable endotoxin cannot be demonstrated. • No evidence of patient safety issue. IVT Micro Week Meeting Cheryl Platco 4 14-16 Jun2017 Phila PA
Task Forces • PDA Parenteral Drug Association – Vendor members – Pharma members – FDA members – Academia members • BPOG Biological Phorum Operations Group – Pharma members • LAL user’s Group (Pharm Micro Forum) – Pharma members IVT Micro Week Meeting Cheryl Platco 5 14-16 Jun2017 Phila PA
Issue • FDA issued G uidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers , June 2012, U.S. Department of Health and Human Services, Food and Drug Administration. • Item #3: requires that manufacturers determine the stability of “assayable endotoxin” in their products. • Most products have no “ assayable endotoxin ” or native endogenous endotoxin • Intent was NOT to contaminate an otherwise clean product with surrogate contaminant, but one firm did. 6 IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA
Controversy • FDA requiring use of CSE as surrogate because its activity is defined. • IF FDA acceptance of NOE – (NOE:RSE/CSE standardization) makes LER a non- issue. LER then IS LLR. • CSE activity rapidly affected in direct contact with many undilute product matrices. • Natural endotoxin is demonstrating no/little activity reduction (no/low masking). – Aggressive matrices may destroy activity IVT Micro Week Meeting Cheryl Platco 7 14-16 Jun2017 Phila PA
Control Standard Endotoxin (CSE) versus Natural Occurring Endotoxin (NOE) • CSE is a chemically purified LPS (mostly). • CSE activity is used to calibrate the Lysate of the LAL test against RSE, also LPS. • CSE is for confirmation of test validity (inhibition/enhancement, PPC recovery). • NOE is Gram negative microbe parts and pieces. IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA 8
Note: LPS must exist as aggregates to be biologically active. IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA
IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA
Mechanisms by Humpty Dumpty Something prevents activation of lysate by Lipid A of LPS molecule(s) Masking: may be reversible if can unmask without destroying activity of analyte Destruction: not reversible, LPS molecule activity destroyed, LPS may no longer be intact “all the kings horses........” Aggregation state changes: may be reversible, have to re-aggregate for activity, may not reaggregate to same activity as original PDA Arlington VA Cheryl Platco 27October 2016 11
mAb Platforms Buffer CSE NOE Histidine + + Citrate - + Phosphate +/- + 12 IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA
Success ! ? ! • Depends on mechanism causing failure to recover activity • Issue is all about sample pretreatment prior to testing. • Try simple solutions first – Buffers – Cation replacement – Experimental design • Determine what dilution of cation replacement buffer has good enhancement/inhibition profile (varies among vendors) • Prepare the product dilutions (contaminated with CSE/LPS) in the cation buffer as diluent. Vortex intermittently for 10 minutes. IVT Micro Week Meeting Cheryl Platco 13 14-16 Jun2017 Phila PA
