Materials

Equipment and Consumables

A Pilot PAK end-to-end continuous purification system from PAK BioSolutions (Virginia, US) was used to perform all purification process steps. Along with the Pilot PAK system were four Pilot PAK flow kits from PAK BioSolutions (Virginia, US) that were used to link unit operations together. MabSelect PrismA® and Sephadex G-25 Coarse® size exclusion chromatographic (SEC) resins were purchased from Cytiva (Massachusetts, US). The three chromatography column housing units were Vantage L Laboratory Column VL 44 x 250 from MilliporeSigma (Massachusetts, US). The Opticap Sterile 0.2µm filter and the Mustang Q Membrane Capsule were purchased from MilliporeSigma (Massachusetts, US) and Pall (New York, US) respectively. Tryptic Soy Agar plates were purchased from VWR (Pennsylvania, US).

Buffers and Solutions

The buffer system utilized for dual-column capture chromatography (MabSelect PrismA®) included six buffers: (1) 50 mM Tris, pH 7.4; (2) 50mM Tris, 0.5 M NaCl, pH 7.4; (3) 25mM sodium acetate, pH 3.6; (4) 100mM acetic acid (5) 1N NaOH and (6) 2% benzyl alcohol, 100 mM sodium acetate, pH 5.0. For the Viral Inactivation (VI) step, the solution system utilized 1M acetic acid and 1M tris base. All buffers were tested for bioburden and only qualified for use with negative results.

Protein

The monoclonal antibody (mAb1) used in the continuous process studies was a humanized IgG1 produced from Chinese hamster ovary (CHO) cells by AstraZeneca. This protein has a molecular weight of 146 kDa, as determined experimentally by mass spectroscopy and has a theoretical isoelectric point of 7.15, as determined by amino acid sequencing.
The monoclonal antibody (mAb2) used in viral clearance studies was a humanized IgG1 produced from Chinese hamster ovary (CHO) cells by AstraZeneca. The mAb2 intermediate (17 mg/mL, pH 4.5) purified by protein A chromatography was used in the virus inactivation study as starting material.