Speeding Up the Analysis of Therapeutic Monoclonal Antibody Fragments Using Supermacroporous Reversed-Phase Particles

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Monoclonal antibody (mAb) treatment is among the most effective method of diagnosis and treatment of a broad range of diseases, including autoimmune disorders, cardiovascular diseases, infectious diseases, cancer, and inflammation. There is an extensive development pipeline for protein and mAb therapeutics, emphasizing the need for innovative and efficient analytical tools that result in faster analysis times. This article explores the use of supermacroporous particles for the analysis of mAbs.

Protein and monoclonal antibody (mAb) biopharmaceuticals form a major part of the growing biologics drug market, with the mAb market being predicted to grow around 15% between 2012 and 2018.¹ Monoclonal antibodies (mAbs) have very complex structures with many possible site-specific variations. With such potential for post-translational modifications (PTMs), quality control and stability assessment are very challenging tasks. Producing mAb drugs therefore requires accurate, reliable, and robust methods to detect, characterize, and quantify structural variants and modifications for final biopharmaceutical product approval and subsequent manufacturing process. This is the key to demonstrating safety and efficacy and is required by the FDA and EMA as well as other regulatory bodies. Reliability in results is essential, but researchers and scientists also need to achieve faster separations and higher throughput, to more quickly establish whether the product is fit for release.    

A Faster Screening Solution

The continuous growth of the mAb market suggests that mAb-based therapies and derivatives will remain the focus of the biopharmaceutical and biotechnology industries for years to come. Over the last decade these industries have been transforming, and technological advancements mean the capability to analyze, stabilize, and manufacture has greatly improved. However, analysis can be slow and the use of inappropriate methods can result in low resolution and low throughput. 

The analysis of mAbs is very challenging because of their complex structures and many possible site-specific variations and PTMs. However, these changes can result in biological consequences, meaning routine analysis is critical. There is a growing trend to obtain intact mass information as well as the glycan profile in the quality control of mAbs using high-resolution mass spectrometers (MS). 

Liquid chromatography (LC) has established itself as an essential tool to characterize mAbs and other protein biotherapeutics. The most widely used liquid chromatography–mass spectrometry (LC–MS) method is to analyze mAbs using reversed-phase LC coupled with MS detection. Intact mAb analysis provides information such as molecular weight, drug-antibody ratio, and glycan profiles. Furthermore, MS analysis of mAb fragments, such as light chain (LC), heavy chain (HC), crystallizable fragment, and antigen-binding fragment (Fab), can quickly reveal the location as well as nature of the modification.²

This article explores a fast separation method for intact mAbs rituximab, trastuzumab, infliximab, and bevacizumab, and their fragments, using a novel supermacroporous reversed-phase column. These therapeutic mAbs were chosen because of their heterogeneous biological products that result from post-translational modifications. Additional modifications such as oxidation can also be introduced during the manufacturing process (methionines located at the Fc region are susceptible to oxidation) and the results of this analysis are also included. 

Experimental

Chemicals and Reagents: The following chemicals and reagents were used: deionized (DI) water, 18.2 MΩ-cm resistivity (Fisher Chemical) Optima LC–MS acetonitrile (MeCN) [C₂H₃N; P/N A955-1]; Optima LC–MS formic acid (FA) [CH₂O₂; P/N A117-1AMP]; and Optima LC–MS trifluoroacetic acid were all purchased from Fisher Scientific. Dithiothreitol (DTT) and papain were purchased from Sigma-Aldrich; FabRICATOR (IdeS protease) (Genovis); Rituximab, trastuzumab, infliximab, and bevacizumab were from the original manufacturers.

The samples initially underwent reduction of the inter-chain disulphides in the mAbs (4 mg/mL), which was achieved by incubating the mAbs with 20 mM dithiothreitol (DTT) at 37 °C for 30 min. Next, digestion was carried out by incubating mAb (2 mg/mL) with papain (0.04 mg/mL) in 100 mM Tris-HCL, pH 7.6, 4 mM EDTA, and 5 mM cysteine buffer at 37 °C for 4 h. Finally, IdeS protease was added at 1 unit enzyme per 1 µg of mAb ratio. The digestion was carried out in 50 mM sodium phosphate, 150 mM NaCl (pH 6.6) buffer at 37 °C for 30 min.

