The Benefits of Ion-Exchange Chromatography to Monitor Charge Heterogeneity in Monoclonal Antibodies

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The Column spoke to Richard Shannon from AstraZeneca about his work characterizing monoclonal antibodies (mAbs), why ion-exchange chromatography (IEC) is his technique of choice for analyzing mAbs, and offers his advice for anyone wanting to use the technique.

Q. What specific considerations are needed for the characterization of monoclonal antibodies (mAbs)?

A: Monoclonal antibodies (mAbs) are glycosylated multi-subunit proteins containing four subunits covalently joined together by disulphide bonds; there are two identical heavy chains (with one glycosylation site on each) and two identical light chains. Once the mass and sequence of the heavy and light chains have been confirmed using mass spectrometry (MS), the main considerations for characterization of product variants include size variants and charge variants. The size variants can be larger than the monomeric mAb (aggregates) or smaller (fragments). The charge variants, caused by single amino acid modifications or charged glycosylation, can have a pI lower than the parent mAb (acidic species) or higher (basic species). Importantly, for mAbs, it is impossible to characterize and/or quantify every single size and charge variant. A mAb has a molecular weight of approximately 150,000 Daltons with more than 1300 individual amino acid residues. Several amino acids are susceptible to chemical modification during manufacture and storage; for example, deamidation of asparagine, isomerization of aspartic acid, and oxidation of methionine and tryptophan. There may be more than 100 sites of possible chemical modification in the whole molecule, but not all of these will be modified-the reactivity at each site is determined by neighbouring amino acids, solvent accessibility, and flexibility of the peptide backbone. Instead of trying to quantify every modification, we monitor groups of species (aggregates, fragments, acidic species, basic species) and focus our characterization efforts on modifications that affect the functional activity, safety, or stability of the product.

Q. Why is ion-exchange chromatography your technique of choice to monitor mAbs? Why did you choose this approach and what are the advantages over other techniques?

A: Ion-exchange chromatography (IEC) is one of three common techniques used to monitor the charge variants of mAbs. Capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) are the other two. In fact, IEC is not our method of choice to monitor mAbs-cIEF is more routinely used to monitor charge variants of mAbs, particularly in early stages of preclinical and clinical development. However, IEC does have some specific advantages over cIEF that make it a particularly useful technique. CZE is less commonly used.

cIEF is usually faster and higher resolution than IEC, and a fit-for-purpose method can be developed more rapidly. These advantages make it the method of choice in early stages of preclinical and clinical development. The key advantage of IEC is that you can collect fractions and analyze the fractions off-line by MS, tryptic peptide mapping, or some other high-resolution method, to find out exactly what modifications are causing the different charge states. Even when cIEF is the preferred method for release testing, IEC with fraction collection is often used to characterize the different charge states present in the cIEF profile.

As a characterization method, IEC is particularly useful because the protein remains in a native state. This enables collected fractions to be tested in a bioactivity assay to establish how a particular modification (or group of modifications) affect the functional activity of the mAb.

In addition, because separation in IEC is due to interactions of the native protein surface with the column stationary phase, changes in surface charge distribution may cause significant changes in retention times. Therefore, IEC may be better than other methods at separating charge (and conformational) variants that alter the protein surface charge distribution. This is important because the functional activity of a mAb is also due to surface charge distribution, but in a much more specific way. Three binding regions known as complementarity determining regions (CDR1, CDR2, and CDR3) interact very specifically with the target protein. These regions are fairly flexible and solvent accessible. Modification to amino acids in these regions can seriously impair the binding of the mAb to its target. As separation of charge variants in IEC is determined by surface charge distributions-including those that influence target binding-you could say that IEC is well suited to separate charge variants that may have altered functional activity.

Even when a chemical modification, such as isomerization of aspartic acid, causes a conformational change, rather than a net charge difference, a change in surface charge distribution may cause a shift in retention time by IEC. IEC may therefore be more able to separate conformational variants that may have reduced functional activity.

I think another advantage of IEC is the flip side to one of its disadvantages. IEC has a lower resolving power than cIEF, but this can contribute to the robustness of IEC as an analytical method. A final charge method is developed to monitor key modifications or attributes that may affect function, safety, or stability of the product. It may not be necessary to monitor all the variants that are separated by a more resolving method, such as cIEF. A less-resolving method may be a more robust method.

Q. Do you have any advice for scientists wanting to use ion-exchange chromatography?

A: One advantage of IEC is that all you need to get started is a high performance liquid chromatography (HPLC) system, an HPLC column, and an HPLC scientist! After that, it’s just the usual fun of developing HPLC methods. There is, however, one important factor to determine before you begin: the pI of the molecule of interest. This will then inform your choice of IEC column-anion exchange or cation exchange, weak or strong ion exchanger-and your separation conditions. Separation can be based on a salt gradient, keeping the pH of the mobile phase the same, or a pH gradient. Salt gradients are more common, but pH gradients are gaining in popularity. Whether you use a salt gradient or a pH gradient, the molecule of interest must bind to the column under the starting conditions. For cation exchange chromatography (usually used for mAbs), the pH of the mobile phase must be lower than the pI of the molecule, so that the molecule is positively charged and binds to the negatively charged column. For an anion exchange column, the converse is true.

Q. Please could you talk a little on the development of the antibody–drug conjugate (ADC) platform with inserted cysteine residues (1).

