title: 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.
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: Denise L
authors.initials: DL
authors.lastname: Ekstrom
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Kirpotin
authors.forename: Jianwei
authors.initials: J
authors.lastname: Zhu
authors.forename: Robert
authors.initials: R
authors.lastname: Andersson
authors.forename: Trevor L
authors.initials: TL
authors.lastname: Broadt
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Ouellette
authors.forename: Shelley C
authors.initials: SC
authors.lastname: Perkins
authors.forename: John M
authors.initials: JM
authors.lastname: Roach
authors.forename: Daryl C
authors.initials: DC
authors.lastname: Drummond
authors.forename: Keelung
authors.initials: K
authors.lastname: Hong
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
authors.forename: John W
authors.initials: JW
authors.lastname: Park
authors.forename: Steven L
authors.initials: SL
authors.lastname: Giardina
mesh_terms: D000595:Amino Acid Sequence; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000302:isolation & purification*; D008081:Liposomes / Q000302:isolation & purification* / Q000378:metabolism*; D008823:Micelles; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000276:immunology; D013997:Time Factors
chemical_list: D007128:Immunoglobulin Fragments; D008081:Liposomes; D008823:Micelles; D010710:Phosphates; D018719:Erb-b2 Receptor Tyrosine Kinases