Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (Rmax) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2. Graphical abstractᅟ Electronic supplementary materialThe online version of this article (10.1007/s00216-018-1414-y) contains supplementary material, which is available to authorized users.
A variable-wavelength Kretschmann configuration surface plasmon resonance (SPR) apparatus with angle scanning is presented. The setup provides the possibility of selecting the optimum wavelength with respect to the properties of the metal layer of the sensorchip, sample matrix, and biomolecular interaction of interest. Monitoring SPR curves over a wide angular range (39 •) permits simultaneous determination of the total internal reflection angle (TIR), the resonance angle, and the intensity and width of the SPR dip, which are essential parameters for measuring binding events and achieving optimum sensitivity. The new apparatus was evaluated by recording full SPR curves at different wavelengths ranging from 600 to 890 nm using sensor surfaces of silver, gold and gold with deposited silicon oxide, aluminum oxide, titanium oxide and indium tin oxide which were exposed to air and an aqueous solution of sodium chloride. Clear wavelength dependencies of sensor-material resonance angles and SPR-dip widths were demonstrated. In order to investigate the capability of the system to probe molecular binding to different sensor surfaces, the layer-by-layer adsorption of charged polyelectrolytes was monitored in angular scanning mode at 600, 670, 785, and 890 nm. Although at longer wavelengths lower angular shifts were observed as result of layer deposition, the sharper dip, wider detection window and better signal-to-noise ratios at these wavelengths can be beneficial for binding studies. The applicability for biosensing was tested by immobilizing human serum albumin (HSA) on an aluminum-oxide-coated gold sensor using a new procedure and measuring the binding of anti-HSA antibodies at the optimal wavelength (890 nm) in angular-scanning and fixed-angle mode. The HSA biosensor showed negligible non-specific interaction and yielded almost ten times better sensitivity than obtained with a conventional gold-dextran-based sensor operated at 670/785 nm. Analysis of anti-HSA samples pre-incubated with different concentrations of HSA allowed measurement of the IC50 value. The reported data demonstrate the usefulness of the presented variable-wavelength angle-scanning SPR instrument, permitting continuous recording of full SPR curves in time at any selected wavelength in the 600-890 nm range using a sensor material of choice.
An angle-scanning Kretschmann configuration SPR instrument allowing multiplexed analysis is presented. Laser light was guided through optics that converted the collimated light into a line-shaped beam, which was directed to a prism, illuminating the gold sensor surface over a 1 × 10 mm area. The reflected light was led to a CCD detector providing simultaneous readout of individual analysis spots along the laser line at a selected angle (fixed-angle detection) or in scanning-angle mode (width of 35°). Full SPR curve could be measured every 3.6 s for each illuminated spot on the sensor surface. Two in-house manufactured flow cell designs were used for evaluating multiplexed angular-scanning SPR. The first comprised six parallel channels with the laser line perpendicular to the flow direction in order to allow interrogation of the sensor surface in the six channels. Refractive index changes by varying solution composition, and adsorption of different concentrations of albumin to the sensor surface could be correctly monitored simultaneously in each of the channels. In the second flow-cell design the laser line was coinciding with the flow path, allowing recording of SPR curves along a 10-mm length of the sensor surface. Adsorption of layers of positively and negatively charged polyelectrolytes could be consistently measured for sixteen selected positions along the channel. As a proof of principle, several target proteins were immobilized on different positions along the sensor and the binding of various antibodies with these proteins was monitored simultaneously, showing excellent selectivity and reproducibility for probing antibodyprotein interactions in a multiplexed fashion.
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