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

Abstract

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 drug'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 degrees 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.

Antitumor activity of an epithelial cell adhesion molecule–targeted nanovesicular drug delivery system

“…The successful large-scale production of HER2-specific immunoliposomes (19) has finally paved the way also for the clinical use of tumor-targeted nanovesicles. In addition to the aforementioned targets, EpCAM also holds promise as a target for liposomal drug delivery because it is abundantly expressed in many solid tumors and shows limited expression in normal epithelial Other treatment groups included HEPES-buffered saline, nontargeted liposomal doxorubicin (SL-Dox) given at the same dose, and free doxorubicin given at its MTD of 7.5 mg/kg.…”

“…Promising results were achieved with immunoliposomes targeting CD19, HER2/ neu, epidermal growth factor receptor, disialoganglioside (GD 2 ), and prostate-specific membrane antigen expressed on various tumor cell types (8, 9, 16 -18). Based on the promising preclinical data and advances made in terms of large-scale production (19), HER2-specific immunoliposomes are on their way to clinical trials.…”

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

“…This method is more conducive to scale-up of manufacturing of immunoliposomal drugs because an antibody-lipid conjugate can easily be inserted into an approved liposomal anticancer drug [81,82]. In addition, for antibody constructs with low storage stability (e.g., scFv), coupling to PEG-lipid micelles may increase stability of the antibody constructs and facilitate retaining activity during storage (see below).…”

“…Although this method will not increase the intrinsic stability of an scFv construct, it is postulated that aggregation of other scFv constructs could be prevented by conjugation to micelles, allowing for scale-up of their production. In fact, an anti-HER2 scFv fragment that is intended for clinical trials of immunoliposomal anticancer drugs is manufactured and coupled to PEG-DSPE micelles, and stored as the scFv-PEG-DSPE conjugate [81].…”

“…Despite the reduced amount of scFv needed for targeting of DDS compared with use as a monotherapy, it can be expected that an amount in the hundred grams to kilograms range will be needed for clinical trials and clinical use. Storage stability is also a major issue that needs to be addressed, although great advances have been made [81,82].…”