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Baseline jumps
- Gert
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5 years 8 months ago #1
by Gert
Baseline jumps was created by Gert
Since 6 months we have encountered some problems with our routine biacore assay. We run this Biacore assay as a QC release test in a QC lab for several years.
Our assay is executed on a Biacore 3000 system and consists out of an amine based immobilization of a CM5 sensor chip with a 25 kDA protein followed by 10 preconditioning cycles and the injections of the analyte diluted at 4 different concentrations in duplicate. In total we analyze 8 samples on the immobilized chip which results in 81 cycles (10 pre conditioning, 3 blanks, 8 x 4 concentration x 2 replicates + 4 controls at end). There is a very strong binding of the analyte to the ligand immobilized on the chip (almost no dissociation occurs) after which the chip is regenerated (40 mM NaOh) before the next sample is injected (picture 2). Our running buffer and sample dilution buffer is HBS-EP buffer with 0.5M NaCl. We always make use of a reference flow cell in which no ligand was immobilized in our assays.
Recently we see randomly unexpected high response (RU) and jumps in the baseline (picture 2). Our regeneration procedure is able to regenerate much higher responses as we use in our assay. So it is strange that sometimes in the run the regeneration is not complete. Dilution errors have been ruled out. Non-specific binding has been ruled out. We perform monthly system checks and they always pass. Biacore service engineer came on site several times but cannot find an instrument issue. We don’t believe in sample aggregation as never seen before. Biacore assay is executed by well trained people and in a controlled GMP QC Lab.
All ideas are welcome.
Our assay is executed on a Biacore 3000 system and consists out of an amine based immobilization of a CM5 sensor chip with a 25 kDA protein followed by 10 preconditioning cycles and the injections of the analyte diluted at 4 different concentrations in duplicate. In total we analyze 8 samples on the immobilized chip which results in 81 cycles (10 pre conditioning, 3 blanks, 8 x 4 concentration x 2 replicates + 4 controls at end). There is a very strong binding of the analyte to the ligand immobilized on the chip (almost no dissociation occurs) after which the chip is regenerated (40 mM NaOh) before the next sample is injected (picture 2). Our running buffer and sample dilution buffer is HBS-EP buffer with 0.5M NaCl. We always make use of a reference flow cell in which no ligand was immobilized in our assays.
Recently we see randomly unexpected high response (RU) and jumps in the baseline (picture 2). Our regeneration procedure is able to regenerate much higher responses as we use in our assay. So it is strange that sometimes in the run the regeneration is not complete. Dilution errors have been ruled out. Non-specific binding has been ruled out. We perform monthly system checks and they always pass. Biacore service engineer came on site several times but cannot find an instrument issue. We don’t believe in sample aggregation as never seen before. Biacore assay is executed by well trained people and in a controlled GMP QC Lab.
All ideas are welcome.
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- Arnoud
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5 years 8 months ago #2
by Arnoud
Replied by Arnoud on topic Baseline jumps
Hi Gert,
This is a difficult one since you ruled out pretty much all the things that can go wrong.
If I understand correctly the baseline jump only occurs during/after the regeneration with the NaOH. I know that high concentrations of NaOH can form sodiumcarbonate precipitation with atmospheric CO2. In addition storage of NaOH in glass can give some precipitation. It might be possible that small crystals are introduced in the system. Maybe you can view a sensor chip with a microscope if you can see something on the surface.
In addition, filter the regeneration solution through a 0.22 µM fiter just in case.
Kind regards
Arnoud
This is a difficult one since you ruled out pretty much all the things that can go wrong.
If I understand correctly the baseline jump only occurs during/after the regeneration with the NaOH. I know that high concentrations of NaOH can form sodiumcarbonate precipitation with atmospheric CO2. In addition storage of NaOH in glass can give some precipitation. It might be possible that small crystals are introduced in the system. Maybe you can view a sensor chip with a microscope if you can see something on the surface.
In addition, filter the regeneration solution through a 0.22 µM fiter just in case.
Kind regards
Arnoud
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