Monoclonal Antibody Fragment Separation and Characterization Using Size Exclusion Chromatography Coupled with Mass Spectrometry

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1 Monoclonal ntibody Fragment Separation and haracterization Using Size Exclusion hromatography oupled with Mass Spectrometry uthors Haiying hen Katherine McLaughlin Sepax Technologies, Inc. 5 Innovation Way Newark, DE US pplication Note ZM-18 Protein Separation bstract Monoclonal antibodies have increasingly become a major part of protein therapeutics. Monoclonal antibody fragments (such as and F(ab ) 2 ) offer advantages over using intact Mbs such as reducing nonspecific antigen binding from. Size exclusion chromatography has been widely used in protein analysis. ggregates, monomers and degradation products of monoclonal antibodies can be separated on size exclusion columns based on their molecular weights under native conditions. In general, protein native buffer conditions such as salts at neutral ph are not mass spectrometry friendly. In this study, we investigated antibody fragments such as heavy and light chains, /, and F(ab ) 2 using SE separation. Heavy and light chains were also analyzed by SE coupled with mass spectrometry with volatile mobile phases. The effect of different percentages of TF, formic acid and acetonitrile in mobile phases on the antibody fragments separation was also explored.

2 PPLITION NOTE ZM-18 Introduction Size exclusion chromatography (SE) has been applied successfully to separate different sizes of proteins under native conditions. It has been routinely used for the characterization and quality control of monoclonal antibody therapeutics in the pharmaceutical industry (1). However, with normal SE salt containing mobile phases, direct mass spectrometry analysis of antibody fragments is not feasible. Here we investigated salt and volatile mobile phase effects on the separation of monoclonal antibody fragments analyzed by Zenix TM -SE. Figure 1 shows the schematic illustrations on the generation of monoclonal fragments. Monoclonal antibodies were reduced to heavy and light chains using DTT. and fragments were generated by papain digestion while the F(ab ) 2 fragment was obtained by pepsin digestion. With an optimized volatile mobile phase, the direct molecular weight analysis of a monoclonal antibody was achieved using online SE with mass spectrometry. Sepax Zenix TM SE columns are based on uniform, hydrophilic, and neutral nanometer thick films chemically bonded on high purity and mechanically stabilized silica. Zenix TM SE- 3 is specifically designed for protein and large peptide separations with a molecular exclusion limit of 1,25, Da. In this application note, we applied Zenix TM SE-3 for the separation of heavy light chains, and, and F(ab ) 2 for the characterization of a monoclonal antibody. Mobile phases including salts and different concentrations of TF, formic acid and acetonitrile were evaluated for the fragment separations. Experimental HPL system gilent 1 HPL with binary pump Q-TOF mass spectrometer Waters Q-Tof Ultima. The scan range is from 35 to 3 m/z. The instrument settings are: source temperature = 8 o, desolvation temperature = 15 o, capillary voltage = 4.44 kv SE column and L method Zenix TM SE-3 (3 m, 3 Å, 7.8 x 3 mm and 4.6 x 3 mm) was used for intact Mb and Mb fragment separations. Mobile phases include different percentages of TF, formic acid and acetonitrile, the regular SE mobile phase is 15 mm sodium phosphate buffer, ph 7.. Flow rates were.5 ml/ and.2 ml/ for 7.8 x 3 mm and 4.6 x 3 mm columns, respectively. SE-MS The Shimadzu HPL with Sepax s Zenix TM SE x3 mm column was used to separate the heavy and light chains. Online Waters Q- TOF mass spectrometer was used to analyze the heavy and light chains. The mobile phase was.1% TF,.1% formic acid and % acetonitrile. The flow rate was.2 ml/. 5 g of reduced Mb 321 was injected for analysis. hemicals and Reagents Recombinant monoclonal antibody (Mb 321) was produced by transfected hinese Hamster ovary (HO) and purified by a local biotechnology company. Papain from papaya latex and pepsin from porcine gastric mucosa were purchased from Sigma ldrich. 4-12% is-tris gels and reagents were purchased from Invitrogen. Sample preparation Reduction DTT reduction was carried out on Mb 321. Mbs were diluted to 1mg/mL with 15 mm phosphate buffer, ph 7.. ntibodies were reduced with a final concentration of mm DTT at 65 o for 15 utes. Various amounts of reduced Mbs were injected. SE runs were monitored using 28 nm UV absorbance. Papain digestion Papain digestion was modified from the procedure reported previously (2). The digestion was carried out by incubating Mb 321 (1 mg/ml) in 1 mm Tris-Hl, ph 7.6, 2 mm EDT and 5 mm ysteine. The digestion was started by adding 1 mg/ml papain. The papain/mb ratio was at 1:1. The digestion 2

