4. Analysis of monoclonal antibody IgG1 by SEC using MS-friendly mobile phases

The use of mass spectrometry is becoming increasingly popular for scientists dealing with biomolecule separation to identify the individual peaks by molecular weight. The liquid chromatography-MS (LC–MS) system available nowadays is very robust and useful for routine mass determination. Reversed phase LC–MS or SEC-MS using organic solvents such as acetonitrile can be used for the mass spectrometric characterization of mAbs. But mAbs get denatured under these conditions.

There is a growing interest in the analysis of mAbs by online-SEC-MS under native conditions. Conventional SEC analysis of mAbs use phosphate buffers at pH 6.7—for example, the most common one is composed of 100 mmol/L phosphates (monobasic + dibasic) as buffering salts +100 mM Na2SO4 as neutral salt to adjust the ionic strength +0.05% NaN3 (as antibacterial agent). Both the buffering salts (phosphates) and neutral salt (sodium sulfate) are helpful in preventing secondary interaction of the proteins with the stationary phase. The concentration of these salts may need further optimization depending on the individual properties of the mAbs. But phosphate buffer is not suitable for the mass spectrometer and yields substantial noise and damage the MS system. So online SEC-MS is not possible in the presence of phosphate and other non-volatile salts. Use of volatile salts at lower concentration, which do not interfere with the MS system, can be applied and the method needs to be optimized as well. SEC columns should not exhibit particle shedding which will interfere with the MS signal.

The data below illustrate the effective use of MS-friendly mobile phase compositions in the online SEC-MS analysis of a monoclonal IgG1, IgG2 antibody, ADC and Bi-specific mAb using volatile salt environments (Figure 22).

(ThermoFisher Scientific) coupled to a Shimadzu Nexera XR UHPLC system. Samples were injected onto a TSKgel UP-SW3000 column (2 μm, 4.6 mm ID x 30 cm) and isocratically separated at 0.350 ml/min for 15 min with a mobile phase comprising 20 mM ammonium acetate and 10 mM ammonium bicarbonate, pH 7.2. A 15-min blank isocratic gradient was run between sample injections. No carryover was observed in the blank runs. Eluted proteins were analyzed by the mass spectrometer set to repetitively scan m/z from 800 to 6000 in a positive ion mode. The full MS scan was collected at 17,500 resolution, with spray voltage 4 kV, S-Lens RF 75, and in-source CID 80 eV. Protein mass deconvolution was performed using ProMass (Novatia). The (1) total ion chromatogram, (2) mass spectrum, and (3) deconvoluted mass

Figure 22. Separation of an ADC, two IgG1 and IgG2 mAbs, and the corresponding bi-specific mAb using a TSKgel UP-

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SW3000 column (2 μm, 4.6 mm ID x 30 cm).

The online LC–MS compatible chromatographic conditions used for the analysis of IgG1, IgG2, ADC, and a Bi-specific mAb is shown below.

Following the development of an optimized separation, liquid chromatography mass spectrometry (LC–MS) analysis was performed using a Q Exactive Plus mass spectrometer


to particular additives. Retention time may shift when the additive is added and the resolution may change, expectedly to a better resolution but these values should remain constant and the analysis should be reproducible. It is always better to dedicate the SEC column if the column is subjected to additives, since we still have no clear idea how the pore characteristic of the stationary phase may behave with and without additives. Analysts should check the operational and conditions (OCS) sheet for the column as provided by the vendor to make sure that additives are compatible with the stationary phase. If compatible the analyst should be aware of the percentage of organic solvent the column is compatible with. Generally, when the organic solvent is used, the column may need a slower ramping rate for proper equilibration of the column with the mobile phase containing the additive, generally by using gradual

4. Analysis of monoclonal antibody IgG1 by SEC using MS-friendly

The use of mass spectrometry is becoming increasingly popular for scientists dealing with biomolecule separation to identify the individual peaks by molecular weight. The liquid chromatography-MS (LC–MS) system available nowadays is very robust and useful for routine mass determination. Reversed phase LC–MS or SEC-MS using organic solvents such as acetonitrile can be used for the mass spectrometric characterization of mAbs. But mAbs get

There is a growing interest in the analysis of mAbs by online-SEC-MS under native conditions. Conventional SEC analysis of mAbs use phosphate buffers at pH 6.7—for example, the most common one is composed of 100 mmol/L phosphates (monobasic + dibasic) as buffering salts +100 mM Na2SO4 as neutral salt to adjust the ionic strength +0.05% NaN3 (as antibacterial agent). Both the buffering salts (phosphates) and neutral salt (sodium sulfate) are helpful in preventing secondary interaction of the proteins with the stationary phase. The concentration of these salts may need further optimization depending on the individual properties of the mAbs. But phosphate buffer is not suitable for the mass spectrometer and yields substantial noise and damage the MS system. So online SEC-MS is not possible in the presence of phosphate and other non-volatile salts. Use of volatile salts at lower concentration, which do not interfere with the MS system, can be applied and the method needs to be optimized as well. SEC columns should not exhibit particle shedding which will interfere with the MS signal.

