Biopharmaceuticals, with monoclonal antibodies (mAbs) at the forefront, constitute a relatively young and thriving branch of products, which in most aspects differ substantially from the small molecule counterparts.
Manufacturing biosynthetic products is a complex and variable process because the molecules are large and undergo numerous post-translational modifications, yielding thousands of variants, and makes separating and identifying each variant quite challenging. Failure to remove unwanted variants can compromise drug efficacy and patient safety, so you need a thorough analytical method to guarantee product quality.
Mass spectrometry (MS) data is a valuable tool you can use to distinguish structurally similar mAb variants but is not easily accessible in many cases due to the non-volatility of liquid chromatography (LC) buffer systems. This MS incompatibility often means you need to carry out additional steps, like manual fraction collection, desalting and reinjection, to collect the essential data.
Finding ways to overcome the manual purification process is what every modern biopharma lab needs.
And that’s where two-dimensional (2D-LC) technology shines.
How 2D-LC went from “expert only” to routine
2D-LC is a technique that successively uses two complementary LC modes for a multi-dimensional separation. Traditionally such systems are used for additional selectivity to help resolve complex or difficult separations or to deplete interfering matrices.
With that said, 2D-LC provides a way to automate laborious manual processes like fraction collection and desalting for MS introduction, allowing busy labs to scale complex tasks by reducing the manual workload and analysis time.
The resulting gain in analytical throughput is why 2D-LC is evolving from a “for experts only” technique into a more routine application.
Boost your analytical capabilities with modern 2D flow paths
While 2D-LC systems can support diverse analytical needs, you can further increase functionality and application flexibility by utilizing instruments with modern flow path designs.
One example is the combination of the 2D functionality of a system with independent accessibility of the particular LC flow paths (Figure 1). These setups are beneficial for the development of new 2D-LC-MS methods or for a fuss-free switch to standard LC or LC-MS instrument usage.
One common application of 2D-LC to highlight here is the hyphenation of MS-incompatible LC methods with MS, like in the desalting of strong-cation exchange (SCX) chromatography fractions for mAb charge variant profiling by reversed-phase chromatography (RP).
A multiple heart-cut 2D-LC method for intact mass analysis of Infliximab charge variants
The SCX-UV profile of the mAb infliximab shows several acidic and basic variants in three major and five minor peaks (Figure 2). With multiple heart-cut 2D-LC the storage, desalting, and MS analysis of up to five SCX fractions is enabled.
After convenient independent method development of either dimension, the first 2D-LC/MS experiment consisted in the collection and transfer of three SCX fractions — one for each major peak respectively — to the desalting RP dimension and consecutive intact MS analysis.
By spectrometric deconvolution, the three major compounds were identified as infliximab variants with zero, one, and two C-terminal lysine residues (Figure 2, blue bars). Baseline resolved glycoform patterns reveal the major glycoforms as G0F/G0F, G0F/G1F, G1F/G1F, G1F/G2F, and G2F/G2F.
Sensitivity boost by fraction enrichment
After identification of the three major compounds, the five minor peaks were subject to the same procedure with just a five-fold increase in injection volume in the SCX dimension. However, the MS signal intensity for one of the minor peaks was still not sufficient for proper deconvolution.
In that case adequate MS data (Figure 3) were attained by a five-fold fraction enrichment by loading all five storage loops with the same fraction in repeated SCX runs. After deconvolution, all minor SCX peaks could be tentatively attributed to some deamidation and sialylation products of the major components (Figure 2, yellow bars).
Multiple LC dimensions offer incredible capabilities
All details of the Infliximab example can be looked up in our new application note, and even better: you can download all methods, configuration files, and a dedicated instrument control ePanel for free.
However, the possibilities with two or even multiple coupled LC dimensions in facilitating biotherapeutic analytics are countless and exceed the “simple” application extension to the desalting of other LC modes like RP or hydrophobic interaction chromatography by far.
To name only a few directions this has already taken:
- Resolution of complex separation problems by slicing main peaks into multiple fractions and transfer these to an orthogonal separation to uncover overlapping hidden impurities
- Process analytics: online clean-up of cell culture samples from bioreactors by affinity capture and subsequent characterization of the purified product e.g. by SEC (aggregation) or IEX (purity)
- Automated online sample preparation including steps like reduction or enzymatic digestion in 2D-, 3D- or 4D-LC/MS workflows
Learn more about 2D-LC technology
The big application potential, plus improvements in commercial hardware and software availability, makes 2D-LC not only attractive in your research and development lab but also gains attention in regulated environments for quality assurance and control.
If you want to learn more about our 2D-LC technologies, visit our product page here.