ABSTRACTWith the recent growth of the biopharmaceutical industry, sensitive and fast methods are required to monitor microheterogeneity and PTMs during all stages of process development to guarantee product safety and efficacy. Therapeutic mAbs, such as rituximab, trastuzumab, infliximab, and bevacizumab, are mostly produced from mammalian cells. These biological products are heterogeneous, containing multiple charge variants and glycosylation forms. Additional modifications such as oxidation can be introduced during the manufacturing process. In the current study, mAbs are broken down into several large fragments using reduction reagent and IdeS enzyme. A fast LC/MS separation method is employed for the following two applications: 1) monitoring mAb fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation.

INTRODUCTIONThe monoclonal antibody (mAb) therapeutics market is growing at a rapid rate owing to increasing demand for targeted treatments. Therapeutic mAbs are mostly produced from mammalian cells. These biological products are heterogeneous due to post-translational modifications (Figure 1). Additional modifications such as oxidation can be introduced during the manufacturing process. A comprehensive characterization of mAb purity, aggregates, and variants is required for the final biopharmaceutical product approval and subsequent manufacturing processes. There is a growing trend to obtain intact mass information as well as the glycan profile in the QC of mAbs using reverse phase chromatography coupled with high resolution mass spectrometry detection. LC/MS analysis of mAb fragments such as light chain (LC), heavy chain (HC), Fc, Fab, scFc and F(ab')2, can accurately reveal the location, as well as nature, of the modification. Moreover, in most QC environments, LC/UV analysis of mAb fragments has been established as a high throughput assay.The reversed phase column (MAbPac RP) used in the fast LC/MS assays is based on wide-pore 4 μmpolymer particles that are stable at extreme pH (0–14) and high temperature (up to 110 °C). The wide-pore size of polymeric particles (1,500 Å) enables efficient separation of large protein molecules with low carry-over. This study focuses on two application areas: 1) monitoring scFc fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation of HC.

Figure 3. LC/UV analysis of mAb fragments containing lysine charge variants.

Figure 4. LC/MS analysis of mAb fragments containing lysine charge variants.

mAb Oxidation Variant AnalysisMet oxidation is one of the critical quality attributes required to be closely monitored. The two Met residues in the CH2-CH3 domain interface of recombinant humanized and fully human IgG1 antibodies were found susceptible to oxidation [1]. It is desirable to monitor the progress of the Met oxidation without complete digestion of mAb. Infliximab was treated with H2O2, resulting in the oxidation of LC and HC. Figure 7 shows the separation of scFc, LC, and Fd’ fragments as well as its oxidized forms on a MAbPac RP column. Figure 8 shows a lesser separation of oxidized fragments from the unmodified fragments when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 9 shows the mass spectra of oxidized scFc at +10 charge state. Figure 10 shows the mass spectra of oxidized LC and Fd’ at +10 charge state. Deconvoluted spectra of scFc and LC show the mass difference between the oxidized form and the unmodified fragment is 16 Da, corresponding to the residue mass of an oxygen (Figure 11 and Figure 12).

CONCLUSIONS mAb fragments scFc, LC, and Fd’ are baseline separated on a 3.0 × 50 mm MAbPac RP column.

Spatial separation of lysine variant and oxidation variant from the unmodified scFc is achieved.

LC/MS analysis of the scFc, LC, and Fd’ fragments successfully pinpoints the location of lysine and oxidation modifications.

A workflow has been developed to successfully remove N-glycan from mAb in 15 min. TrastuzumabHC and its deglycosylated form are baseline separated on a longer (2.1 × 100 mm) MAbPac RP column.

REFERENCES1. Liu H., Gaza-Bulseco G., and Zhou L. Mass Spectrometry Analysis of Photo-InducedMethionine

Oxidation of a Recombinant Human Monoclonal Antibody, J. Am. Soc. Mass. Spectrom., 2009, 20, 525–528.

TRADEMARKS/LICENSING© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

mAb Deglycosylation AnalysisA fast workflow has been developed to remove N-linked glycans in 15 min. Figure 13 shows the separation of LC and HC of trastuzumab before and after deglycosylation. The deglycosylated HC elutes later (retention time at 16.238 min) than the glycosylation HC (retention time at 15.821 min). Figure 14 shows the mass spectra of glycosylated HC and deglycosylated HC. Deconvoluted spectrum of deglycosylated HC shows a single polypeptide of 48,157 Da (Figure 15). The deconvoluted result agrees well with the calculated MW based on the sequence of trastuzumab HC.

