Sponsored Content by SartoriusFeb 10 2023Reviewed by Alex Smith
Protein-based therapeutics play a crucial role in drug development and require the identification of therapeutic targets.
Drug development using protein-based therapies is crucial in medical research but faces challenges in cost and time with a low approval rate by regulators.
Accurate kinetics and affinity data are crucial in drug development, helping to identify and isolate therapeutic molecules for their target.
Drug development involves identifying therapeutic targets. Accurate kinetics/affinity and critical quality attributes are essential for isolating therapeutic molecules. Systems must be high throughput and sensitive to minimize sample usage.
Early and accurate data speed up drug development, leading to quicker decisions on lead candidates.
Modern label-free analytical techniques enable high-resolution real-time monitoring of kinetic interactions, which, when combined with high-throughput capabilities, can significantly shorten the time to discovery, streamlining the selection of optimal drug candidates with the best chances of success downstream.
During screening, drug candidates are typically prepared at one concentration and evaluated for binding levels only by injecting across the target.
Drug screening with single-concentration drug candidates can only determine binding levels, not kinetic parameters, leading to limited information. The results are limited to a "yes/no" binding conclusion based on the response falling within a set range without considering non-specific interactions.
Single-concentration drug screening has limitations as it only assesses binding levels and provides no kinetic data. This can result in a time-consuming characterization process and delay the selection of optimal drug candidates.
Using SPR in screening is limited to hit validation, as screening multiple drug candidate concentrations for kinetic parameters is time-consuming. This method can only provide binding information and not quantitative kinetic data.
Limitations in SPR use in drug screening include time and sample preparation, lack of quantifiable kinetic information, and limitations on target regeneration cycles and sample accommodation.
Because of these constraints, there has always been a compromise between throughput and accuracy, but this can be overcome with OneStep® injections, a feature unique to the Octet® SF3 SPR system.
OneStep® injections save time, sample, and buffer compared to standard MCK by providing kinetics and affinity from a single-concentration injection profile.
OneStep® injections help obtain accurate kinetics and affinity data for high-affinity interactions with a single measurement, reducing the necessary data compared to traditional methods. This increases sample throughput and saves sample material.
OneStep® injections enhance SPR as a tool for large screens, enabling rapid progression and more efficient assay development of potential therapeutics.
Methods
Instrument and reagents
This information tells us that the Octet® SF3 SPR system was used to conduct all experiments, with the HBS-EP+ buffer as the running buffer. The experiments were performed at 37 °C unless otherwise specified.
Assays were performed on the Octet® SF3 SPR system. HBS-EP+ buffer was used with all assays at 37 °C. Recombinant HER2, VEGF 165/121a purchased from Sino Biological. Herceptin and anti-VEGF were obtained from Midwinter Solutions and Absolute Antibody, respectively.
Reagents used were purchased from various sources: Sino Biological (recombinant biotinylated HER2, VEGF 165 and 121a), Midwinter Solutions (Herceptin), Absolute Antibody (anti-VEGF), Thermo Fisher Scientific (EDC), and Sigma Aldrich (all others). All reagents apart from EDC were prepared in-house.
Kinetics and affinity determination
HER2
The immobilization of recombinant streptavidin on an Octet® SPR CDL Sensor Chip was carried out using a standard amine coupling process.
A mixture of 50% 0.4 M EDC and 50% 0.1 M NHS was administered to flow cells 1, 2, and 3 for seven minutes at a 10 µl/min flow rate. Subsequently, the recombinant streptavidin (5 µg/mL in sodium acetate at pH 4.0) was introduced to the same flow cells at a flow rate of 10 µl/min for another seven minutes.
The surface was then treated with 1 M ethanolamine HCl (pH 8.5) at a flow rate of 10 µl/min for seven minutes, leading to the immobilization of approximately 400 RU of recombinant streptavidin on each flow cell.
Biotinylated HER2 (bHER2) was prepared to a final concentration of 1.25 µg/mL in HBS-EP+.
Using the manual mode function of the Octet® SF3, bHER2 was delivered to flow cell 1 at a flow rate of 10 µl/min until an approximate capture level of 150 RU was achieved. The interaction between Trastuzumab and bHER2 was then evaluated through standard multi-cycle kinetics and OneStep® injections, which are specific to the Octet® SF3 instrument.
Trastuzumab was prepared with a final concentration of 25 nM in HBS-EP+ running buffer, and a series of six different concentrations was created by diluting 1:3 into HBS-EP+.
