title: Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis.
abstract: Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 degrees 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 investigations.
authors.affiliation: SAIC-Frederick, Inc., National Cancer Institute at Frederick, PO Box B, Frederick, Maryland 21702, USA.
authors.forename: David F
authors.initials: DF
authors.lastname: Nellis
authors.forename: Steven L
authors.initials: SL
authors.lastname: Giardina
authors.forename: George M
authors.initials: GM
authors.lastname: Janini
authors.forename: Shilpa R
authors.initials: SR
authors.lastname: Shenoy
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
authors.forename: Richard
authors.initials: R
authors.lastname: Tsai
authors.forename: Daryl C
authors.initials: DC
authors.lastname: Drummond
authors.forename: Keelung
authors.initials: K
authors.lastname: Hong
authors.forename: John W
authors.initials: JW
authors.lastname: Park
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Ouellette
authors.forename: Shelley C
authors.initials: SC
authors.lastname: Perkins
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Kirpotin
mesh_terms: D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008245:Lysophospholipase / Q000378:metabolism; D008823:Micelles; D008954:Models, Biological; D008970:Molecular Weight; D010316:Particle Size; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000276:immunology; D020539:Sequence Analysis, Protein; D013696:Temperature; D013997:Time Factors
chemical_list: D007128:Immunoglobulin Fragments; D008081:Liposomes; D008245:Lysophospholipase; D008823:Micelles; D018719:Erb-b2 Receptor Tyrosine Kinases