The data below illustrate the effective use of MS-friendly mobile phase compositions in the online SEC-MS analysis of a monoclonal IgG1, IgG2 antibody, ADC and Bi-specific mAb using

The online LC–MS compatible chromatographic conditions used for the analysis of IgG1, IgG2,

Following the development of an optimized separation, liquid chromatography mass spectrometry (LC–MS) analysis was performed using a Q Exactive Plus mass spectrometer

solvent changes using a shallow gradient at low flow rate.

mobile phases

156 Antibody Engineering

denatured under these conditions.

volatile salt environments (Figure 22).

ADC, and a Bi-specific mAb is shown below.


Figure 22. Separation of an ADC, two IgG1 and IgG2 mAbs, and the corresponding bi-specific mAb using a TSKgel UP-SW3000 column (2 μm, 4.6 mm ID x 30 cm).

(ThermoFisher Scientific) coupled to a Shimadzu Nexera XR UHPLC system. Samples were injected onto a TSKgel UP-SW3000 column (2 μm, 4.6 mm ID x 30 cm) and isocratically separated at 0.350 ml/min for 15 min with a mobile phase comprising 20 mM ammonium acetate and 10 mM ammonium bicarbonate, pH 7.2. A 15-min blank isocratic gradient was run between sample injections. No carryover was observed in the blank runs. Eluted proteins were analyzed by the mass spectrometer set to repetitively scan m/z from 800 to 6000 in a positive ion mode. The full MS scan was collected at 17,500 resolution, with spray voltage 4 kV, S-Lens RF 75, and in-source CID 80 eV. Protein mass deconvolution was performed using ProMass (Novatia). The (1) total ion chromatogram, (2) mass spectrum, and (3) deconvoluted mass

spectrum of one mAb was evaluated. A main peak can be seen at m/z 149,264; adjacent peaks at m/z 149,426 and 149,592 correspond to different glycoforms.

Here we report the use of a TSKgel® UP-SW3000, 2 μm column for the separation of a bispecific antibody and the two parent mAbs (IgG1) followed by MS analysis. The Bispecific T cell Engager (BiTE®) technology was used in this study. BiTE is a fusion protein consisting of two single-chain variable fragments (scFvs)–CD19, a biomarker for normal and neoplastic B cells and CD3 (on T cells) – recombinantly linked by a nonimmunogenic five-amino-acid chain (Figure 23). BiTE is approximately 55 kDa in size. SEC/MS analysis was performed by the Wistar Proteomics and Metabolomics Facility (Philadelphia, PA) using a Nexera® XR UHPLC system (Shimadzu) coupled to a Q Exactive™ Plus mass spectrometer (Thermo Fisher Scientific) (Figures 24–26).

Prior to analysis, a blank injection was run in order to assess column particle shedding. The total ion chromatogram of a blank injection was run on a new TSKgel UP-SW3000 column. MS data indicate that there is no shedding from the TSKgel UP-SW3000 column prior to sample injection. Additionally a blank injection was run between each of the sample injections in order to monitor sample carryover.

Each mAb is different, and a method with the use of volatile salts needs to be optimized for reproducibility. There was a difference between the retention time of mAb1 under the isocratic mobile phase 20 mM ammonium acetate and 10 mM ammonium bicarbonate, pH 7.2 compared to 100 mM phosphate buffer containing 100 mM Na2SO4 and 0.05% NaN3 pH 6.8. In an attempt to look for the condition where a MS compatible buffer yields a retention time similar to phosphate buffer, a comparison of elution profiles under 100 mM phosphate buffer and 100 mM ammonium acetate buffer both at pH 6.8 is shown below (Figure 27 and Table 4).

Monomer peak areas remain constant under both conditions with high reproducibility of all the peak parameters. % RSD deviations of all the peak parameters were low. Mass spectrometric analysis under this chromatographic condition will be reported elsewhere.

Figure 24. SEC/MS analysis of the CD19 X CD3 BiTE antibody.

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Figure 23. Scheme of a BiTE and corresponding original mAb 1 and mAb 2.

