Moving biologics to market with automated biopharma workflow solution
Vicki Pandey, Agilent Global Marketing Communications
Biologic recombinant proteins are highly complex molecules that require specific purification techniques, complex sample preparation, and often long analytical methods to achieve resolution and detection. High-throughput platforms and automated workflows are two ways to improve efficiency, increase throughput, and ensure precision. In this article, we review some examples of automated Agilent systems that can improve your workflows as you move biologics to market.
Figure 1. Agilent MassHunter BioConfirm offers automated peptide mapping data acquisition and analysis workflows.
A peptide mapping experiment may contain hundreds of peptides with various modifications. Automatic data processing improves experimental efficiency for data mining. Figure 1 shows the peptide mapping workflow provided by Agilent MassHunter BioConfirm software. After data acquisition, compounds are extracted automatically based on the chromatography characteristics. The table of compounds is then matched to the list of theoretical peptide digests with potential modification groups. The MS/MS spectrum of each peptide is interpreted by the b/y fragments of the peptides. A sequence coverage map is then compiled and the peptide mapping report automatically generated at the end of the workflow.
Agilent AssayMAP Bravo allows parallel processing of samples with low variability
An ever-increasing number of potential biotherapeutic proteins must be rapidly and accurately characterized so that data-driven decisions can be made to move the best candidates efficiently through drug discovery and development processes. This demands reductions in sample bottlenecks that in turn require more rapid, scalable, and reproducible methods to supplant current low-throughput and laborious manual methods with common workflows. These workflows include protein purification and quantification, whole protein mass determination, sequence coverage analysis, and characterization of posttranslational modifications.
The Agilent AssayMAP Bravo platform addresses these challenges by enabling the parallel processing of samples through a variety of LC/MS sample prep workflows, with low variability and minimal hands-on time.
Figure 2. Workflow for antibody sample preparation.
Figure 2 illustrates a sample prep workflow on AssayMAP Bravo. A full plate of 96 samples, each containing 15 µg of mAb, was processed using affinity purification, in-solution digestion, and peptide cleanup. LC/MS produced extracted ion chromatograms for six proteotypic peptides, from which relative protein abundance could be inferred. CVs of 5 to 8% were calculated from EIC peak areas for all 96 samples, which indicated that reproducibility of the entire workflow was very high.
Agilent columns offer high-resolution analysis of complex glycan
Detailed characterization of the glycan profile of biopharmaceuticals, such as recombinant EPO (rhEPO), is essential as differences in glycosylation can affect their pharmacodynamics and pharmacokinetic behavior. The method of choice is typically hydrophilic interaction chromatography (HILIC) after labelling with 2-aminobenzamide (2AB) for sensitive fluorescence detection.
Whereas HILIC efficiently separates glycans according to hydrodynamic radius, it is insufficient to fully resolve the complex mixture of branched glycan structures that are present in samples such as EPO or fetuin. Fortunately, weak/strong anion-exchange chromatography (WAX/SAX) provides a highly orthogonal separation that depends on the number and arrangement of acidic monosaccharides in the glycan. To improve resolution and enhance peak capacity, a combination of WAX and HILIC separation can be used. First, a comprehensive WAX/HILIC 2D-LC setup was tested using fetuin. A 110 minute WAX gradient with an Agilent Bio WAX column was used for the first dimension, followed by a 30 second, second-dimension comprehensive HILIC run using a 4.6 × 50 mm HILIC column.
Figure 3. Comprehensive HILIC/WAX 2D-LC separation of fetuin and EPO, showing highly orthogonal separation. The ion exchange chromatography in the second dimension reveals the charge pattern of the glycans.
The comprehensive HILIC/WAX separation was used in an online 2D-LC setup, maintaining the highly orthogonal separation. Figure 3 shows the 2D-LC image from a HILIC/WAX 2D run. The 2D separation provides high peak capacity and resolution, and many of the coeluting peaks from the HILIC dimension are well separated by the WAX column.
Agilent solutions accelerate biopharma discovery and development
Whether you are looking to increase sample throughput, accelerate method development, automate sample preparation, or solve challenging biopharma applications, the highly modular Agilent approach gives you the flexibility you need to succeed. Explore a wide range of LC tools and solutions, as well as LC/MS instruments, for the automation of virtually any pharma-related liquid chromatography application. To discover more, contact your Agilent Representative who can help you identify the right tools for your application.
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