Sophisticated Antibody Analysis by GPC/SEC with RALS
PSS Polymer Standards Service GmbH
Monoclonal antibodies (mABs) are increasingly growing in importance for the diagnosis and therapy of various diseases, including cancer and autoimmune and inflammatory disorders. One essential parameter to define their quality is the content of aggregates (dimers, trimers, and higher aggregates). These aggregates can be formed during processing and purification or are the result of long-term storage. As a result of aggregation, antibodies lose their pharmaceutical efficacy and can facilitate an immunology response.
Antibody fragments which lack the Fc region can be used for the treatment of diseases. They can also be the result of degradation of full length antibodies. Therefore, a GPC method, which offers the opportunity to analyse antibodies and their aggregates, as well as antibody fragments simultaneously, with superior resolution and high sensitivity is invaluable.
Experimental
GPC/SEC analysis was performed on a PSS SECcurity GPC system, equipped with a PSS SECcurity SLD1000 light scattering detector, using the following conditions:
Results
Figure 1 shows an overlay of elugrams obtained for a full length antibody and antibody fragments analysed on a single set of columns.
Figure 1: Separation range of the column combination. The red curve shows the UV signal of a full length antibody and its dimers plotted against the elution volume. The blue curve is the elugram of antibody fragments and their high level aggregates.
All three detector signals for the analysis of a monoclonal antibody are shown in Figure 2. The light scattering signal shows improved sensitivity for high aggregates compared to the other signals.
Figure 2: Sensitive analysis of antibody aggregates. The light scattering signal for the dimer is relatively high compared to that of the mABs because of molar mass dependency and provides improved sensitivity for the detection of high aggregates (inset).
Conclusion
The GPC/SEC method including UV, RI, and RALS can be used for the simultaneous determination of aggregate content of monoclonal antibodies as well as antibody fragments. The column combination covers the separation range for all three types and provides a high resolution for the determination of the dimer content. Because of its molecular weight dependency, the PSS SLD1000 RALS detector offers high sensitivity for very small quantities of high aggregates and also allows the determination of the absolute molecular weight of the antibodies. In addition, it has a unique feature for a light scattering detector as the wavelength can be altered to increase the sensitivity.
PSS Polymer Standards Service GmbH
In der Dalheimer Wiese 5, D-55120 Mainz, Germany
Tel: + 49 6131 962390 fax: + 49 6131 962390 11
E-mail: info@pss-polymer.com
Website: www.pss-polymer.com
The Next Frontier for Mass Spectrometry: Maximizing Ion Utilization
January 20th 2025In this podcast, Daniel DeBord, CTO of MOBILion Systems, describes a new high resolution mass spectrometry approach that promises to increase speed and sensitivity in omics applications. MOBILion recently introduced the PAMAF mode of operation, which stands for parallel accumulation with mobility aligned fragmentation. It substantially increases the fraction of ions used for mass spectrometry analysis by replacing the functionality of the quadrupole with high resolution ion mobility. Listen to learn more about this exciting new development.
Liquid Chromatography to Analyze Vitamin D Proteins in Psoriasis Patients
January 21st 2025Can a protein involved in delivering Vitamin D to target tissues have an altered serum profile in psoriasis patients with cardiovascular disease? Researchers used liquid chromatography (LC) to help find out.
The Complexity of Oligonucleotide Separations
January 9th 2025Peter Pellegrinelli, Applications Specialist at Advanced Materials Technology (AMT) explains the complexity of oligonucleotide separations due to the unique chemical properties of these molecules. Issues such as varying length, sequence complexity, and hydrophilic-hydrophobic characteristics make efficient separations difficult. Separation scientists are addressing these challenges by modifying mobile phase compositions, using varying ion-pairing reagents, and exploring alternative separation modes like HILIC and ion-exchange chromatography. Due to these complexities, AMT has introduced the HALO® OLIGO column, which offers high-resolution, fast separations through its innovative Fused-Core® technology and high pH stability. Alongside explaining the new column, Peter looks to the future of these separations and what is next to come.
A Guide To Finding the Ideal Syringe and Needle
January 20th 2025Hamilton has produced a series of reference guides to assist science professionals in finding the best-suited products and configurations for their applications. The Syringe and Needle Reference Guide provides detailed information on Hamilton Company’s full portfolio of syringes and needles. Everything from cleaning and preventative maintenance to individual part numbers are available for review. It also includes selection charts to help you choose between syringe terminations like cemented needles and luer tips.