Instrument Conditions mAb and mAb FragmentsMass range m/z 1,000–4,000

Spray voltage 3.9 kV

Sheath gas 45 arb. units

Auxiliary gas 15 arb. units

Capillary temperature 320 °CS-lens level 55

In-source CID 40 eV

Microscans 10

AGC target 3 × 106

Maximum IT 200 ms

Resolving power 17,500

Probe temperature 300 °C

Figure 1. Structure of IgG and typical forms of heterogeneity.

Figure 2. mAb reduction and IdeS digestion flowchart.

Column: MAbPac RP, 4 µmFormat: 3.0 × 50 mmMobile phase A: H2O/FA/TFA (99.88 : 0.1 : 0.02

v/v/v)Mobile phase B: MeCN/ H2O/FA/TFA (90 : 9.88 :

0.1 : 0.02 v/v/v/v)Gradient:

Time (min) %A %B0.0 80 201.0 80 2011.0 55 4512.0 55 4514.0 80 2015.0 80 20

Temperature: 80 ºCFlow rate: 0.5 mL/minInj. volume: 2 µL MS Detection: positive-ion mode Mass Spec: Q Exactive™ PlusSample: Infliximab+IdeS+DTT

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

7.89

8.33

7.006.93

scFc

scFc- Lys

LC

Fd´

Figure 5. Mass spectra of scFc, LC, and Fd’ fragments.

+10scFc-Lys

scFc

LC

Fd’

+10

+10

+10

2400 2420 2440 2460 2480 2500 2520 2540 2560 2580 2600 2620 2640 2660 2680 2700m/z

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

0

20

40

60

80

100 2533.75

2549.79

2520.77 2566.462498.572520.91

2536.95

2498.38 2553.472516.75

2485.85 2653.622604.75

2609.532467.57 2600.16 2618.282424.23

2565.30

2569.092561.132443.44 2459.95

Figure 6. Deconvoluted spectra of infliximab scFc fragments containing lysine charge variants.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.588

25489.688

25199.288 25285.08825099.088 25123.688 25651.888

25199.42125361.521

25327.521 25524.02125221.62125157.42124995.621 25383.921

Mass

Lysine

Figure 7. LC/UV analysis of mAb fragments containing oxidation variants.

Figure 8. LC/MS analysis of mAb fragments containing oxidation variants.

Figure 9. Mass spectra of scFc fragments. Figure 10. Mass spectra of LC and Fd’ fragments.

+10

+10

+10

+10

Oxidized scFc

scFc

Oxidized scFc-lys

Twice OxidizedscFc-lys

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002537.20

2553.15

2577.242524.142427.30 2644.16 2662.24

2535.26

2551.472522.72

2512.79 2561.72

2522.46

2533.56

2538.84

2501.92 2566.022520.86

2537.17

2498.18 2553.14 2583.71

+10

+10

+10

+10

Oxidized Fd’

Fd’

LC

Oxidized LC

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002606.55

2613.102641.212466.04 2600.002439.26

2604.71

2611.792467.92 2598.41 2645.882566.80

2572.822444.57 2558.23

2565.17

2571.362677.81

Figure 11. Deconvoluted spectra of infliximab scFc fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.61425215.214

25489.91425377.814

25345.31425011.514 25237.114 25651.81425285.81425172.814 25447.41425124.414 25508.714

25199.66625361.666

25237.66624971.266 25156.766 25523.76625400.76625318.866 25482.16625013.466 25126.966 25643.966

Mass

O

Figure 12. Deconvoluted spectra of LC fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

23450.164

23406.964

23434.234

23472.53423417.334

Mass

O

Figure 13. LC/UV analysis of trastuzumabLC and HC fragments before and after deglycosylation.

Figure 14. Mass spectra of trastuzumab HC before and after deglycosylation.

control

+PNGaseF

1320 1340 1360 1380 1400 1420m/z

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

1332.66

1373.04 1406.691336.951368.69

1341.15 1415.691364.69 1387.731377.35

1330.651421.74

1404.72

1358.701319.69 1347.72

1366.45 1405.481329.65

1369.721326.68 1418.721402.75

1387.721358.681348.711336.731315.68

1413.73

Figure 15. Deconvoluted spectra of trastuzumab HC before and after deglycosylation.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

49157.554

49138.354 49321.654

50763.886

50454.786

Mass

Deglycosylated HC

HC

Table 1. MS conditions.

