Preclinical Manufacture of an Anti-HER2 scFv-PEG-DSPE, Liposome-Inserting Conjugate. 1. Gram-Scale Production and Purification

A GMP‐compliant process is described for producing F5cys‐PEG‐lipid conjugate. This material fuses with preformed, drug‐loaded liposomes, to form ”immunoliposomes” that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drugapos;s therapeutic index. The soluble, single‐chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high‐density cultures. Affinity adsorption onto horizontally tumbled Streamline rProtein‐A resin robustly recovered F5cys from high‐pressure‐disrupted, whole‐cell homogenates. Two product‐related impurity classes were identified: F5cys with mid‐sequence discontinuities and F5cys with remnants of a pelB leader peptide. Low‐pressure cation exchange chromatography, conducted at elevated pH under reducing conditions, enriched target F5cys relative to these impurities and prepared a C‐terminal cysteine for conjugation. Site‐directed conjugation, conducted at pH 5.9 ± 0.1 with reaction monitoring and cysteine quenching, yielded F5cys‐MP‐PEG(2000)‐DSPE. Low‐pressure size exclusion chromatography separated spontaneously formed, high‐molecular‐weight conjugate micelles from low‐molecular‐weight impurities. When formulated at 1–2 mg/mL in 10 mM trisodium citrate, 10% sucrose (w/v), at pH 6.4 (HCl), the conjugate was stable when stored below −70 °C. Six scale‐up lots were compared. The largest 40‐L culture produced enough F5cys to manufacture 2,085 mg of conjugate, enough to support planned preclinical and future clinical trials. The conjugate was 93% pure, as measured by polyacrylamide gel electrophoresis. Impurities were primarily identified as product‐related. Residual endotoxin, rProtein A, and genomic DNA, were at acceptable levels. This study successfully addressed a necessary step in the scale‐up of immunoliposome‐encapsulated therapeutics.