Recommendations for LLR Studies • Best using kinetic testing. Gel clot results difficult to interpret. • Run enh/inh test and determine non interfering concentration of uncontaminated product. • Add concentrated CSE/LPS directly to aliquots of undilute product at a volume not to exceed 1% total. – CSE stock activity should be at about 1000 EU/mL. – The resultant activity after dilution for testing should be at approximately ½ to ¾ of the standard curve range. • Contaminate product starting with the longest time first, then down to T=0 so that all testing can occur at T=0. • Store samples at 2-8C between contamination events. • Do not overvortex or use same vessel repeatedly for testing. • Run set of contaminated water controls in same manner as above. • Compare activity data from product to activity data from water controls. IVT Micro Week Meeting Cheryl Platco 14 14-16 Jun2017 Phila PA
Study Design Example (7 day test) Place 5mL of product (neat, 1:1) into each of 7 vials Prepare same set for water controls (Prepare a set for CSE and NOE) Sample PFW Day 7 Day5 Day3 Day2 Day1 4hour Time=0 IVT Micro Week Meeting Cheryl Platco 15 14-16 Jun2017 Phila PA
Time for Some Math! Artificially contaminate each vial starting with the longest time. Store 2-8C between contamination events. Desired level after product dilution (1:10) : midpoint of 5.0 to 0.05 EU/mL standard curve) Want product contamination to be at approximately 5- 10 EU/mL. Let’s use 10. How many mcL of stock contaminant to add for 10EU/mL contaminated product which “ after dilution ” for testing will read 1.0 to 0.5 EU/mL from standard curve? CSE stock 1000 EU/mL NOE stock 768 EU/mL 5mL product Formula: V 1 X C 1 = V 2 X C 2 CSE V 1 X 1000 EU/mL = 5mL X 10 EU/mL V 1 = 0.05mL or 50mcL NOE V 1 X 768 EU/mL = 5mL X 10 EU/mL V 1 = 0.065mL or 65mcL IVT Micro Week Meeting Cheryl Platco 16 14-16 Jun2017 Phila PA
Vortex CSE and NOE (use same stock vial source) before contaminating samples. Store samples 2-8C. Minimize vortexing contaminated samples until T=0 test day. CSE Sample NOE CSE PFW NOE Day 7 Day5 Day3 Day2 Day1 4hour Time=0 Dec 01 Dec 03 Dec 05 Dec 06 Dec 07 Dec 08 Dec 08 Test day is Dec 08 IVT Micro Week Meeting Cheryl Platco 17 14-16 Jun2017 Phila PA
Test day! T=0 Immediately dilute T=0 after contamination. Vortex and dilute all timepoints. Try to test samples all on same test run. PPC values are not valuable (already proven that 1:10 dilution PPC are valid) IVT Micro Week Meeting Cheryl Platco 18 14-16 Jun2017 Phila PA
Results (EU/mL) 2.0 1.8 2.3 1.5 4.2 7.8 7.2 Sample 8.9 8.8 9.2 11.5 8.0 9.8 9.2 CSE PFW 13.0 12.9 10.0 11.5 13.3 12.0 9.9 Sample NOE 12.0 11.9 10.5 13.5 11.3 11.0 12.8 PFW Day 7 Day5 Day3 Day2 Day1 4hour Time=0 Dec 01 Dec 03 Dec 05 Dec 06 Dec 07 Dec 08 Dec 08 IVT Micro Week Meeting Cheryl Platco 19 14-16 Jun2017 Phila PA
IVT Micro Week Meeting Cheryl Platco 20 14-16 Jun2017 Phila PA
IVT Micro Week Meeting Cheryl Platco 21 14-16 Jun2017 Phila PA
IN-VITRO / IN-VIVO ANALYSES WITH NOES Tween/Citrate matrix Approx. 20 EU/mL Cumulative Temp USP 3-Rabbit LAL Results Ligand Results NOE Increase (º C) Pass/Fail EU/ml EU/ml Dose 1mL/kg 17hr binding E. cloacae 1.4 Fail 22.1 0.2 E. coli 2.1 Fail 15.5 20.7 S. marcescens 1.4 Fail 23.7 0.1 R. pickettii 1.3 Fail 26.3 1.1 P. aeruginosa 0.8 Pass 11.1 2.1 Tween/Citrate/ 0.1 Pass <0.5 <5.0 Saline Control USP defines a 3 rabbit failure as having a single rabbit with a temperature increase of 0.5 degrees or greater. IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA 22
Rabbits cannot respond to inactive LPS(RSE) 24 Hours TIME ZERO TIME RABBIT DATA RABBIT DATA 50 EU/mL 1mL/kg permission from J. Dubczak, CRE 23 IVT Micro Week Meeting Cheryl Platco 14-16 Jun2017 Phila PA
IVT Micro Week Meeting Cheryl Platco 24 14-16 Jun2017 Phila PA
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