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spectrum of one mAb was evaluated. A main peak can be seen at m/z 149,264; adjacent peaks

Here we report the use of a TSKgel® UP-SW3000, 2 μm column for the separation of a bispecific antibody and the two parent mAbs (IgG1) followed by MS analysis. The Bispecific T cell Engager (BiTE®) technology was used in this study. BiTE is a fusion protein consisting of two single-chain variable fragments (scFvs)–CD19, a biomarker for normal and neoplastic B cells and CD3 (on T cells) – recombinantly linked by a nonimmunogenic five-amino-acid chain (Figure 23). BiTE is approximately 55 kDa in size. SEC/MS analysis was performed by the Wistar Proteomics and Metabolomics Facility (Philadelphia, PA) using a Nexera® XR UHPLC system (Shimadzu) coupled to a Q Exactive™ Plus mass spectrometer (Thermo Fisher Scien-

Prior to analysis, a blank injection was run in order to assess column particle shedding. The total ion chromatogram of a blank injection was run on a new TSKgel UP-SW3000 column. MS data indicate that there is no shedding from the TSKgel UP-SW3000 column prior to sample injection. Additionally a blank injection was run between each of the sample injections in order

Each mAb is different, and a method with the use of volatile salts needs to be optimized for reproducibility. There was a difference between the retention time of mAb1 under the isocratic mobile phase 20 mM ammonium acetate and 10 mM ammonium bicarbonate, pH 7.2 compared to 100 mM phosphate buffer containing 100 mM Na2SO4 and 0.05% NaN3 pH 6.8. In an attempt to look for the condition where a MS compatible buffer yields a retention time similar to phosphate buffer, a comparison of elution profiles under 100 mM phosphate buffer and 100 mM ammonium acetate buffer both at pH 6.8 is shown below (Figure 27 and Table 4).

Monomer peak areas remain constant under both conditions with high reproducibility of all the peak parameters. % RSD deviations of all the peak parameters were low. Mass spectromet-

ric analysis under this chromatographic condition will be reported elsewhere.

Figure 23. Scheme of a BiTE and corresponding original mAb 1 and mAb 2.

at m/z 149,426 and 149,592 correspond to different glycoforms.

tific) (Figures 24–26).

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to monitor sample carryover.



Figure 24. SEC/MS analysis of the CD19 X CD3 BiTE antibody.

Figure 26. Analysis of blank injections in order to assess column particle shedding using the TSKgel UP-SW3000 column.

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Figure 27. Comparison of elution profiles of IgG1 under 100 mM phosphate buffer and 100 mM ammonium acetate

buffer, pH 6.8 using a TSKgel UP-SW3000 column.

Figure 26. Analysis of blank injections in order to assess column particle shedding using the TSKgel UP-SW3000 column.


Figure 27. Comparison of elution profiles of IgG1 under 100 mM phosphate buffer and 100 mM ammonium acetate buffer, pH 6.8 using a TSKgel UP-SW3000 column.

Figure 25. SEC/MS analysis of the original IgG1 mAb1.

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As shown in the figure below when individual peaks F (ab) 2 and Fab + Fc from SEC separation (panel A) were applied to the reversed-phase chromatographic (RPC) column, a number of hydrophobic variants eluted (panel B) in the increasing order of their hydrophobicity (Figure 28). Mechanism of papain digestion is discussed in Section 2.6. Though papain digestion yields primarily the Fab fragments, F(ab')2 fragment can be generated if the papain is first activated with 10 mM cysteine. Following the completion of the reaction, the excess needs to be removed by gel filtration. Size exclusion chromatography cannot differentiate these heterogenic impurities or hydrophobic variants, which are not sufficiently different in the size or hydrodynamic radii from each other. Similarly, a number of other chromatographic modes, other than RPC, can also be used as an orthogonal technique. The extent of the heterogeneity present in the SEC peak can

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Similarly, a reversed-phase chromatography column can also be used as a complimentary chromatography column along with SEC as shown below (Figure 29). The elution order of

The PEG-conjugated species were more strongly retained by RPC, than the different forms of intact lysozyme. The order of elution in RPC is opposite to the order of size-based separation in

Figure 29. Separation of PEG (MW 5000)-lysozyme and PEG (MW 30,000)-lysozyme on a SEC TSKgel SuperSW3000 column (A) followed by chromatography of the SEC fractions 1–4 using a reverse phase TSKgel protein C4–300 column (B).

only be confirmed by an orthogonal analysis.

SEC [35].

elution of the peaks is simply reversed as expected.

Table 4. Analysis of retention time, peak area, peak height, as (peak asymmetry) and N (theoretical plates) of the monomers. The average (Avg), standard deviation (Std) and relative standard deviation (RSD) are also shown.
