Geoffrey Rule, Ph.D. - Principal Scientist, MilliporeSigma, Bellefonte, PA, A business of Merck KGaA, Darmstadt, Germany
Kevin Ray, Ph.D. - Senior Manager, Analytical R&D MilliporeSigma, St. Louis, MO, A business of Merck KGaA, Darmstadt, Germany
Thorough characterization of protein therapeutics is essential to ensure that drugs developed and manufactured meet all safety, efficacy, and quality requirements. Here we provide an overview of approaches for breaking mAbs into subunits, performing chromatographic separation of those subunits, and then obtaining high resolution spectra of the subunits to verify mass, characterize glycosylation and other post-translational modifications (PTMs).
Creating mAb fragments for analysis
A middle-up analysis describes a process where the mAb of interest is broken down into smaller pieces prior to analysis. In the MS experiment focus is on getting accurate mass measurements of the subunit pieces. In a middle-down experiment the same subunits may be formed but these pieces are then further broken down as ions in the gas phase, within the mass spectrometer, to obtain amino acid sequence information and localize PTMs.
There are two common ways of creating antibody subunits. One is to make use of a chemical reductant, such as dithiothreitol (DTT), that breaks disulfide bonds. This creates two heavy chains (HC) and two light chains (LC) by breaking interchain disulfide bonds. Another is the use of an enzyme that cleaves at a specific site of the IgG molecule. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes, or IdeS, is one commonly used enzyme that cleaves IgG at a specific location in the hinge region. This creates two pieces of the Fc domain (Fc/2) and a single piece comprised of both Fab binding domains, denoted as F(ab’)2.
These two strategies are frequently combined to yield two smaller Fd’ fragments in addition to the Fc/2 and LC fragments. When combining the methods, it is important that the second, chemical reduction step be performed with heat and a denaturing agent such as guanidine hydrochloride solution. Otherwise, reduction of intrachain disulfide bonds can be incomplete resulting in more complex chromatography and MS spectra from partially reduced protein fragments.
One advantage of doing middle-up instead of intact mass analysis is that the protein is made into smaller pieces thereby improving mass accuracy of the measurement. It also allows for interrogation of the subunits separately so that PTMs can be somewhat localized, though not precisely located. Both qTOF and Orbitrap-type instruments are well suited for this type of mass measurement.
The chromatographic approaches
Three forms of chromatography are commonly used in middle-up mAb analysis: reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and size-exclusion chromatography (SEC). In each case the objective is to separate the fragments and perform high-resolution MS analysis on each to evaluate mass and PTMs.
SEC can be used for separation of chemically reduced mAb HC and LC as these are substantially different in size at 50 and 25 kDa, respectively. This entails relatively simple sample preparation and a basic chromatography set-up where generic conditions will work for most every mAb. Separations of HC from LC on 30 cm columns can be completed in 6-8 minutes. Newer SEC columns with smaller 2 um particles can provide the same separation on shorter columns and in less time. Another potential advantage of SEC over other types of chromatography is that the protein can be retained in its native conformation, using aqueous volatile buffers, since organic solvents need not be used. This provides lower charge states, at high mass to charge ratios, and therefore an improvement in mass accuracies after deconvolution.
When looking for additional information requiring IdeS digestion and reduction the fragments are much closer in size, approximately 23 - 27 kDa each, so a better separation is required.
Better separations for additional information
RPLC is a common approach for separation of protein subunits prior to MS analysis. Specialized sorbents available for these types of separations make use of wide pores, short chain alkyl phases (C4) and fused-core particles. These can readily separate the Fd’, LC, and Fc/2 fragments generated by IdeS digestion and reduction. This is where complete reduction of all intrachain disulfide bonds becomes important for clean chromatography and spectra. If reduction is incomplete, additional peaks may be found with slightly different masses (-2 Da per bond) from the fully reduced form.
Recent research has shown HILIC separation of IdeS-digested and chemically reduced fragments to be viable as well. In this case, the Fc/2 fragments generally elute later than the other fragments as the more polar glycans impart stronger retention with HILIC. Different glycoforms of the Fc/2 fragment can even be chromatographically separated to provide data complementary to that obtained by RPLC.
A host of strategies is therefore available for middle-up analysis of monoclonal antibodies. Creation of mAb subunits by digestion with enzymes such as IdeS, Lys-C, or papain, or with chemical reduction, can be used alone or in combination. Choice of chromatographic technique then depends on the type of information required. Recently, we have developed several useful workflows, including for intact and middle-up mass analysis of reduced and non-reduced mAbs by SEC-MS1 .
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