Preclinical Manufacture of Anti-HER2 Liposome-Inserting, scFv-PEG-Lipid Conjugate. 2. Conjugate Micelle Identity, Purity, Stability, and Potency Analysis David F. Nellis, † Steven L. Giardina,* ,† George M. Janini, † Shilpa R. Shenoy, § James D. Marks, | Richard Tsai, | Daryl C. Drummond, ‡,⊥ Keelung Hong, ‡,⊥ John W. Park, | Thomas F. Ouellette, † Shelley C. Perkins, † and Dmitri B. Kirpotin ‡,⊥,# 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, Molecular Targets Development Program, Center for Cancer Research, NCI-Frederick, Frederick, Maryland 21702, Departments of Medicine and Anesthesia, University of California-San Francisco, San Francisco, California 94143, and Hermes Biosciences, Inc., South, San Francisco, California 94080 Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein- lipopolymer conjugate are presented. The apparent micelle molecular weight, deter- mined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorim- etry, were near 82 °C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot- to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody- directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investiga- tions. Introduction The development and manufacture of a protein phar- maceutical requires state-of-the-art analytical method- ologies to characterize protein structure and to ensure preparation homogeneity, purity, and stability. Tradi- tional chromatographic methods, such as SEC, IEX, and electrophoretic methods, such as SDS-PAGE and IEF, have been used extensively to analyze proteins, especially in the field of proteomics (1). However, it is often not possible to apply an existing procedure to the analysis of a new product without modifying the procedure to fit the size and complex nature of that product. This paper presents analytical procedures that are specifically op- timized for the analysis of micelle-forming F5cys-MP- PEG(2000)-DPSE conjugate, termed F5-L, a novel re- agent used in the manufacturing of HER2-targeted immunoliposomes carrying anticancer drugs (2). Traditional methods for identification by AAA and lot comparison by SEC (micelle size distribution) and DSC (protein thermal stability) were compatible with micellar conjugate. However, traditional methods of purity analy- sis by cIEF and IEX were deemed inapplicable because DSPE lipid-lipid interactions interfered with antibody characterization methods that required monodisperse mixtures. Use of detergents in cIEF and either enzymatic sample pretreatment or organic cosolvent elution for IEX enabled conjugate analysis. The antigen-binding func- * To whom correspondence should be addressed. Ph: +1-301- 846-1821. Fax: +1-301-846-6886. Email: giardina@mail.ncifcrf.gov. † SAIC-Frederick, Inc. ‡ California Pacific Medical Center Research Institute. § Center for Cancer Research. | University of California-San Francisco. ⊥ Hermes Biosciences, Inc. # Senior author. Ph: +1-650-873-2583. Fax: +1-650-873-2501. Email: dkirpo@hermesbio.com. 221 Biotechnol. Prog. 2005, 21, 221-232 10.1021/bp049839z CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 12/04/2004