Middle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationShanhua Lin1, Zoltan Szabo1, Yury Agroskin1, Terry Zhang2, Jonathan Josephs2, and Xiaodong Liu1, 1Thermo Fisher Scientific, Sunnyvale; 2Thermo Fisher Scientific, San Jose

RESULTSmAb Charge Variant Analysis

A workflow to generate smaller mAb fragments scFc, LC, and Fd’s is shown in Figure 2. Each of these fragments has approximate 25 KDa molecular weight. LC and Fd’ are single polypeptide chain. scFccontains the N-glycan modification site and therefore more heterogenerous. mAb charge variants often include C-terminal lysine variant. Figure 3 shows the baseline separation of scFc, LC, and Fd’ of infliximab on a MAbPac RP column. In addition, there is sufficient spatial separation of lysine containing scFc fragment from the unmodified scFc when using mobile phase containing 0.1% TFA ion-pairing reagent. Figure 4 shows a lesser separation of lysine modified scFc and unmodified scFc when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 5 shows the mass spectra of scFc and scFc-Lys at +10 charge state. Deconvoluted spectra show the mass difference between scFc-Lys and scFc is 128 Da, corresponding to the residue mass of a lysine (Figure 6).

MATERIALS AND METHODSChemicals and ReagentsFabRICATOR™ (IdeS) protease was purchased from Genovis (Lund, Sweden). PNGase F was purchased from Prozyme (Hayward, CA) .Sample Preparation

Reduction: reduction of inter-chain disulfides in a mAb (4 mg/mL) was achieved by incubation of mAbwith 20 mM DTT at 37 °C for 30 min.IdeS Digestion: 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.Deglycosylation: glycoprotein solutions were prepared at 10 mg/mL concentration in PBS buffer. Prior to deglycosylation, buffer containing HEPES at pH 7.9, reductant (TCEP) and detergent (proprietary) were added to the glycoprotein solutions. This was followed by heat denaturation at 95 °C for 5 min. PNGaseF was added to the mixture followed by incubation at 50 ⁰C for 15 min. Deglycosylated proteins were injected onto Thermo Scientific™ MAbPac™ RP analytical columns without further sample cleanup.Oxidation: dilute the mAb solution (5 mg/mL) in half with the 2X oxidation buffer (360 mM sodium chloride, 10 mM sodium acetate, pH 5.0). Then add H2O2 to a final concentration of 0.01% (v/v) and incubate the sample for 24h at room temperature.High Performance Liquid Chromatography

Thermo Scientific™ Dionex™ UltiMate™ 3000 BioRS system was used for mAb fragment separation. The column used was the MAbPac RP analytical column (3.0 x 50 mm, P/N 088645; 2.1 x 100 mm, P/N 088647). The following mobile phase A and B are used for LC/UV experiment: H2O/TFA (99.9 : 0.1 v/v) and MeCN/ H2O/TFA (90 : 9.9 :0.1 v/v/v). The following mobile phase A and B are used for LC/MS experiment: H2O/FA/TFA (99.88 : 0.1 : 0.02 v/v/v) and MeCN/ H2O/FA/TFA (90 : 9.88 : 0.1 : 0.02 v/v/v/v).

Mass Spectrometry Conditions

The Thermo Scientific™ Q Exactive™ Plus Orbitrap™ mass spectrometer was used for this study. Intact mAb or mAb fragments were analyzed by ESI-MS. H-ESI II probe was used. The resolution was set at 17.5 k (FWHM) at m/z 200, see Table 1.

Data Analysis

Full MS spectra of intact mAbs and mAb fragments were analyzed using Thermo Scientific™ Protein Deconvolution™ software 4.0 that utilizes the ReSpect algorithm for molecular mass determination.

Middle-down Approach for Monitoring Monoclonal Antibody Variants and Deglycosylation Shanhua Lin1, Zoltan Szabo1, Yury Agroskin1, Terry Zhang2, Jonathan Josephs2, and Xiaodong Liu1, 1Thermo Fisher Scientific, Sunnyvale; 2Thermo Fisher Scientific, San Jose

Po

ster No

te 720

17

ABSTRACTWith the recent growth of the biopharmaceutical industry, sensitive and fast methods are required to monitor microheterogeneity and PTMs during all stages of process development to guarantee product safety and efficacy. Therapeutic mAbs, such as rituximab, trastuzumab, infliximab, and bevacizumab, are mostly produced from mammalian cells. These biological products are heterogeneous, containing multiple charge variants and glycosylation forms. Additional modifications such as oxidation can be introduced during the manufacturing process. In the current study, mAbs are broken down into several large fragments using reduction reagent and IdeS enzyme. A fast LC/MS separation method is employed for the following two applications: 1) monitoring mAb fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation.