3 PPLITION NOTE ZM-18 mixture was incubated for 2, 3, 3.5 and 4 hours at 37 o. Pepsin digestion Pepsin digestion was performed with a method modified from the previously reported (3,4). Mb 321 was incubated at a final concentration of 1 mg/ml in mm sodium acetate, ph 4. with a pepsin to Mb 321 ratio of 1:. The digestion was carried out at 37 o for 15.5 hours. The reaction was stopped by adding 2 M TRIS to increase the ph to 8.. Papain digestion and Pepsin digestion mixtures were stored at - o until use. Freshly reduced Mb 321 was prepared each time before use. ll samples were stored in the chilled autosampler for sequenced HPL runs. Results Monoclonal antibody fragment separations on Zenix SE-3 with.1% TF,.1% formic acid and % acetonitrile Reduced Mb Separations of heavy chain and light chain were achieved using both 4.6 x 3 mm and 7.8 x 3 mm Zenix TM SE-3 columns. HPL profiles in figure 2 and 3 indicate that the elution times of heavy and light chains were later than that of intact Mb. Heavy and light chains were separated based on their molecular elution order. 4-12% is-tris gel image shows the correct molecular weight for each collected fraction containing heavy (5 kd) and light (25kD) chains. There was a small fraction of the dimerized fragments in each peak according to the gel bands with the dimer molecular weights. With a 7.8 x 3 mm column at a.2 ml/ flow rate and directly coupled to online mass spectrometry, identities of the heavy and light chains were confirmed. Heavy chain and light chain were separated. Sample were directly introduced to the mass spectrometer without a flow split (L profile not shown, similar to figure 3 with later retention time due to slower flow rate). Figure 4 represents the deconvoluted mass spectra of heavy and light chain peaks. Heavy chain has a major fragment with a mass of 5,598 Da. nother proent peak shown in the spectra was 5,76 Da with an additional mass of 162 Da. The possible difference between these two peaks is the mass of galactose due to the different glycosylation on the Mb. Light chain fragment has a mass of 23,441 Da. Papain digestion / separation Papain digestion generated and fragments from the intact Mb. time course of papain digestion at 37 o yielded optimum 3.5 hours of incubation time. Time point material was taken and subjected to SE analysis similar to figure 5 (3.5 hour time point). t 3 hours of incubation, based on peak integration from a 7.8 x 3 mm run with g digested Mb injection, there was a.28% intact Mb and.5% / dimer present. Intact Mb, incomplete digestion fragments, and were all well separated on Zenix TM SE-3 (data not shown here). Figure 5 shows an overlay profile of individually injected intact Mb 321, papain digested Mb 321 and papain blank on a 4.6 x 3 mm column. and fc maintained the baseline separation with 5 g injection. The gel image of the collected peaks indicates the correct molecular weight order elution. Pepsin digestion F(ab ) 2 Pepsin digestion generated the F(ab ) 2 fragments and many small fc fragments. Figure 6 panel shows the digested Mb separation on Zenix TM SE x 3 mm. F(ab ) 2 eluted as a single peak with a small shoulder due to a smaller fragment which is indicated by the gel image shown in panel. This smaller fragment had an estimated molecular weight of 7 kd. There was also a small fraction of undigested intact Mb 321, indicated by a tiny visible peak before the F(ab ) 2 peak and a faint gel band at 16 kd in the gel image. omparison of mobile phases with different TF concentrations in volatile buffer.1% formic acid with % acetonitrile and salt mobile phase Figures 7, 8 show chromatogram overlays for heavy/light chains, / and F(ab ) 2 separations with.2%,.5% and.1% TF in.1% formic acid and % acetonitrile on a 4.6 x 3 mm column, respectively. The results indicate that when TF concentration is reduced to.5%, all fragments still can be baseline separated with slightly broader peak width in comparison to.1% TF. t.2%, all fragments in each sample could not be separated and were merged into the buffer peaks. 3

