SPIKE RECOVERY


Table 3. Recovery for Liquid mAb B Vial --formulation includes Polysorbate + Citrate. Tested by Kinetic Turbidimetric


Day 0 Series Dilution EU/mL Recovery %PPC % spike recovery Liquid Sample 1 2 3 4 5 6


1:50


1:100 1:50


1:100 1:50


1:100


Day 1 1 2 3 4 5 6


Day 4 1 2 3 4 5 6


Day 5 1 2 3 4 5 6


1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100 1:50 0.110


0.0448 0.125


0.0565 0.111


0.0526 0.108


0.0488 0.117


0.0537 0.114


0.0555


0.0845 0.0461 0.0845 0.0452 0.0921 0.0497


0.0991 0.0391 0.0939 0.0435 0.0977 0.0479


Day 7 (week later) 1 2 3 4 5 6


1:100 1:50


1:100 1:50


1:100


0.0737 0.0341 0.0852 0.0391 0.0835 0.0393


mAb B-Test 1 (Table 2)


Diffi culties with both logistics and spike recovery were experienced in MAb B-Test 1 (Table 2). In terms of logistics, early tests to establish the amount of spike to add and the amount of subsequent dilution to use relative to MVD showed poor and inconsistent recovery (0-60%). One wants to add a low but assayable amount of spike, while allowing enough dilution to overcome product interference. The interference properties of some drugs may require dilution that necessitates very low recovery of spike. Logistics should be worked out to ensure that recovery achieved is a metric of


6 American Pharmaceutical Review | Endotoxin Supplement 2013


71 90 98


97.1 79.2


110.4


115 99.8 100 98.0 106 92.9


65 88 91


95.6 86.2


103.4


110 94.9 89


113 98.1


83 77


74.6 81.4


107 74.6 100 79.9 101 81.4 109 87.8


65 88 97


87.5 69.1 83.0


113 76.9 67


86.3 124 84.6


68 86 95


65.1 60.2 75.3


108 69.1 92


73.8 110 69.4


protein binding rather than due to interference from an under-diluted test. Results shown for mAb B were improved from earlier recovery eff orts, yet the recovery still dropped off moderately at day 5 and drastically at day 7. The reduction in spike over time may be due to protein aggregation and/or the generation of surfactant-generated peroxides [6].


mAb B-Test 2 (Table 3)


The fi nal set of study results show improved recoveries due to changes in diluent combinations while using almost half the previous spike


Snapshot of % recoveries obtained from Tables 1, 2, and 3 over 7 day test period. 100.7


Sample Reconstitution / Spike / Diluents / Dilution Scheme


20 mL in original vials. Prepared separate vials for sets1/2, 3/4, and 5/6. Vial for 1/2 added 50 microliters of surfactant and others did not (3/4 and 5/6). Spike


1.2 mL of 100 EU/mL CSE = 120 EU /21.2mL = 5.66 EU/mL Spike Theory 1:1 = 5.66 EU/mL 1:10 = 0.566 EU/mL 1:50 = 0.1132 EU/mL 1:100 = 0.0566 EU/mL


Test Variations


Tests 1, 2 -50 microliters surfactant added to sample vial only at Day 0. Tested 1:50 and 1:100 as diluted in 1% surfactant


3, 4 -No Surfactant added to vial - Diluted in 1% surfactant cut to 0.5% using equal volume 100 mM Tris buffer 5, 6 -No surfactant added to vial - Diluted in 1% surfactant prepared in 10 mM MgCl2


Storage


All samples refrigerated at 5˚C between runs and contained in a box (dark). Vortexing is 1 minute between dilutions and for undiluted solutions.


Dilution Scheme


1:10 is 0.25 mL sample into 2.25 mL diluent 1:50 is 0.5 of 1:10 into 2.0 mL diluent 1:100 is 1 mL of 1:50 into 1.0 mL diluent


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