A: Antibody–drug conjugates (ADCs) have the potential to be powerful therapies for the treatment of cancer. A cytotoxic chemical is covalently attached (conjugated) to a mAb via a linker. The mAb directs the cytotoxic drug to the tumour cell, where it is released into the tumour environment, therefore reducing the systemic effects otherwise associated with chemotherapy. The first ADCs were prepared by conjugation of a linker bearing the cytotoxic drug to the mAb, using existing accessible lysine and cysteine amino acid residues in the sequence. This type of conjugation can lead to heterogeneous conjugates, which could have variable numbers of drug molecules per antibody. One approach to reduce this heterogeneity is site specific conjugation, where a reactive cysteine or lysine residue is engineered into the mAb sequence, thus controlling the number and sites of linker conjugation. At AstraZeneca (MedImmune), a cysteine residue is inserted into the sequence in the heavy chain, at a position that does not affect the activity, safety, or stability of the product. A more homogenous ADC product is manufactured, which consistently has two drug molecules per antibody.

Q. Why is an antibody intermediate with inserted cysteine residues more complex than a regular mAb? (2)

A: The inserted cysteine residues are reactive. During manufacture of the mAb intermediate (the mAb before conjugation), the inserted cysteine residues can react with other sulphide-containing compounds present in the bioreactor, such as L-cysteine or glutathione, and become capped. Or they can remain in the uncapped free thiol form. The capped state of inserted cysteines does not impact the manufacture of the final ADC because the initial step of the conjugation reaction involves reduction of the inserted cysteines to their free thiol form. Also, the function of the final ADC is not affected by whether the inserted cysteines in the mAb intermediate were capped or not. However, the capped forms add to the heterogeneity of the mAb intermediate and complicate the charge profile. Glutathione has a negative charge, so glutathione capping can add one or two extra negative charges to the mAb intermediate. This can be observed as two distinct additional acidic peaks in the cIEF and IEC profile. The covalent attachment of L-cysteine to the inserted cysteine does not add a charge in the same way as glutathione, but the difference in pKa between a free thiol and a disulphide bond is enough of a difference to see additional peaks by cIEF, and possibly IEC. In our laboratory, we have observed additional peaks for the L-cysteine capped species using cIEF, but not for IEC. Therefore, as the capped state was not important for the final ADC product quality, the IEC method was preferred for lot release over the cIEF method because the profiles were less complex and the method more robust.

The peaks from capping of the inserted cysteines may interfere with other peaks that are more important to monitor, such as charge variants from deamidation of asparagine and glutamine residues. In this case, samples can be pretreated with a mild reducing agent that removes the capping species, leaving the rest of the molecule intact. However, an analytical method with sample modification should be employed with caution, partly because some analytical data about process consistency will be lost.

Q. How challenging is the analysis of heterogeneity related to charged variants?

A: The number of possible charge variants present in a mAb is too large to routinely monitor each one. The use of mass spectrometry and tryptic peptide mapping does enable us to take a snapshot of all chemical modifications in the molecule, but these are not routine quality control (QC)-friendly assays. Targeted MS methods using HPLC with simple mass detectors are becoming more common as release methods, but the charge heterogeneity method (IEC or cIEF) is still the primary lot release method used to monitor chemical modifications in mAbs.

Most of the chemical modifications of a mAb will not result in compromised product quality or safety, so the main challenge, already discussed, is to identify the key modifications that affect product quality and design the method to measure these.

Q. What does the future hold?

A: A key advantage of IEC is fraction collection to enable characterization of charge variants. However, this is a laborious task, so how about hooking up a mass spectrometer directly to the IEC column? This has been reported by several groups who have utilized a pH gradient with low concentrations of volatile salt compatible with electrospray mass spectrometry (3). As vendors begin to promote certain columns and mobile phases for this purpose, it may become more routine. However, fraction collection will always be needed because it provides the only means to measure the functional activity of different charge variants.

References

1. N. Dimasi, R. Fleming, H. Zhong, B. Bezabeh, K. Kinneer, R.J. Christie, C. Fazenbaker, H. Wu, and C. Gao, Mol. Pharm.14(5), 1501–1516 (2017).
2. R. Shannon, “Analysis of Charge Heterogeneity in Monoclonal Antibodies with Introduced Cysteines,” paper presented at the 8th Annual World ADC Berlin, 2018.
3. F. Füssl, K. Cook, K. Scheffler, A. Farrell, S. Mittermayr, and J. Bones, Anal. Chem.90(7), 4669–4676 (2018). doi: 10.1021/acs.analchem.7b05241

Richard Shannon studied chemistry at the University of Sheffield (UK) and stayed on to complete a Ph.D. studying functional supramolecular assemblies. His first industry position was in the analytical department at Pfizer, based in Sandwich (UK), where he worked mainly on HPLC analysis of small molecules. He then took up a position at the MRC Mitochondrial Biology Unit in Cambridge (UK) and worked on the isolation, purification, and analysis of proteins, specifically the Complex I protein from bovine mitochondria. He then trained to be a secondary school science teacher and worked for five years in Cambridgeshire, before returning to research at the University of Cambridge Division of Neurosurgery, where he used HPLC to measure the levels of amino acids and neurotransmitters in brain extracellular fluid from neurotrauma patients at Addenbrookes Hospital. In 2014 he returned to the pharmaceutical industry, taking a position at MedImmune, the biologics unit of AstraZeneca, where he has worked in the Analytical Sciences group on a range of projects including mAbs, peptides, bi-specifics, and ADCs.

E-mail:shannonr@medimmune.com