Sample preparation is a crucial first step in mass spectrometry (MS) analysis, with the power to strongly influence final results.
Liquid chromatography is a common sample preparation technique used prior to MS, especially when characterizing molecules that are biotherapeutic targets such as monoclonal antibodies and antibody-drug conjugates (ADCs).
Discussed here is liquid chromatography technology currently used for preparing MS samples in the analysis and characterization of biotherapeutics.
Simplifying complex samples
Sample preparation often includes reducing the complexity of biotherapeutics prior to MS analysis. Commonly used stationary phases for this purpose include size-exclusion chromatography (SEC), ion-exchange (IEX) chromatography, hydrophobic interaction chromatography (HIC) and reverse-phase (RP) chromatography.
Thermo Fisher Scientific offers a range of Thermo Scientific™ MabPac™ columns in these modes for analyzing intact biotherapeutics. MAbPac columns can be used to separate components prior to subsequent ultra-HPLC separation on the Thermo Scientific Vanquish UHPLC platform. Thermo Fisher Scientific has been working with the National Institute for Bioprocessing Research and Technology (NIBRT) in Ireland on SEC and has optimized setup with the Vanquish UHPLC system. Additionally, the Thermo Scientific™ Q Exactive™ BioPharma platform is optimized for analyzing native intact proteins.
Waters has developed an online SEC-MS approach with biotherapeutics in mind. The company’s diol-coated BEH SEC columns can be used to analyze higher-order structures in their native form. “This is also used for cys-modified antibody-drug conjugates that may have the disulfides holding the two heavy chains disrupted by the drug-conjugation process, and would fall apart under denaturing conditions prior to MS detection,” says Scott Berger, senior marketing manager for biopharmaceutical business operations at Waters. The BEH SEC columns can also be used to separate subunits of denatured, intact targets.
Liquid Chromatography Systems
Find, compare and review different
liquid chromatography systemsSearch
Two-dimension liquid chromatography (2D-LC)—an initial LC analysis, followed by a second LC analysis of the first’s eluent—can also provide greater separation of components prior to MS. Agilent Technologies offers the Agilent 1290 Infinity II 2D-LC system for researchers looking for an integrated 2D-LC for MS solution. “With 2D-LC, researchers can speed up their total analysis workflow without having first to collect individual fractions and then re-inject the samples into an LC-MS system,” says Michael Yap, marketing manager for biopharma development at Agilent Technologies. “2D-LC MS also offers a higher separation dimension and greater resolution, leading to the better characterization of complex biotherapeutic samples.”
Several vendors, including Agilent Technologies, are helping to smooth the workflow from LC to MS. Some LC buffers (such as those used in IEX or SEC) are high in salt, rendering them incompatible with downstream MS—high-salt mobile phases interfere with the electrospray MS ionization process. Agilent Technologies uses a desalting strategy with RP cartridges or a trapping column, which enables direct online 2D-LC MS analysis. Agilent also offers the automated AssayMAP Bravo platform, which is designed for reproducible sample prep before LC-MS analysis.
A new, multidimensional sample-preparation solution is also offered by Shimadzu Scientific Instruments. In collaboration with Perfinity Biosciences, Shimadzu developed the automated Perfinity Platform to prepare antibodies for multidimensional MS analysis. The platform includes affinity selection, trypsin digestion, desalting and a final separation. “The method provides a 10-minute cycle time, is a reproducible immuno-MS platform for quantitation of IgG peptides and can be applied to many other peptide and proteins applications,” says Gurmil Gendeh, marketing manager for pharmaceuticals at Shimadzu Scientific Instruments.
Taking targets apart
Biotherapeutics are often studied by taking them apart—for example, with peptide-mapping analysis, in which target proteins are digested and their resulting peptides analyzed by MS. The Thermo Scientific™ SMART Digest™ Kits were designed to increase reproducibility and streamline digestion for peptide mapping of biotherapeutics. Rowan Moore, pharma and biopharma vertical marketing manager for chromatography and mass spectrometry at Thermo Fisher Scientific, recommends using the Thermo Scientific™ Acclaim™ Vanquish™ C18 UHPLC column for subsequent peptide separations. “It works in harmony with the Vanquish UHPLC system to deliver high resolution of low-level peptides,” she says. “We are really excited about work looking at the analysis of host cell proteins and running the Vanquish UHPLC in ‘tandem’ mode to increase peptide-mapping throughput with this column.”
Shimadzu Scientific Instruments recently released the nSMOL Antibody Bioanalysis (BA) Kit, designed to quantify antibody drugs in biological fluids by LC-MS/MS. The kit reagents enable selective proteolysis of the Fab region of monoclonal antibodies in blood or other biological samples. “Fab-derived peptide fragments produced by limited proteolysis can be accurately quantified by MRM measurements using a triple-quadrupole HPLC mass spectrometer,” says Gendeh. An advantage of using this approach for large molecules like antibodies is that the selective fragmentation produces fewer peptide fragments, which helps to suppress background noise and ion suppression, according to Gendeh.
Glycoproteins and glycopeptides
Another way to take biotherapeutics apart is to study the sugars attached to proteins or peptides. One approach is to cleave the sugars from the target and then analyze the released free glycans. Another tack is to study intact glycoproteins or glycopeptides with their sugars attached. Agilent Technologies offers HIC columns for glycan analysis of free N-glycans, as well as glycopeptides and glycoproteins. Waters recently developed a new, larger-pore HILIC chemistry, available in its BEH HILIC Glycoprotein Columns for online LC-MS analysis of glycoproteins and glycopeptides, as well as for optimal analysis of larger free, labeled glycan structures.
Because glycans are hydrophilic, they influence the interaction of a relatively hydrophobic peptide or protein with the column during LC.
“Separation of glycoforms is typically not as effective using traditional RP-LC-MS methods, as the hydrophobic nature of the peptide portions of the molecules dominates the column interactions,” says Berger. “Our large-pore HILIC approach enables researchers to construct analyses focusing on studying antibody and other biotherapeutic glycosylation, without interference from most nonglycosylated peptides.”
For analysis of free glycans, the Thermo Scientific™ GlycanPac™ LC Columns use mixed-mode column chemistries to achieve more than one type of separation simultaneously. They are capable of resolving native or labeled glycans by size, charge, polarity and isomer type, which is “really useful for the analysis of glycans released from highly charged, sialylated biologics such as etanercept,” says Moore.
The Thermo Scientific™ Accucore™ 150 Amide HILIC LC Column is better suited for glycan analysis of monoclonal antibodies, which typically have a low level of charged glycans. “Whilst some commercially available kits do exist for the amplification of glycan signals by MS, I think there are still significant advances to be seen in this area,” says Moore.
Waters has also developed the ionKey-MS system, a ceramic-based microfluidics device for microscale RP separations. This is valuable for biotherapeutic researchers working with minute quantities or extremely limited sample materials—such as when screening early clones, grown in small-volume cell-culture systems—and aiming for optimal production of a biotherapeutic protein. The ionKey-MS technology is also used in preclinical studies, “where biofluid samples are small, and high sensitivity is required to follow the molecule and key variants from high levels at dosing to much lower levels as the biotherapeutic is cleared,” says Berger.
One thing is for sure: Scientists can’t choose just one tool. Biotherapeutics are complex enough to require analysis of intact molecules, their subunits and individual peptide levels or free glycans. By combining all these levels of analysis, scientists are quickly moving the development of biotherapeutics toward new discoveries and therapeutic applications.
Image: Shutterstock Images