INTRODUCTIONThe monoclonal antibody (mAb) therapeutics market is growing at a rapid rate owing to increasing demand for targeted treatments. Therapeutic mAbs are mostly produced from mammalian cells. These biological products are heterogeneous due to post-translational modifications (Figure 1). Additional modifications such as oxidation can be introduced during the manufacturing process. A comprehensive characterization of mAb purity, aggregates, and variants is required for the final biopharmaceutical product approval and subsequent manufacturing processes. There is a growing trend to obtain intact mass information as well as the glycan profile in the QC of mAbs using reverse phase chromatography coupled with high resolution mass spectrometry detection. LC/MS analysis of mAb fragments such as light chain (LC), heavy chain (HC), Fc, Fab, scFc and F(ab')2, can accurately reveal the location, as well as nature, of the modification. Moreover, in most QC environments, LC/UV analysis of mAb fragments has been established as a high throughput assay.The reversed phase column (MAbPac RP) used in the fast LC/MS assays is based on wide-pore 4 μmpolymer particles that are stable at extreme pH (0–14) and high temperature (up to 110 °C). The wide-pore size of polymeric particles (1,500 Å) enables efficient separation of large protein molecules with low carry-over. This study focuses on two application areas: 1) monitoring scFc fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation of HC.

Figure 3. LC/UV analysis of mAb fragments containing lysine charge variants.

Figure 4. LC/MS analysis of mAb fragments containing lysine charge variants.

mAb Oxidation Variant AnalysisMet oxidation is one of the critical quality attributes required to be closely monitored. The two Met residues in the CH2-CH3 domain interface of recombinant humanized and fully human IgG1 antibodies were found susceptible to oxidation [1]. It is desirable to monitor the progress of the Met oxidation without complete digestion of mAb. Infliximab was treated with H2O2, resulting in the oxidation of LC and HC. Figure 7 shows the separation of scFc, LC, and Fd’ fragments as well as its oxidized forms on a MAbPac RP column. Figure 8 shows a lesser separation of oxidized fragments from the unmodified fragments when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 9 shows the mass spectra of oxidized scFc at +10 charge state. Figure 10 shows the mass spectra of oxidized LC and Fd’ at +10 charge state. Deconvoluted spectra of scFc and LC show the mass difference between the oxidized form and the unmodified fragment is 16 Da, corresponding to the residue mass of an oxygen (Figure 11 and Figure 12).

CONCLUSIONS mAb fragments scFc, LC, and Fd’ are baseline separated on a 3.0 × 50 mm MAbPac RP column.

Spatial separation of lysine variant and oxidation variant from the unmodified scFc is achieved.

LC/MS analysis of the scFc, LC, and Fd’ fragments successfully pinpoints the location of lysine and oxidation modifications.

A workflow has been developed to successfully remove N-glycan from mAb in 15 min. TrastuzumabHC and its deglycosylated form are baseline separated on a longer (2.1 × 100 mm) MAbPac RP column.

REFERENCES1. Liu H., Gaza-Bulseco G., and Zhou L. Mass Spectrometry Analysis of Photo-InducedMethionine

Oxidation of a Recombinant Human Monoclonal Antibody, J. Am. Soc. Mass. Spectrom., 2009, 20, 525–528.

TRADEMARKS/LICENSING© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

mAb Deglycosylation AnalysisA fast workflow has been developed to remove N-linked glycans in 15 min. Figure 13 shows the separation of LC and HC of trastuzumab before and after deglycosylation. The deglycosylated HC elutes later (retention time at 16.238 min) than the glycosylation HC (retention time at 15.821 min). Figure 14 shows the mass spectra of glycosylated HC and deglycosylated HC. Deconvoluted spectrum of deglycosylated HC shows a single polypeptide of 48,157 Da (Figure 15). The deconvoluted result agrees well with the calculated MW based on the sequence of trastuzumab HC.

