Preclinical Manufacture of an Anti-HER2 scFv-PEG-DSPE, Liposome-Inserting Conjugate. 1. Gram-Scale Production and Purification David F. Nellis, † Denise L. Ekstrom, † Dmitri B. Kirpotin, ‡,| Jianwei Zhu, † Robert Andersson, † Trevor L. Broadt, † Timothy F. Ouellette, † Shelley C. Perkins, † John M. Roach, † Daryl C. Drummond, ‡,| Keelung Hong, ‡,| James D. Marks, § John W. Park, § and Steven L. Giardina* ,† SAIC-Frederick, Inc., National Cancer Institute at Frederick, P.O. Box B, Frederick, Maryland 21702, Liposome Research Laboratory, California Pacific Medical Center Research Institute, San Francisco, California 94115, Departments of Medicine and Anesthesia, University of California-San Francisco, San Francisco, California 94143, and Hermes Biosciences, Inc., South, San Francisco, California 94080 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 °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. Introduction Studies over the past decade have demonstrated the in vivo advantages of targeted anti-cancer drug delivery via immunoliposomes for both hematological malignan- cies and solid tumors (1-3). In this approach, antigens enriched on carcinoma surfaces are identified and mono- clonal antibodies are generated to target them. One such antigen is the p185 HER2/neu glycoprotein (HER2), a member of the epidermal growth factor receptor family (EGF-R) of receptor tyrosine kinases encoded by the HER2/neu (c-erb-2) proto-oncogene (4, 5). This antigen is suitable for targeting because although HER2 is expressed in healthy tissue, overexpression is unique to malignancy in some human breast cancers (25-30% of cases), gastric, colon, ovarian, and non-small-cell lung carcinoma (6). The F5 single-chain Fv antibody fragment was selected using a phage-display library: human IgM and IgG variable region fragments were screened for internalization into carcinoma upon binding the HER2 cell surface receptor (7-9). The resulting antibody frag- ment, F5, has been shown to specifically stimulate uptake of attached therapeutic agents into carcinoma (10). The F5cys derivative of F5 is expressed in E. coli strain RV 308 (11), under the control of an E. coli K12 alkaline phosphate promoter (PhoA), with soluble F5cys directed to the periplasmic space by a fused pelB leader peptide (12, 13), which is removed upon translocation across the periplasmic membrane. This expression system is com- patible with high-density bacterial cell culture (14). The F5cys construct contains immunoglobulin heavy- and light-chain variable regions linked by a [-(Gly) 4 -Ser-] 3 * To whom correspondence should be addressed. Ph: +1-301- 846-1821. Fax: +1-301-846-6886. E-mail: giardina@mail.ncifcrf.gov. † SAIC-Frederick, Inc. ‡ California Pacific Medical Center Research Institute. § University of California-San Francisco. | Hermes Biosciences, Inc. 205 Biotechnol. Prog. 2005, 21, 205-220 10.1021/bp049840y CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 12/04/2004