Charge variant analysis is a demanding application for applied liquid chromatography systems due to the use of highly corrosive buffer salts in combination with very shallow gradients for optimal separation.

The evaluation of different salt gradients was performed on the Agilent 1290 Infinity II Bio LC System and analyzed for resolution as well as reproducibility.

The 1290 Infinity II Bio LC including High-Speed Pump, with its completely iron-free flow path, is optimally suited for the conditions used in biochromatography-avoiding potentially corrosive damage to the system.

Excellent reproducibility even for highly challenging shallow gradients was determined, confirming the 1290 Infinity II Bio LC as the next generation of Agilent high-end liquid chromatography systems for high confidence in generated data.

The application demonstrates the superior resolution in size exclusion chromatography (SEC) protein separation made possible by the Agilent 1290 Infinity II Bio LC System equipped with the Agilent Advance Bio SEC column and ultralow dispersion capillaries.

The biocompatible UHPLC system enables analysis using corrosive salty buffers and therefore saves maintenance expense.

The resolution in SEC analysis can be compared for capillaries of different inner diameter.

A protein standard mixture and monoclonal antibodies (mAbs), including aggregates, were separated and compared for resolution.

In addition, molecular weight is determined by the Agilent OpenLab GPC/SEC add on software in one software solution, enabling a one-step workflow.

Product-related impurities such as low molecular weight (LMW) or high molecular weight (HMW) species are considered critical quality attributes (CQAs) in therapeutic monoclonal antibody (mAb) products and need to be monitored across the drug production process.

This application note developed an RPLC method based on the excellent performance of the Agilent 1290 Infinity II Bio LC System combined with the PEEK-lined Agilent PLRP-S column.

By analyzing the reduced heavy and light chains of the NISTmAb, excellent relative retention time and area deviations were observed, even with extremely shallow gradient slopes.

After method development, all relevant LMW fragments, such as two heavy chains (H2) or two heavy chains and one light chain (H2L), could be separated and detected.

Due to the sequential coupling of the UV and MS detector, this method can be used in several areas of the biopharmaceutical production chain.

The method also stands as an alternative to SDS-PAGE/CE-SDS with the possibility to analyze two CQAs - LMW species and post-translational modifications (PTMs) - in one run.

Charge variants separation of monoclonal antibodies can be a challenging task for the chromatographer.

Due to the microheterogeneity of the analyzed monoclonal antibody, extensive method development can be necessary to find the optimal desired resolution.

Outperforming many traditional salt gradients, the resolving power of pH gradients enables the separation of charge variants in a very efficient way.

This application note demonstrates high-resolving and reproducible charge variant analysis of two monoclonal antibodies, trastuzumab and NIST mAb, with different types of pH gradients.

The Agilent 1260 Infinity II Prime Bio LC System, with a completely iron-free flow path and featuring an Agilent 1260 Infinity II Bio Flexible Pump, enables the use of Agilent Buffer Advisor Software to facilitate dynamic mixing of solvents from only four stock solutions.

The analysis of critical process parameters (CPP) and critical quality attributes (CQA) plays an important role in monitoring and ensuring optimal process yields and product quality during the production of biopharmaceuticals.

HPLC methods are often used for the analysis of these parameters.

However, cost, lab space, and workload are crucial for the implementation of such tools.

This application note demonstrates the use of an Agilent 1260 Infinity II Bio-Inert LC System coupled to an Agilent Infinity Lab LC/MSD iQ with an automated column selection valve for spent media analysis and protein A-based titer determination.

By applying hydrophilic interaction liquid chromatography (HILIC) with smart MS detection, excellent linearity, sensitivity, and selectivity for important cell culture medium components were achieved.

The quaternary pump used in the 1260 Infinity II Bio-Inert LCS system enabled the subsequential analysis of the monoclonal antibody (mAb) product with UV detection, without manual reconfiguration of the instrument.

All necessary maintenance operations such as column switching, washing, or equilibration could be performed using Agilent OpenLab CDS 2 software, which can be fully qualified to meet the requirements of the FDA regarding 21 CFR part 11 compliance.

The determination of the drug-to-antibody ratio (DAR) is typically performed using hydrophobic interaction chromatography (HIC).

The eluents for this mild, nondenaturing analysis method contain high concentrations of corrosive salts, which challenge the liquid chromatography (LC) system.

The Agilent 1290 Infinity II Bio LC System including High-Speed Pump, with its completely iron-free flow path, is optimally suited for the conditions used in biochromatography-avoiding potential corrosive damage to the system.

This application note demonstrates the DAR determination of brentuximab vedotin using HIC.

The DAR was calculated to 3.7 drug molecules per antibody.

Excellent reproducibility was found, demonstrating that the 1290 Infinity II Bio LC belongs to the next generation of Agilent high-end liquid chromatography systems for the highest confidence in generated data.

"Blank subtraction", as a software feature of Agilent OpenLab CDS, removes drifting baselines due to less pure ammonium sulfate, enabling smooth integration.

The addition of organic modifiers like isopropanol in hydrophobic interaction chromatography (HIC) can be an important parameter to decrease the retention of hydrophobic antibody drug conjugates (ADCs) as well as to adjust selectivity.

This application note demonstrates the drug-to-antibody (DAR) determination of brentuximab vedotin using a ternary gradient with isopropanol as organic modifier inthethirdchannel.

Excellent reproducibility was found for this challenging combination of high-salt-containing buffer and organic solvent, making the Agilent 1260 Infinity II Prime Bio LC the next generation of Agilent high-end liquid chromatography systems for the highest confidence in generated data.

This application note demonstrates the reliable separation of 2-AB- and Instant PC-labeled glycans with the Agilent 1290 Infinity II Bio LC System under resolution- and speed-optimized conditions.

The reliability is showcased by calculation of performance values such as retention time precision, area precision, and resolution, all of which obtained excellent values.

For detection, the Agilent 1260 Infinity II Fluorescence Detector (FLD) with bio-inert flow cell was used.

The 1290 Infinity II Bio LC System has low internal volume and the binary pump provides highly precise solvent compositions and flow rates, which enables method transfers from high-resolution to speed-optimized methods with comparable results.