Instrument Conditions mAb and mAb FragmentsMass range m/z 1,000–4,000

Spray voltage 3.9 kV

Sheath gas 45 arb. units

Auxiliary gas 15 arb. units

Capillary temperature 320 °CS-lens level 55

In-source CID 40 eV

Microscans 10

AGC target 3 × 106

Maximum IT 200 ms

Resolving power 17,500

Probe temperature 300 °C

Figure 1. Structure of IgG and typical forms of heterogeneity.

Figure 2. mAb reduction and IdeS digestion flowchart.

Column: MAbPac RP, 4 µmFormat: 3.0 × 50 mmMobile phase A: H2O/FA/TFA (99.88 : 0.1 : 0.02

v/v/v)Mobile phase B: MeCN/ H2O/FA/TFA (90 : 9.88 :

0.1 : 0.02 v/v/v/v)Gradient:

Time (min) %A %B0.0 80 201.0 80 2011.0 55 4512.0 55 4514.0 80 2015.0 80 20

Temperature: 80 ºCFlow rate: 0.5 mL/minInj. volume: 2 µL MS Detection: positive-ion mode Mass Spec: Q Exactive™ PlusSample: Infliximab+IdeS+DTT

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

7.89

8.33

7.006.93

scFc

scFc- Lys

LC

Fd´

Figure 5. Mass spectra of scFc, LC, and Fd’ fragments.

+10scFc-Lys

scFc

LC

Fd’

+10

+10

+10

2400 2420 2440 2460 2480 2500 2520 2540 2560 2580 2600 2620 2640 2660 2680 2700m/z

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

0

20

40

60

80

100 2533.75

2549.79

2520.77 2566.462498.572520.91

2536.95

2498.38 2553.472516.75

2485.85 2653.622604.75

2609.532467.57 2600.16 2618.282424.23

2565.30

2569.092561.132443.44 2459.95

Figure 6. Deconvoluted spectra of infliximab scFc fragments containing lysine charge variants.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.588

25489.688

25199.288 25285.08825099.088 25123.688 25651.888

25199.42125361.521

25327.521 25524.02125221.62125157.42124995.621 25383.921

Mass

Lysine

Figure 7. LC/UV analysis of mAb fragments containing oxidation variants.

Figure 8. LC/MS analysis of mAb fragments containing oxidation variants.

Figure 9. Mass spectra of scFc fragments. Figure 10. Mass spectra of LC and Fd’ fragments.

+10

+10

+10

+10

Oxidized scFc

scFc

Oxidized scFc-lys

Twice OxidizedscFc-lys

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002537.20

2553.15

2577.242524.142427.30 2644.16 2662.24

2535.26

2551.472522.72

2512.79 2561.72

2522.46

2533.56

2538.84

2501.92 2566.022520.86

2537.17

2498.18 2553.14 2583.71

+10

+10

+10

+10

Oxidized Fd’

Fd’

LC

Oxidized LC

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002606.55

2613.102641.212466.04 2600.002439.26

2604.71

2611.792467.92 2598.41 2645.882566.80

2572.822444.57 2558.23

2565.17

2571.362677.81

Figure 11. Deconvoluted spectra of infliximab scFc fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.61425215.214

25489.91425377.814

25345.31425011.514 25237.114 25651.81425285.81425172.814 25447.41425124.414 25508.714

25199.66625361.666

25237.66624971.266 25156.766 25523.76625400.76625318.866 25482.16625013.466 25126.966 25643.966

Mass

O

Figure 12. Deconvoluted spectra of LC fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

23450.164

23406.964

23434.234

23472.53423417.334

Mass

O

Figure 13. LC/UV analysis of trastuzumabLC and HC fragments before and after deglycosylation.

Figure 14. Mass spectra of trastuzumab HC before and after deglycosylation.

control

+PNGaseF

1320 1340 1360 1380 1400 1420m/z

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

1332.66

1373.04 1406.691336.951368.69

1341.15 1415.691364.69 1387.731377.35

1330.651421.74

1404.72

1358.701319.69 1347.72

1366.45 1405.481329.65

1369.721326.68 1418.721402.75

1387.721358.681348.711336.731315.68

1413.73

Figure 15. Deconvoluted spectra of trastuzumab HC before and after deglycosylation.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

49157.554

49138.354 49321.654

50763.886

50454.786

Mass

Deglycosylated HC

HC

Table 1. MS conditions.