4 PPLITION NOTE ZM-18 When the mobile phase was switched to a 15 mm sodium phosphate buffer, only the papain digested sample was analyzed; / fragments were eluted as one peak with a delayed retention time as shown in figure 9. Effect of formic acid concentration on monoclonal antibody fragment separations With.2% TF,.1% formic acid and % acetonitrile, there is no separation of heavy/light, / and F(ab ) 2. When the concentration of formic acid was increased to 1% in.2% TF and % acetonitrile, all fragments were separated again on the same column. Figure 1 and 11 show the heavy/light, /, and F(ab ) 2 separations under three different mobile phases (..2% TF in 1% formic acid and % acetonitrile;. 1% formic acid in % acetonitrile;..1% TF in.1% formic acid and % acetonitrile). When.2% TF was omitted from the mobile phase, heavy and chains were baseline separated while F(ab ) 2 exhibited a much broader peak width, indicating a stronger retention on the column. oth and peaks were eluted earlier and there was a slight overlap between the two peaks without the.2% TF as shown in figure 11. Table 1 summarizes the column performances using the three different mobile phases..2% TF in 1% formic acid and % acetonitrile has similar effect on the separation of and as.1% TF in.1% formic acid and % acetonitrile. Effect of acetonitrile concentration on / separations When the percentage of acetonitrile was changed to 5% from % in.2% TF and 1% formic acid, both heavy/light and F(ab ) 2 separation maintain a similar separation as with % acetonitrile. ll fragment peaks were eluted with earlier retention times (data not shown). However with / separation as shown in figure 12, 5% acetonitrile can t separate and fragments. % acetonitrile, as shown in figure 13. The other fragment peak, next to the peak, was visible at.1 g. onclusion Zenix TM SE x 3 mm can successfully separate Mb fragments including heavy/light chains, / and F(ab ) 2 from their reaction mixtures, respectively. Volatile mobile phases were optimized for the separation of Mb fragments. To imize the TF MS signal suppression effect, TF concentrations can be reduced to lower than.5%. t.2% TF, 1% formic acid and % acetonitrile, all fragments can be separated from their reaction mixtures with similar separation efficiency as.1% TF. Even without TF, all Mb fragments can be separated with some reduced column performance. oth mobile phases are suitable for the Mb fragment separation and SE-MS online analysis. With low sample loading and mass spec friendly mobile phases, the on line SE MS method can be applied to general protein separation and mass detection. For more information on Zenix TM - SE products, please visit Sepax website: or contact us at SEPX-US Sample loading test on Zenix TM SE x 3 mm Increasingly low sample loadings are required in the characterization and Q process of a monoclonal antibody and its fragments. When the injection of papain digested Mb 321 was reduced to.1 g, and remained baseline separated with.2% TF, 1% formic acid and 4

5 PPLITION NOTE ZM-18 2 x 5 kd Papain digestion Intact Mb 15 kd Pepsin digestion F(ab ) 2 11 kd + + DTT reduction 5 kd 2 x Light chain 25 kd + 2 x Heavy chain 5 kd small fragments Figure 1. Schematic illustrations of monoclonal antibody fragment generation Intact Mb 321 Mb 321 Heavy chain Light chain uffer peaks 6 Reduced Mb 321 DTT blank Figure 2. Reduced Mb 321 separation on Zenix TM SE-3, 4.6 x 3 mm. Mobile phase was.1% TF,.1% formic acid with % acetonitrile. Flow rate was at.2 ml/. UV detection was set at 28 nm. 5 g of intact Mb 321 and g of reduced Mb 321 were injected. 5

