Preclinical Manufacture of Anti-HER2 Liposome-Inserting, scFv-PEG-Lipid Conjugate. 2. Conjugate Micelle Identity, Purity, Stability, and Potency Analysis
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- 作者：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 Dmitr
- 刊名：Biotechnology Progress
- 出版年：2005
- 出版时间：February 2005
- 年：2005
- 卷：21
- 期：1
- 页码：221 - 232
- 全文大小：383K
- 年卷期：v.21,no.1(February 2005)
- ISSN：1520-6033
文摘
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 F5cysantibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 
C. Traditional methods for characterizing monodisperse proteinspecies were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2)and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) afteraddition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation withphospholipase B selectively removed DSPE lipid groups and dispersed the conjugateprior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchangechromatography provided gentler, nonenzymatic dispersion, resulting in well-resolvedpeaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) forconjugate lots, described previously. The F5cys amino acid content was confirmed afterconjugation. The expected conjugate avidity for immobilized HER-2/neu was measuredby bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were madeby conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Togetherthese developed assays established that the manufacturing method as described inthe first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE havingsufficient purity, stability, and functionality for use in preclinical toxicology investigations.
C. Traditional methods for characterizing monodisperse proteinspecies were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2)and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) afteraddition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation withphospholipase B selectively removed DSPE lipid groups and dispersed the conjugateprior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchangechromatography provided gentler, nonenzymatic dispersion, resulting in well-resolvedpeaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) forconjugate lots, described previously. The F5cys amino acid content was confirmed afterconjugation. The expected conjugate avidity for immobilized HER-2/neu was measuredby bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were madeby conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Togetherthese developed assays established that the manufacturing method as described inthe first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE havingsufficient purity, stability, and functionality for use in preclinical toxicology investigations.