SPIKE RECOVERY


1. Screening for BET Spike Recovery from Undiluted Protein Solutions


Development tests were performed on two diff erent mAb solutions to answer the question: “Is there a spike recovery problem with this drug solution?” As purifi ed endotoxin has long been considered worst case in terms of clinging and binding to container/closure surfaces due to its hydrophobic lipid A/fatty acid end, likewise its recovery from undiluted products has historically represented a worst case recovery scenario as the hydrophobic end is not associated with cellular debris as is naturally


occurring, non-purifi ed endotoxin (NOE).


The screens below involved


adding CSE at low levels (<10 EU/mL) to undiluted drug product and testing for recovery over time. Results are shown using a lyophilized (reconstituted) mAb (Table 1) and a liquid mAb (Tables 2/3).


mAb A (Table 1)


The data for mAb A showed no diffi culty in recovering low level endotoxin spikes over the test series using the addition of and continued dilution in dilute surfactant (Table 1).


Table 1. Reconstituted (lyophilized) mAb endotoxin spike into undiluted product. Formulation includes polysorbate. Tested via Kinetic Turbidimetric.


Test Parameter Recovery in EU/mL %PPC (in-plate spike)


% of spike recovered (from undiluted product) Reconstitution method


20 mL vial recon - 20 mL Sterile Water for Injection (SWFI). Added 50 microliters undiluted surfactant post spike and reconstitution.


Day 0 0.265 112 106


Spike Theory


1.0 mL 50 EU/mL CSE = 50EU / 21mL 1:1 = 2.38 EU/mL 1:10 = 0.238 EU/mL


Day 1 0.256 78


107.6


Day 3 0.257 78


107.9 Dilution


Tested 1:10 in 0.5% surfactant


Day 4 0.259 68


108.8 Storage


Samples refrigerated at 5˚C between runs and held in a box (dark). All vortexing is 1 minute.


Day 7 (week later) 0.221 63


92.8


Tested via Kinetic Turbidimetric


Day 0 Series


1 2 3 4


Day 1 1 2 3 4


Day 4 1 2 3 4


Day 5 1 2 3 4


Dilution 1:50


1:100 1:50


1:100


Table 2. Liquid mAb endotoxin spike into undiluted product. Formulation includes Polysorbate + Citrate. Sample Reconstitution / Spike / Diluents / Dilution Scheme


EU/mL recovery


0.144


0.0686 0.146


0.0694 %PPC 98


109 90


110


% spike recovery


80


76.2 81.1 77.1


Liquid sample


No surfactant added. 10 mL in original vial. CSE Spike Theory


1mL of 100 EU/mL CSE into 10 mL = 100 EU per 11mL= 9 EU/mL 1:10 = 0.9 EUmL 1:50 = 0.18 EU/mL 1:100 = 0.09 EU/mL Test Iterations


Tests 1, 2 1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100 1:50


1:100


Day 7 (week later) 1 2


1:10 1:50


0.121


0.0625 0.122


0.0625


0.113 0.098


0.0855 0.0556


0.102


0.0914 0.0576 0.0527


0.0755 0.0146


91


109 88


107


92 94 88


107


100 104 111 110


113 106


67.2 69.4 67.8 69.4


62.8


108.8 47.5 61.8


56.6


101.6 27.7


58.55


8.3 8.1


Note that by day 7 dwindling recovery tested at 1:10 and 1:50 in 0.5% surfactant (3 and 4 failed at days 4 and 5).


Test 1:50 and 1:100 diluted in 0.5% surfactant. Tests 3, 4 Test 1:50 and 1:100 diluted in 1% surfactant 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 Storage - Samples refrigerated at 5˚C between runs and held in a box (dark). Vortexing is 1 minute between dilutions and for undiluted solutions.


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