2.5.1 SURFACE PLASMON RESONANCE SPECTROSCOPY
Measurements were performed on a Biacore X-instrument (GE Healthcare Europe GmbH, Freiburg, Germany). Immobilization of rh-GCSF was carried out on a CM-5 research chip following standard immobilization procedures described by Biacore Life Sciences. The surfaces of research grade CM5 chips were activated by a 6-min injection of a solution containing 0.2 M N-ethyl-N9-(dimethylaminopropyl) carbodiimide and 0.05 M N-hydroxy-
succinimide. After immobilization of the protein and deactivation of the reference cell with ethanolamine approximately 2000 RU remained on the chip (difference between the steady state response before and after immobilization of the protein). Therefore, taking into account the CDs’ molecular weights a theoretical maximal response of 229 RU for SBEβCD and 104 RU for α CD can be calculated.
As running buffer 20mM Acetate (pH = 4) was used. Cyclodextrins were dissolved in the exact same kind of buffer. In order to examine the effect on binding of pH in later measurements 20 mM Phosphate buffer (pH = 7) was applied to dissolve cyclodextrins. If not stated otherwise measurements were performed at a temperature of 25°C.
The flow rate was set to 30 µL/min and 60 µL of cyclodextrin solution were injected in various concentrations. This means that the contact between immobilized protein and cyclodextrin solution lasted for 2 minutes. The response was monitored as difference of the responses of the cell containing the immobilized protein and the reference cell in order to avoid measuring a simple bulk effect.
For the determination of the steady state - affinity between cyclodextrins and proteins cyclodextrin solutions of various concentrations showing a response in this experimental setup were injected consecutively. Using the software tool Biaevaluation® the average maximum response was calculated for every cyclodextrin solution injected. These maximum responses were then plotted against the concentration of each CD-solution. From the best fit the average steady state affinity could then be calculated. From ESI-MS experiment hints were available that binding occurs in a 1:1-stochiometry and therefore a simple 1:1 (Langmuir)-binding model was assumed for the calculation of the steady-state affinity.
For the mAb and rh-GH the immobilization procedure and materials as well as the monitoring of CD-binding were exactly the same, only the amount of protein attached to the chip varied, as indicated in the respective data chapter.
2.5.2 FLUORESCENCE SPECTROSCOPY MAB
Concentrated stock solutions of CD-derivatives were titrated into 2 ml of a 0.24 mg/ml solution of the mAb in His 20 mM buffer and changes in intrinsic steady-state fluorescence
Materials and Methods
spectra were monitored. Each titration was carried out three times and after recording the spectra were corrected for dilution. The rest of the experimental conditions were identical to those described above for the assessment of changes in the apparent melting temperature by fluorescence spectroscopy.
RH-GCSF
Fluorescence titration was carried out at an excitation wavelength of 280 nm and at an emission wavelength of 337 nm on a Varian Cary Eclipse fluorescence spectrometer (Varian Inc., Darmstadt, Germany). Protein concentration was 1 µg/ml and the protein was buffered in 20 mM sodium phosphate buffer at pH = 4. SBEβCD was titrated to the solution to yield a final concentration of 8 mM.
2.5.3 SURFACE ACOUSTIC WAVE SENSOR
Surface acoustic wave sensors use piezoelectric materials to generate an acoustic wave. The amplitude and/or the velocity of the surface acoustic wave is strongly influenced by coupling to any medium contacting the surface. In contrast to SPR, SAW sensors are not sensitive to changes in the bulk refractive index thereby providing useful complementary information to the SPR results.
The experiments were carried out on the commercially available S-sens® K5 (Biosensor GmbH, Bonn, Germany) instrument. The central measurement unit consists of a read-out system into which the gold-coated quartz sensor is placed and the detected signals of the five measurement cells are recorded independently in real-time. Changes in phase and amplitude of the surface acoustic wave (in this a case a Love-wave) are triggered by changes in the bound mass and viscosity, respectively.
The gold-coated sensor chip was incubated overnight in a solution of mercaptoundecanoic acid thereby allowing for later coupling of proteins to carboxylic groups on the chip. After activation of the carboxylic groups with a mixture of EDC/NHS, rh-GCSF (dissolved in phosphate buffer) was immobilized to the surface of the chip. Unsaturated carboxylic functions were afterwards deactivated by Ethanolamine.
In order to be able to discriminate between phase shifts due to changes in bound mass and shifts due to changes in viscosity, 80 µL of an aqueous solution of glycerol (5 % m/m) were injected. The subsequent change in the binding signal can be solely attributed to a change in viscosity and using this information the Biosens K12 software can later correct the phase shift of the protein immobilization for changes in viscosity.
Increasing concentrations of cyclodextrins were injected onto the immobilized rh-GCSF and the binding signals were recorded. Using the Biosens K12 software and assuming a simple 1:1 binding model a kinetic analysis of the binding events was carried out. The association
Chapter 2
constant ka and the dissociation constant kd were fitted to the binding curves and from the ratio of kd and ka the equilibrium binding constant was finally calculated.
2.5.4 ELECTROSPRAY-IONIZATION MASS SPECTROMETRY
All measurements were performed on a Bruker Daltonics Esquire 3000plus 3D-ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) fitted with an orthogonal electrospray (ESI) ion source under the following conditions: capillary voltage, 4.0 kV (positive ions) and -4 kV (negative ions; in the case of the Captisol®-sample) and curtain gas temperature 300°C.
Pure protein samples (rh-IFNα-2a, rh-GCSF, rh-GH, Lysozyme) were initially analyzed by infusion of a 1 pmol/μl solution of methanol : water = 1 : 1 containing 0.1 % glacial acetic acid (Merck, Darmstadt, Germany) at a flow rate of 3 μl/min with a Cole-Parmer syringe pump (Core-Parmer, Vernon Hills, IL, USA).
Pure cyclodextrins and derivatives thereof were analyzed by infusion of a 1 mg/ml solution of methanol : water = 1 : 1 at a flow rate of 3 μl/min.
In order to detect complexes between the proteins and cyclodextrins and its derivatives a purely aqueous solution (without adding acetic acid) of a molar ratio of protein : carbohydrate = 1 : 10 was chosen according to S. Cao et al.17. Additionally, similar experiments were run after adding 10 mM ammonium acetate to these aqueous solutions (pH 4 for rh-GCSF and pH 5 for rh-GH). Furthermore by lowering in steps the molar amount of carbohydrate to a molar ratio of protein : carbohydrate to 1 : 1 and even to 10 : 1 the selectivity of the complex formation was tested. Control experiments were conducted using the linear carbohydrates maltoheptaose, maltopentaose, sucrose and trehalose in order to evaluate whether the existence of the CD-cavity is a necessary prerequisite for binding. Furthermore, in order to evaluate the influence of basicity control experiments with the amino acids L-tryptophan and L-tyrosine as well as their derivatives N-Acetyl-L-tryptophanamide and N-Acetyl-L- tyrosinamide were conducted. Further information regarding molar ratios of the solution- components as well as absolute concentrations can be taken from the respective figures in the data chapter.