Materials and processing

A preparation of recombinant infliximab, from a single batch of bulk drug substance was kindly donated to WHO (see Acknowledgement) and a commercial batch of Remicade® (Janssen) was purchased to act as a comparator.

Trial fills were conducted using two different formulations; A) 10 mM Tris, pH 7.4, 4% D-mannitol, 1% sucrose, 0.2% HSA; and B) 25 mM sodium citrate tribasic dihydrate, pH 6.5, 150 mM sodium chloride, 1% HSA. The biological activity of the lyophilized preparations was compared with the bulk material in a cytotoxicity assay and a reporter gene assay.

Final lyophilizations of the candidate and comparator B were carried out at NIBSC using WHO guidelines.⁴ For this, buffers and excipients, were prepared using nonpyrogenic water and depyrogenated glassware and solutions filtered using sterile nonpyrogenic filters (0.22 µm Stericup filter system, Millipore, USA) where appropriate.

The two preparations were coded as described in the supplementary material, Table S2. The mass content of the protein in the ampoules, given as ‘predicted µg’ is calculated from the dilution of the bulk material of known protein mass content as provided by the manufacturer.

For both preparations, a solution of infliximab at a theoretical protein concentration predicted to be 50 µg/ml was distributed in 1 ml aliquots into 5 ml ampoules and lyophilised under optimised and controlled conditions. The glass ampoules were sealed under dry nitrogen by heat fusion and stored at −20°C in the dark until shipment at room temperature.

For each fill, a percentage of ampoules were weighed, residual moisture of each preparation was measured by the coulometric Karl-Fischer method (Mitsubishi CA100) and the headspace oxygen content was determined by frequency modulated spectroscopy using the Lighthouse FMS-760 Instrument (Lighthouse Instruments, LLC). The mean fill weights, moisture content and headspace oxygen content, which is a measure of ampoule integrity, are reported in the supplementary material, Table S2. Testing for microbial contamination using the total viable count method did not show any evidence of microbial contamination.