6 PPLITION NOTE ZM Zenix mL/ UV28nm.1%TF,.1% formic acid, % N 3 g reduced Mb321 Peak1 Heavy Peak2 Light kd Figure 3. Panel shows a separation profile of reduced Mb 321 on Zenix TM SE-3, 7.8 x 3 mm. Peak 1 and Peak 2 were each collected and speed-vac dried. Dried samples were then dissolved in Invitrogen LDS sample buffer. Panel. shows the 4-12% is-tris gel image of reduced Mb sample and Peak 1, 2 fractions. Lane 1 protein marker; lane 2 reduced Mb sample mixture; lane 3 peak 1 heavy chain; lane 4 peak 2 light chain. 1 heavy chain 5598 light chain Light chain 162 Da mass difference-galactose % 576 % mass mass Figure 4. Online MS analysis of heavy and light chain from SE separation of reduced Mb x 3 mm Zenix SE-3 column was used to separate the heavy and light chains. Flow rate was.2 ml/ with.1% TF,.1% formic acid and % acetonitrile as the mobile phase. Samples were directly introduced to online Q-TOF after SE. Deconvoluted mass spectra of peaks were shown as in heavy chain and light chain. 6

7 PPLITION NOTE ZM Intact Mb 321 Papain digested Mb 321 Papain blank Mb kd Figure 5. Panel shows the papain digested Mb 321 (3.5 hour incubation time) -/ separation on Zenix TM SE-3, 4.6 x 3 mm. Mobile phase was.1% TF,.1% formic acid with % acetonitrile. Flow rate was.2 ml/. UV detection was set at 28 nm. 5 g of intact Mb 321 and 5 g of papain digested Mb 321 were injected. Panel shows the 4-12% is-tris gel image of collected and fractions separated on a Zenix TM SE-3, 7.8 x 3 mm column at.5 ml/ (L profile not shown) Mb 321 Undigested Mb. Pepsin digestion blank. Pepsin digested Mb 321. Intact Mb 321 F(ab) 2 Small fragments and buffer peaks kd a b c Figure 6. Panel showed the pepsin digested Mb 321-F(ab ) 2 separation on Zenix TM SE-3, 4.6 x 3 mm. Mobile phase was.1% TF,.1% formic acid with % acetonitrile. Flow rate was.2 ml/. UV detection was set at 28 nm. 5 g of intact Mb 321 and 15 g of papain digested Mb 321 were injected. Panel shows 4-12% is-tris gel image. 5 g of each sample were loaded. Lane 1 protein markers; lane 2 undigested Mb 321; lane 3 pepsin digested Mb 321. and (a) is undigested Mb, band (b) is F(ab ) 2, and bands (c) are smaller fragments from the digestion. 7

8 PPLITION NOTE ZM Mobile phases:..2% TF,.1% formic acid, % N..5% TF,.1% formic acid, % N..1% TF,.1% formic Heavy acid, % N Light Heavy Light Heavy Light Heavy + light Heavy+light Figure 7. Effect of different TF concentrations on the separation of heavy and light chains on a Zenix TM SE-3, 4.6 x 3 mm column. Flow rate was.2 ml/. UV detection was set at 28 nm. g of reduced Mb 321 was injected. t.5% TF, heavy and light chains can be baseline separated. However, at.2% TF, heavy and light chains are not separated Mobile phases:..2% TF,.1% formic acid, % N..5% TF,.1% formic acid, % N..1% TF,.1% formic acid, % N Mobile phases:..2% TF,.1% formic acid, % N..5% TF,.1% formic acid, % N..1% TF,.1% formic acid, % N F(ab ) Figure 8. Effect of different TF concentrations in.1% formic acid and % acetonitrile on the separation of / and F(ab ) 2 on a Zenix TM SE-3, 4.6 x 3 mm column. Flow rate was.2 ml/. UV detection was set at 28 nm. Panel shows the chromatogram overlays of 5 g of papain digested Mb 321 with the mobile phases indicated. Panel shows the chromatogram overlays of 15 g pepsin digested Mb