Separation Conditions: Instrumentation: UltiMate 3000 BioRS system (Thermo Scientific Dionex); column: 3.0 × 50 mm, 4-µm MabPac RP (Thermo Scientific) (P/N 088645); mobile phase A: H₂O/FA/TFA (99.88:0.1:0.02 v/v/v); mobile phase B: acetonitrile/H₂O/FA/TFA (90:9.88:0.1:0.02 v/v/v/v); flow rate: 0.5 mL/min; column temperature: 80 °C; UV detector wavelength: 280 nm; injection volume: 5 µL; samples: Rituximab, 5 mg/mL; trastuzumab, 5 mg/mL; infliximab, 5 mg/mL; bevacizumab, 5 mg/mL.

Results and Discussion

Comprehensive analysis of the heterogeneous mAbs post-translational modifications, such as deamidation, C-terminal lysine truncation, and N-terminal pyroglutamation, requires complete digestion of the mAbs and sequencing of all the resulting peptides. However, peptide mapping can be time consuming. A simpler and faster method to analyze the mAb variants and locate the modification sites is to measure the mAb fragments.² Light chains (LC) and heavy chains (HC) were generated by the reduction of mAbs with DTT and the Fc and Fab fragments were generated by papain digestion. Single chain Fc (scFc) and F(ab’)² fragments were then generated by digestion via IdeS. 

Rituximab (Figure 1[a]), trastuzumab (Figure 2[a]), infliximab (Figure 3[a]), and bevacizumab (Figure 4[a]) were analyzed using a reverse phase (RP) column containing supermacroporous particles using a 10-min gradient. The column is specifically designed for the separation of intact monoclonal antibodies (mAbs) and mAb fragments. The stationary phase is fully compatible with “mass spectrometry-friendly” organic solvent such as acetonitrile and isopropanol, as well as low pH eluents containing trifluoroacetic acid or formic acid. The column is based on wide-pore 4-μm polymer particles that are stable at extreme pH (0–14) and high temperature (up to 110 °C). The wide-pore size of polymeric particles enables efficient separation of protein molecules with low carry-over. In all cases, in addition to the main mAb peak, minor variant peaks eluting at a later retention time were observed. DTT reduction of mAb produced two copies of LC (25 kDa) and two copies of HC (50 kDa). In addition, the analysis of LC and HC of each mAb (Figures 1[b], 2[b], 3[b], and 4[b]) showed two major peaks, plus small variant peaks eluting after HC. The degree of separation varied, with rituximab LC and HC giving the best resolution, and infliximab LC and HC the least. 

Papain digestion produced one copy of Fc (50 kDa) and two copies of Fab (50 kDa). The separation of Fc and Fab of each mAb (Figures 1[c], 2[c], 3[c], and 4[c]) showed two major peaks, plus small variant peaks eluting after both Fc and Fab peaks. MS analysis identified two polypeptide chains with the later eluting peak corresponding to the bevacizumab Fab, based on the molecular weight calculation. The analysis of scFc and F(ab’)₂ of each mAb (Figures 1[d], 2[d], 3[d], and 4[d]) showed two major peaks, plus small variant peaks eluting after the F(ab’)₂ peak. In the case of infliximab (Figure 3[d]), two co-eluting scFc peaks were observed. Further MS analysis revealed that the early eluting peak had one c-terminal lysine. This approach enables the general locations of the modifications without complete tryptic digestion and peptide mapping.

Conclusion

The mAb market within the biopharmaceutical and biotechnology industries is growing. LC–MS is the established technology of choice, however, current techniques have limitations and can have long throughput times. The use of a supermacroporous reversed-phased chromatography column in the system configuration in this study provide an effective solution for the separation of mAbs and their related variants, as a result of the larger pore sizes that allow for the separation of large proteins. The ability to directly analyze different monoclonal antibody fragments to determine the location and nature of the modification, without the need to run sequencing of all the peptides, means the total run time is significantly reduced and could therefore help increase laboratory throughput, a key advantage for busy biopharmaceutical laboratories. 

References

1. L.V. Marks, The Lock and Key of Medicine: Monoclonal Antibodies and the Transformation of Healthcare (Yale University Press, 2015). 
2. N. Stackhouse, A.K. Miller, and H.S.Gadgil, J. Pharm. Sci.100, 5115 (2011).

Shanhua Lin is an R&D manager at Thermo Fisher Scientific. She manages the development of HPLC consumable products and workflows for biologics characterization.