Quality control of biopharmaceuticals with microplate readers

The variety of applications for microplate readers in the quality control of biopharmaceuticals

Microplate readers have been common laboratory measuring instruments for several decades and are an indispensable instrument in research, development and quality control of biopharmaceuticals. (1) The wide availability of analysis kits enables a broad and flexible use for the measurement of biological, chemical and physical reactions in microplates. The range of applications includes protein, nucleic acid and endotoxin quantification as well as ELISA and interaction assays. Compared to measurements in cuvettes, microplate readers offer considerable advantages. While a photometer can only take one sample, a microplate reader analyses almost 100 samples within a few minutes and requires significantly smaller sample volumes. Additionally, so-called nano quant plates with specially developed quartz optics enable analyses with a sample volume of just 2 µL, which makes NanoDrop applications possible.

How it works

The measuring system of a microplate reader corresponds to that of a conventional photometer. (2) The incident light from a light source is first bundled and focussed into the wells of the microplate. The reader measures the intensity of the light that is transmitted (absorption) or emitted (fluorescence) through the sample. The light signal is then converted into an electrical signal and quantified. For the analysis, the samples are pipetted into the wells of a microplate. A standard plate consists of 96 wells, but plates with up to 384 wells are also commercially available. The system has several detection modes (e.g. fluorescence intensity, absorbance). Which detection mode is used depends on the type of assay. In addition to the detection mode, the assay kits differ in their sensitivity and compatibility with reagents contained in the sample solution (e.g. buffer salts, DTT).

A wide range of applications

Protein quantification is an important step prior to chromatographic or electrophoretic analysis. Using microplate readers, fast and accurate high-throughput quantification can be performed. (3) In UV-based assays, a dye-containing reagent that interacts with the proteins is added to the samples. This leads to a colour change that is directly proportional to the concentration. This principle is used in the Detergent Compatible Bradford Assay (Figure 1). (4,5) It allows proteins to be analysed quickly (incubation time: 10 minutes) and without time-consuming sample preparation. (6) In addition, the assay is compatible with a wide range of detergents and lysis reagents that are commonly used in the production process of biological products. Only 10 µL sample volume is required for the analysis. However, the assay shows high protein-to-protein signal variability, as well as increasing protein precipitation over time.

Figure 1: Quantitative analysis of bovine serum albumin (BSA) using the Detergent Compatible Bradford Assay Kit. BSA concentrations between 0 and 2000 µg/mL were measured. Absorbance measurements were performed using a Tecan Infinite M Nano+ microplate reader at 595 nm. A four-parameter Marquardt fit was used for curve fitting.

Fluorescence-based NanoOrange® Protein Quantification Assay uses reagents that are virtually non fluorescent in aqueous solutions and only undergo a fluorescence enhancement when bound to proteins. Compared to the Bradford assay, the NanoOrange® assay has a significantly lower signal variability (Figure 2). Furthermore, it is more sensitive with a detection range of 10 ng/mL to 10 µg/mL and is not affected by nucleic acids present in the sample solution. Based on chemically more stable reagents, a measurement is possible up to 6 hours after sample preparation.

Figure 2: Quantitative analysis of bovine serum albumin (BSA) using the NanoOrange® Protein Quantification Kit. BSA concentrations between 0.06 and 10 µg/mL were measured. Fluorescence measurements were performed using a Tecan Infinite M Nano+ microplate reader at excitation/emission wavelengths of 485/590 nm. A four-parameter Marquardt fit was used for curve fitting.

In addition to proteins, nucleic acids can also be quantified using assays in microtiter plates. This reduces time and costs in downstream experiments (PCR, transcription, reverse transcription, Northern blot analysis) and provides information on the purification success of a biopharmaceutical. (3) Both UV-Vis and fluorescence measurement can be used. Examples include the Invitrogen Quant-iT assay kits from Thermo Fisher Scientific. These assays use fluorophores that bind to DNA/RNA and form a complex. The fluorescence intensity is proportional to the amount of the target molecule in the sample.

Other applications include enzyme-linked immunosorbent assays (ELISA). (3) The principle of these assays is based on the binding of the analyte via antigen-specific antibodies and its subsequent labelling by an enzyme-linked antibody. The enzyme converts a substrate into a chromogenic or fluorogenic product, which then can be detected by the microplate reader. The signal correlates with the amount of analyte present in the sample. ELISA is an essential method in the life sciences for the quantitative analysis of specific peptides, proteins, antibodies and hormones (e.g. cytokines, phosphoproteins, oncogenes, biomarkers).

More flexibility for your biopharmaceuticals

Microplate readers offer flexibility and high sensitivity for the analysis of your biopharmaceutical products. Based on the various detection modes and analysis kits, you can find a suitable assay for your product and achieve reliable results.

Have we caught your interest? Do not hesitate to contact us for further information. Our experts in laboratory services are always ready to provide you with more information. Let us work together to ensure the safety and efficacy of your products!

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1. Courcol, R.J. et al. (1983): “Automated reading of a microtiter plate: preliminary evaluation in antimicrobial susceptibility tests and Enterobacteriaceae identification”
2. Tecan (2016): “Instructions for Use for Infinite 200 Pro”
3. Thermo Fisher Scientific (2022): “Guide to microplate readers- a one-stop solution for your microplate detection needs”, https://www.google.com/url?sa=t&source=web&rct=j&opi=89978449&url=https://assets.thermofisher.com/TFS-Assets/BID/brochures/guide-microplate-readers.pdf&ved=2ahUKEwiql8SA7NqFAxVl_7sIHSygASEQFnoECBgQAQ&usg=AOvVaw3THPD4IXck8EDCBt1HxHId
4. Molecular Probes (2008): “NanoOrange Protein Quantitation Kit Protocol”, https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fmp06666.pdf
5. Thermo Fisher Scientific (2016): “Pierce Detergent Compatible Bradford Assay Kit Protocol”, https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2F23246_23246S_deter_compat_bradford_UG.pdf
6. Life Technologies (2010): The Molecular Probes Handbook- A Guide to Fluorescent Probes and Labeling Technologies, 11^th Edition, https://www.thermofisher.com/de/de/home/references/molecular-probes-the-handbook/protein-detection-and-proteomics-technology/quantitation-and-selective-purification-of-proteins-in-solution.html)