9 PPLITION NOTE ZM-18 1 uffer peak % TF,.1% formic acid, % N uffer peak. 15 mm phosphate buffer, ph Figure 9. Organic mobile phase vs. salt mobile phase for / separations on Zenix TM SE-3, 4.6 x 3 mm. Flow rate was.35 ml/, 5 g of papain digested Mb 321 was injected for both runs. L profile was obtained with.1% TF,.1% formic acid in % acetonitrile, while profile was generated with 15 mm sodium phosphate buffer, ph % TF, 1% formic acid, % N. 1% formic acid, % N..1% TF,.1% formic acid, % N Heavy Light % TF, 1% formic acid, % N. 1% formic acid, % N..1% TF,.1% formic acid, % N F(ab ) Figure 1. Heavy/light chains, F(ab ) 2 separations on Zenix TM SE-3, 4.6 x 3 mm. Flow rate was.2 ml/ with UV 28 nm detection. Panel shows the chromatogram overlays of reduced Mb 321 separation (heavy and light chains). Panel shows the chromatogram overlays of pepsin digested Mb 321 separation (F(ab ) 2 ). Mobile phase was.2% TF, 1% formic acid, % acetonitrile. Mobile phase was 1% formic acid and % acetonitrile. Mobile phase was.1% TF,.1% formic acid and % acetonitrile. 9

10 PPLITION NOTE ZM % TF, 1% formic acid, % N. 1% formic acid, % N..1% TF,.1% formic acid, % N Figure 11. and separations on Zenix TM SE-3, 4.6 x 3 mm with different mobile phases. Flow rate was.2 ml/, 5 g of papain digested Mb 321 was injected for all three runs. Table 1. Zenix TM SE-3 4.6x3mm column performance dependence on mobile phase compositions for / separations. Peak Mobile phase RT () Plate Tailing Resolution.2% TF, 1% formic acid, % N % formic acid, % N % TF,.1% formic acid, % N % TF, 1% formic acid, % N % formic acid, % N % TF,.1% formic acid, % N

11 PPLITION NOTE ZM % TF, 1% formic acid, % N..2% TF, 1% formic acid, 5% N Figure 12. cetonitrile concentration effect on / separations on Zenix TM SE-3, 4.6 x 3 mm. Flow rate was.2 ml/, 5 g of papain digested Mb 321 was injected for both runs. L profile was obtained with % acetonitrile, while profile was generated with 5% acetonitrile Papain digested Mb loading:.1 g.25 g.5 g 1 g 3 g 5 g g papain digested Mb Figure 13. Panel shows the chromatogram overlays of different sample loadings of papain digested Mb from.1 g to 5 g on Zenix TM SE-3, 4.6 x 3 mm. HPL mobile phase was.2% TF in 1% formic acid and % acetonitrile. Flow rate was.2 ml/ with UV 28 nm detection. Panel shows the separation profile with.1 g loading. and maintained a baseline separation at.1 g loading. Reference: 1. nalysis of Reduced Monoclonal ntibodies Using Size Exclusion hromatography oupled with Mass Spectrometry, J. m. Soc. Mass Spectrom., (9) 2. nalysis of Post-translational Modifications in Recombinant Monoclonal ntibody IgG1 by Reversed-phased Liquid hromatography/mass Spectrometry. Journal of hromatography, 1164, (7) 3. Papain Digestion of Different Mouse IgG Subclasses as Studied by Electrospray as Mass Spectrometry. Journal of Immunological Methods, 237, () 4. Enzyme Digestion of Monoclonal ntibodies from the Protein Protocols Handbook, 2 nd edition by Sarah M. ndrew. 11

12 PPLITION NOTE ZM-18 Order information Part Number Particle Size Pore Size ID Length [1] 3 µm 3 Å 2.1 5mm µm 3 Å 2.1 3mm [1] 3 µm 3 Å 4.6 5mm 2133P-465 [1] [3] 3 µm 3 Å 4.6 5mm µm 3 Å mm µm 3 Å mm µm 3 Å 4.6 3mm 2133P-463 [3] 3 µm 3 Å 4.6 3mm [1] 3 µm 3 Å 7.8 5mm µm 3 Å mm µm 3 Å 7.8 mm µm 3 Å mm µm 3 Å 7.8 3mm [1] 3 µm 3 Å 1. 5mm µm 3 Å 1. 1mm µm 3 Å 1. 15mm µm 3 Å 1. 25mm µm 3 Å 1. 3mm [1] 3 µm 3 Å mm µm 3 Å mm µm 3 Å mm [1] Guard column [2] olumn packed with PEEK tubing 12