The samples were placed in Octet® SPR 0.9 mL vials and assembled in a mixed format sample rack. A 3% sucrose solution, made using HBS-EP+ as the bulk reference, was prepared for OneStep® injections, and 100 mM HCl was used for regeneration injections. The sample rack was then secured with resealable septa and placed in a sample tray set at 15 °C.
The Octet® SF3 system was first flushed three times with HBS-EP+ running buffer, and the sensor chip was hydrated and conditioned using injections of HBS-EP+ and 100 mM HCl.
A short and long assay format was employed (as described in Katsamba 2006), with a common association time of 180 seconds at a flow rate of 50 µL/min for multi-cycle kinetics. The highest concentration used a "long" dissociation time of 3600 seconds, while all other concentrations used a "short" dissociation time of 420 seconds.
The parameters for the OneStep® injections were fixed based on the injection loop volume, which was set to 100%. The same dissociation parameters were used for the multi-cycle kinetics.
A buffer blank injection was carried out for each analyte concentration to produce accurate double-referenced data. The Trastuzumab-bHER2 complex was regenerated using a single injection of 100 mM HCl at a flow rate of 50 µL/min for 60 seconds, followed by a 180-second stabilization period.
The data was analyzed using the standard short and long method, determining the dissociation rate constant (kd) for the highest concentration (3600-sec dissociation) and restricting the lower concentrations (420-sec dissociation) to the same value. The data was fitted locally to a simple 1:1 interaction model.
VEGF 165 and VEGF 121a
The Octet® SPR CDL Sensor Chip was used to immobilize recombinant VEGF 165 and VEGF 121a on flow cells 1 and 3, respectively, through standard amine coupling chemistry.
A solution containing a 50:50 mixture of 0.4 M EDC and 0.1 M NHS was infused across flow cells 1, 2, and 3 at a rate of 10 µl/min for seven minutes.
Recombinant VEGF 165 was then infused across flow cell 1 at a concentration of 0.25 µg/mL in sodium acetate (pH 5.0), while Recombinant VEGF 121a was infused across flow cell 3 at the same concentration. The surface was activated until a response of 35 RU was reached for both ligands.
Afterward, the surface was deactivated by injecting a solution of 1 M ethanolamine HCl (pH 8.5) across flow cells 1, 2, and 3 at a flow rate of 10 µl/min for seven minutes.
The interaction between a bevacizumab biosimilar and VEGF 165 and VEGF 121a was evaluated through standard multi-cycle kinetics and OneStep® injections, which are unique to the Octet® SF3 system.
The bevacizumab biosimilar was synthesized in HBS-EP+ running buffer to a final top concentration of 100 nM, and a six-fold concentration series was prepared using a 1:3 dilution into HBS-EP+.
The samples were put in Octet® SPR 0.9 mL vials in a mixed format sample rack. For OneStep® injections, 3% sucrose was produced using HBS-EP+ as the bulk reference standard, and regeneration injections employed 100 mM HCl.
The sample rack was sealed with resealable septa and put in a 15 °C sample tray. The Octet® SF3 system was primed three times into HBS-EP+ running buffer, and the sensor chip was hydrated and conditioned with HBS-EP+ and 100 mM HCl injections.
The study used both a short and long assay format, with a common association time of 180 sec, and the dissociation time varied from 420 sec to 3600 sec for different concentrations.
OneStep® association parameters are fixed depending on the volume of the injection loop utilized, which was set to 100% in this case, and the identical dissociation parameters used in multi-cycle kinetics.
To provide correct double-referenced data, a buffer blank injection was conducted for each analyte concentration. The bevacizumab VEGF complex was reconstituted by two injections of 100 mM HCl at 50 L/min for 30 seconds, followed by a stabilization period of 180 seconds.
The data was analyzed using the conventional short and long methods, which involved finding the dissociation rate constant (kd) for the greatest concentration (3600-sec dissociation) and restricting the lower concentrations (420-sec dissociation) to the same value. All data was fitted globally to a simple 1:1 interaction model.
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References
- Cannon MJ et al. Anal. Biochem. 2004 1;330(1):98-113
- Karlsson R and Larsson A. Methods Mol Biol. 2004;248:389-415
- Katsamba P et al. Anal. Biochem. 2006 15;352(2):208- 221
- Navratilova I et al. Anal. Biochem. 2007 1;364(1):67-77
- Papalia GA et al. Anal. Biochem. 2006 1;359(1):94-105
- Onell A and Andersson K. J. Mol Recognit. 2005;18(4):307-17
- Quinn J. Anal Biochem. 2012 15
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