title: Ultra scale-down stress analysis of the bioprocessing of whole human cells as a basis for cancer vaccines.
abstract: This study examines the use of a capillary shear device for the rapid characterization of human cell lines in terms of their resistance to hydrodynamic stress. An ultra scale-down (USD) approach is presented to allow the use of small quantities of cells available at the early discovery stage and to expose them to a wide range of hydrodynamic stresses. In this way an indication is gained of the relative properties of different cell lines and the challenge which may be faced during full-scale processing. A design of experiments approach allowed the interaction between a number of key processing factors such as capillary length, flow rate, and number of passes to be studied in a limited number of experiments. Out of this an USD test based on flow rate in a device of fixed geometry was proposed. Based on observations made elsewhere (Ma et al., 2002, Biotechnol Bioeng 80(4): 428-437) a detailed analysis of possible geometries was performed using a combination of USD experiments and computational fluid dynamics analysis of the capillary entry region. This allowed the properties of the cells to be characterized in terms of a critical stress below which there is no significant loss of cell integrity. The results suggested that the OnyCap23 and P4E6 cell lines, used as components of a whole cell prostate cancer vaccine, are resistant to damage below critical elongational shear stress values of 235 and 275 N/m(2), respectively. Above these stress values the loss of intact cells is predicted to be significant; such loss being due to a combination of whole cells becoming permeable to trypan blue and complete breakage of cells into debris at extreme stresses. The sensitivity of cell surface markers CD9, CD44, CD59, CD81, CD147, and MHC-1 to exposure to shear stress was considerably less than for cell membrane integrity. The surface marker levels for recovered whole cells (i.e., both with and without intact cell membranes) were either independent of shear stress or showed a slight decrease with increased shear stress, for example, as for CD9. The results were used to predict successfully a capillary design where no damage would occur at a specified high flow rate; for example, as required for cell dispensing or vialling operations. Equally, the extent of loss of cell integrity was also successfully predicted in a capillary flow system designed to yield high levels of break up as may be required in intracellular analysis without the use of chemical lysing reagents or relying on autolytic damage.
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, London, UK.
authors.forename: J P
authors.initials: JP
authors.lastname: Acosta-Martinez
authors.forename: I
authors.initials: I
authors.lastname: Papantoniou
authors.forename: K
authors.initials: K
authors.lastname: Lawrence
authors.forename: S
authors.initials: S
authors.lastname: Ward
authors.forename: M
authors.initials: M
authors.lastname: Hoare
mesh_terms: D001709:Biotechnology / Q000379:methods; D019496:Cancer Vaccines / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology; D006801:Humans; D057446:Hydrodynamics; D008565:Membrane Proteins / Q000032:analysis; D013312:Stress, Physiological; D054159:Torsion, Mechanical
chemical_list: D019496:Cancer Vaccines; D008565:Membrane Proteins