IMG_4714High Performance Liquid chromatography (HPLC) encompasses a large range of methods in biopharmaceutical research such as RP-HPLC, IEX-HPLC, SEC-HPLC that can be applied in combination with each other as well as in combination with other analytical techniques. This makes HPLC one of the most used analytical techniques in laboratories world-wide, allowing the separation of biomolecule from amino acids to protein complexes. The use of UPLC systems has further increased the speed and resolution of these analyses.

HPLC methods generally allow a qualitative and quantitative analysis of samples with high reproducibility, making it also a method of choice for release testing. At Protagen Protein Services GmbH, we apply HPLC to:

Proteins

Reversed-Phase HPLC is often used in combination with UV or mass spectrometry as a proof of identity and purity. As examples, protein fragmentation or oxidation can be monitored.

Ion Exchange HPLC (IEX) is usually applied to detect protein deamidation or C-terminal lysine variants in monoclonal antibodies.

The main application of Size-Exclusion chromatography (SEC) is to detect protein aggregation and protein fragmentation.

Peptides

For peptide mapping, usually RP-HPLC is applied, either with UV or MS detection.

Amino Acids

Amino acid analysis is carried out using a reversed-phase HPLC with fluorescent detection.

Glycans/Monosaccharides

N- and O-glycans, neutral monosaccharides and sialic acids are detected by HPAEC-PAD or NP-HPLC. The HPAEC-PAD anion exchange chromatography allows the detection of the unlabeled glycans after release from the protein backbone. The NP-HPLC method requires labelling of the glycans but affords a higher sensitivity and resolution.

Also, more unusual monosaccharides such as Mannose-6-Phosphate can be quantified.

Other Methods

Immunoaffinity depletion of serum samples can help to improve the sensitivity of analysis for low abundant proteins in these samples.

Protein A IgG enrichment can be used to make monoclonal antibodies available for analysis from unpurified cell lysates.

Either an existing HPLC method is transferred to Protagen Protein Services GmbH, or a suitable method is developed by our experts.

The general quantitative analysis is carried out by measuring a calibration curve for the analytes of interest and then interpolating the sample concentration from the peak areas. The developed methods are then subjected to a robustness testing before being validated for release testing.

Liquid chromatographic patterns are required for the protein characterization according to ICH Q6B.

Your contact for Liquid Chromatography at Protagen Protein Services GmbH

burkhard fleckenstein

Dr Burkhard Fleckenstein

Director Operations 
Our business development team will be happy to assist you with your project.

Phone: +49 (0) 231 9742-6100
Email: salesproteinservices@ProtagenProteinServices.com