Identification, quantification and characterisation of biomolecules

Medicines that are produced using biotechnological methods must satisfy in Europe the “DNA-recombination technology manufactured products” monograph of the European pharmacopoeia. Analysis methods for the identification, quantification and characterisation of biomolecules are clearly different to those used in the classical analysis of relatively small organic compounds. This has led to the development of a distinct branch in analysis, bioanalysis.

As an FDA-approved and GMP-certified contract laboratory in Switzerland, UFAG LABORATORIEN has established bioanalytical methods for proteins. Our portfolio comprises electrophoretic methods such as SDS-PAGE, IEF and 2D-combinations of these, blotting techniques (western blot) and amino acid composition determination, among other techniques.

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René Wicki

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Bioanalysis versus classical analysis - Peculiarities of biomolecules

Proteins and peptides are comprised of a change of amino acids that are connected with each other by peptide bonds. The sequence of the amino acids determines the so-called primary structure. Regular structural elements within a polypeptide chain such as the a-helix and the b-pleated sheet form the secondary structure while the complete three-dimensional structure of the whole chain constitutes the tertiary structure. If a protein consists of a number of polypeptide chains that are connected to each for example by disulphide bridge, then this is known as a quaternary structure. Non-peptidic components such as elements, lipids or carbohydrate building blocks can also contribute to this structure.

The three-dimensional structure of an active protein is defined precisely and is essential for its function. It is thermodynamically stable only within a defined range of chemical and physical parameters. Changes in the temperature, pH-value or the ionic strength could denature a protein. The protein will lose its activity. This denaturing can be permanent or it can be reversible. Just as in the proteins, the three-dimensional structure of DNA and RNA is also stable only within defined physical and chemical limits. Changes here could lead to reversible or permanent denaturing. The structure, the size and the “sensitivity” of biomolecules are the reason that classical analytic methods are of no use or of only limited use for analysis.

Bioanalysis: Gel electrophoresis
Bioanalysis: Gel electrophoresis

Analysis of biomolecules

Analytical chemistry has a number of questions to answer. They can be roughly divided into four categories:

  • Qualitative analysis of a mixture of compounds (qualitative composition, evidence, purity)
  • Qualitative analyse of a pure compound (identification)
  • Quantitative analysis of a defined compound in a mixture (selective quantification, content, purity)
  • Structural determination of a pure compound (determination of the constitution, configuration, conformation)

A variety of analytical methods in classical analysis take care of these tasks. For biological molecules, these methods are pushed to their limits due to the high molecular weight and their limited stability. Bioanalytical methods which take account of these conditions have been developed and improved over the past few decades. First and foremost the electrophoretic methods. One and two dimensional gel electrophoresis both with and without denaturing (e.g. SDS-PAGE), capillary electrophoresis, Isoelectric focusing (IEF) and the blotting techniques connected with it (e.g. western blot) are nowadays standard.

Proteins as well as DNA and RNA are comprised of a large number of monomers that are connected with each other. Consequently, determination of the sequence is a new parameter that is enormously important for the characterisation of a biomolecule. Methods to do this have been developed and optimised to such an extent that nowadays, laboratory devices carry out these analyses automatically in high throughput.

An overview of the analytical questions

The table gives an overview of the analytical questions and a comparison of the most frequently used methods in classical analysis and bioanalysis. The list is not complete but it makes the differences very clear.

Analytical task

Classical analysis Small molecules

Bioanalysis DNA/RNA

Bioanalysis Proteins

Qualitative analysis of a mixture of compounds

Gas chromatography (GC) Liquid chromatography (HPLC, UPLC)

Capillary electrophoresis Gel electrophoresis 2D gel electrophoresis

MALDI-TOF-MS Gel electrophoresis Isoelectric focusing 2D gel electrophoresis

Qualitative analysis of a pure compound

Mass spectrometry NMR

PCR DNA arrays

Amino acid composition Tryptic digest with subsequent gel electrophoresis MALDI-TOF-MS ESI-MS

Quantitative analysis of a defined compound in a mixture

Gas chromatography (GC) Liquid chromatography (HPLC, UPLC) chemical

Capillary electrophoresis Real-time PCR DNA array Biosensor

Bioassay (e.g. ELISA, RIA) Biosensor

Structural determination of a pure compound

NMR Mass spectrometry Infra-red spectroscopy X-ray crystallography

DNA-sequencing NMR X-ray crystallography Electron microscopy

Amino acid sequencing NMR X-ray crystallography Electron microscopy

Our services in detail:

  • Consultation about bioanalysis
  • SDS-PAGE (polyacrylamide gel electrophoresis with sodium dodecyl sulphate)
  • IEF (Isoelectric focusing)
  • 2d-combination of SDS-PAGE and IEF
  • Western blot
  • Combination of SDS-PAGE / IEF and western blot
  • Amino acid analysis 
  • Capillary electrophoresis 
  • LC-MS
  • Endotoxins
  • Additional services are according to the directory of services or upon request
Electrophoresis in bioanalysis
Electrophoresis in bioanalysis

Infrastructure and Methods

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