1. Atomic Force Spectroscopy between Hansenula polymorpha and Ion Exchange Chromatographic Beads Cristina Chiutu, Vikas Yelemane, Marcelo Fernandez-Lahore, and Jrgen Fritz Jacobs University Bremen Introduction The design of ion exchange chromatographic beads plays a significant role in the performance, fluidity and classification of the downstream processing and chromatography. Atomic Force Microscopy probes the interaction between beads and biomass, cells or proteins at the nanoscale and can lead to a better understanding of the structure and functionality of various beads. In this project, different types of beads, rigid and elastic, positive and negative, are attached on triangular soft cantilevers and characterised against a negatively charged silicon surface via force spectroscopy with a Veeco PicoForce AFM. Furthermore, the beads are analysed in contact with monocellular layer of Hansenula polymorpha (immobilized on Roti Bond glass) in buffer (with different NaCl concentration or on various phases of cell life cycle). Elasticity of the cellular layer can be approximated by fitting the Hertz-Stanndon model. Hansenula polymorpha is employed at commercial-scale in biotechnology for recombinant protein production. Hansenula polymorpha Growth Phases 7 h (Lag) 12 h (Accelerated) 16 h (Exponential) 24 h (Late Exponential) 30 h (Stationary) 48 h (Late Stationary) Approaching Hansenula polymorpha with Negative Beads Beads-Silicon Measurements in Different NaCl Concentrations Elasticity of Hansenula polymorpha in Liquid (Hertz-Stanndon fit) Summary and Outlook Hansenula polymorpha cells were successfully immobilized on Roti Bond glass for liquid imaging and force spectroscopy. Several types of chromatographic beads were investigated and it was found that the rigid negative Source S bead is most reproducible and reliable to perform a larger set of experiments on cells. Different salt concentrations affect the interaction between beads and silicon/cells as well as cells in various phase of their life cycle leads to different result. Elasticity of cellular layer can also be determined. Future measurements will be theoretically characterized with the XDLVO theory calculations. [1] R.R. Vennapusa et al., Separation Science and Technology, 2010, 45, 23352344. [2] G. Helms et al., 2014, submitted. [3] A. Touhami et al. Langmuir 2003, 19, 4539-4543. Attractive interaction or force of adhesion of the positive beads is significantly reduced in buffer with increasing salt concentration. This large magnitude for attraction force on extend was measured only for a limited number of Source Q beads. For negative beads a reduction of the electrostatic force is observed with increasing salt concentration, emphasizing the elastic component of the force. (Air Imaging) 7 h: typical size 2 m, height 0.5 m, small buds growing 12 h: similar size, cells with more small buds 16 h: size of cells increases to 3 m and height to 0.8 m, bigger buds 24 h: cells 2-3 m, big buds, but some cells deflated 30 h: many big buds, many young cells 48 h: many young cells and many deflated Hansenula polymorpha in Air Hansenula polymorpha in Liquid Detail Bud Scar (in Air) Size 2-3 m Height 0.5-0.8 m Size 2-3 m Height 1-1.5 m Size 0.5 m Height 0.2 m Cristina Chiutu, 8th North German Biophysics Meeting, Jan. 2015, Borstel Bead Cantilever Cell Layer Chromatographic Beads Characterization Imaging of Yeast Cells Type of ion exchanger: Strong cation Functional group: Sulfonate R-SO3 Matrix structure: polystyrene/divinyl benzene polymer Spherical, rigid, 30 m monosized Type of ion exchanger: Strong anion Functional group: Quaternary ammonium -CH2N+(CH3)3 Matrix structure: polystyrene/divinyl benzene polymer Spherical, rigid, 30 m monosized Type of ion exchanger: Strong cation Functional group: Sulfopropyl OCH2CHOHCH2O-CH2CH2CH2SO3 Matrix structure: 85-90% agarose (inert) and 10-15% tungsten carbide (inert) Spherical, elastic (soft), 100-200 m Source S Bead Source Q Bead Fastline SP Bead Fastline DEAE Bead Type of ion exchanger: Weak anion Functional group: Diethylaminoethyl OCH2CH2N+(C2H5)2H Matrix structure: 85-90% agarose (inert) and 10-15% tungsten carbide (inert) Spherical, elastic (soft), 100-200 m Both rigid and elastic beads were characterized from interaction with a negatively-charged silicon surface in water or phosphate buffer. The negative beads demonstrated reproducibility and stability regarding the behaviour of electrostatic repulsion and/or elastic forces over time, while the magnitude of attraction and adhesion forces of positive beads decreases in time. Therefore, due to changes in time, the measurements on Hansenula polymorpha focused on the interaction with negative beads. Data shown in this section are similar to other measurements on positive/negative beads performed in our group by G. Helms [1,2]. Imaging on Hansenula polymorpha was performed with MSNL-10 cantilevers in liquid and air. This type of yeast showed good adhesion to the Roti Bond glass (commercially purchased), therefore full monolayer of cells could be obtained. The cells were imaged with high resolution of details such as bud scars or membrane structure (emphasized in the deflection image). However, in liquid the structure of cell membrane can be imaged with poor resolution. Interaction of negative beads (both rigid and elastic) with Hansenula polymorpha cells proved to be reproducible both in water and in phosphate buffer. More difficult is the interpretation of spectra, since a separation between elastic and electrostatic forces is required considering the elasticity of both beads and cells. The measurements on the same spot are fully reproducible, while elasticity or electrostatics varies significantly across the sample for measurements in different points. In different salt concentrations the electrostatic component of force is reduced with increasing salt quantity, while elasticity of cells seems to increase. Source S measurements present similar data to the Toyo SP bead experiments performed by G. Helms [2]. This experiment aims to understand the behavior of cells during the growth phases. Images of cells and force spectra were consecutively recorded in phosphate buffer. While images indicate differences in size and appearance of cells for each phase, force spectra show a change in slope for each phase, but do not present a trend or gradient with the phase as expected. One reason for this could be that in later phases the sample is a mixture of young, old and dead cells. On the other hand, various spots on sample may lead to significant differences in the slope of spectra. -100 0 100 200 300 Separation (nm) Force(nN) -7-6-5-4-3 Contact Point MSNL-10 cantilever on single cell in water: 0.0627 MPa 0 100 200 300 Separation (nm) Force(nN) -4-3-2-10 Contact Point Source S bead on cellular layer in water: 0.0021 MPa For comparison and understanding of spectra analysis with elasticity models, measurements were performed with MSNL-10 cantilever on individual cells. Young modulus values resulted from curve fitting are comparable to the values given by A. Touhami [3] for measurements on Saccharomices cerevisiae cells but slightly softer. Measurements with beads lead to smaller values for the Young modulus resulted from higher elasticity of cellular layer comparing to individual cell. This could be explained in terms of pressure decreasing with increasing contact area.