Middle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationShanhua Lin1, Zoltan Szabo1, Yury Agroskin1, Terry Zhang2, Jonathan Josephs2, and Xiaodong Liu1, 1Thermo Fisher Scientific, Sunnyvale; 2Thermo Fisher Scientific, San Jose

RESULTSmAb Charge Variant Analysis

A workflow to generate smaller mAb fragments scFc, LC, and Fd’s is shown in Figure 2. Each of these fragments has approximate 25 KDa molecular weight. LC and Fd’ are single polypeptide chain. scFccontains the N-glycan modification site and therefore more heterogenerous. mAb charge variants often include C-terminal lysine variant. Figure 3 shows the baseline separation of scFc, LC, and Fd’ of infliximab on a MAbPac RP column. In addition, there is sufficient spatial separation of lysine containing scFc fragment from the unmodified scFc when using mobile phase containing 0.1% TFA ion-pairing reagent. Figure 4 shows a lesser separation of lysine modified scFc and unmodified scFc when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 5 shows the mass spectra of scFc and scFc-Lys at +10 charge state. Deconvoluted spectra show the mass difference between scFc-Lys and scFc is 128 Da, corresponding to the residue mass of a lysine (Figure 6).

MATERIALS AND METHODSChemicals and ReagentsFabRICATOR™ (IdeS) protease was purchased from Genovis (Lund, Sweden). PNGase F was purchased from Prozyme (Hayward, CA) .Sample Preparation

Reduction: reduction of inter-chain disulfides in a mAb (4 mg/mL) was achieved by incubation of mAbwith 20 mM DTT at 37 °C for 30 min.IdeS Digestion: 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.Deglycosylation: glycoprotein solutions were prepared at 10 mg/mL concentration in PBS buffer. Prior to deglycosylation, buffer containing HEPES at pH 7.9, reductant (TCEP) and detergent (proprietary) were added to the glycoprotein solutions. This was followed by heat denaturation at 95 °C for 5 min. PNGaseF was added to the mixture followed by incubation at 50 ⁰C for 15 min. Deglycosylated proteins were injected onto Thermo Scientific™ MAbPac™ RP analytical columns without further sample cleanup.Oxidation: dilute the mAb solution (5 mg/mL) in half with the 2X oxidation buffer (360 mM sodium chloride, 10 mM sodium acetate, pH 5.0). Then add H2O2 to a final concentration of 0.01% (v/v) and incubate the sample for 24h at room temperature.High Performance Liquid Chromatography

Thermo Scientific™ Dionex™ UltiMate™ 3000 BioRS system was used for mAb fragment separation. The column used was the MAbPac RP analytical column (3.0 x 50 mm, P/N 088645; 2.1 x 100 mm, P/N 088647). The following mobile phase A and B are used for LC/UV experiment: H2O/TFA (99.9 : 0.1 v/v) and MeCN/ H2O/TFA (90 : 9.9 :0.1 v/v/v). The following mobile phase A and B are used for LC/MS experiment: H2O/FA/TFA (99.88 : 0.1 : 0.02 v/v/v) and MeCN/ H2O/FA/TFA (90 : 9.88 : 0.1 : 0.02 v/v/v/v).

Mass Spectrometry Conditions

The Thermo Scientific™ Q Exactive™ Plus Orbitrap™ mass spectrometer was used for this study. Intact mAb or mAb fragments were analyzed by ESI-MS. H-ESI II probe was used. The resolution was set at 17.5 k (FWHM) at m/z 200, see Table 1.

Data Analysis

Full MS spectra of intact mAbs and mAb fragments were analyzed using Thermo Scientific™ Protein Deconvolution™ software 4.0 that utilizes the ReSpect algorithm for molecular mass determination.

ABSTRACTWith the recent growth of the biopharmaceutical industry, sensitive and fast methods are required to monitor microheterogeneity and PTMs during all stages of process development to guarantee product safety and efficacy. Therapeutic mAbs, such as rituximab, trastuzumab, infliximab, and bevacizumab, are mostly produced from mammalian cells. These biological products are heterogeneous, containing multiple charge variants and glycosylation forms. Additional modifications such as oxidation can be introduced during the manufacturing process. In the current study, mAbs are broken down into several large fragments using reduction reagent and IdeS enzyme. A fast LC/MS separation method is employed for the following two applications: 1) monitoring mAb fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation.

INTRODUCTIONThe monoclonal antibody (mAb) therapeutics market is growing at a rapid rate owing to increasing demand for targeted treatments. Therapeutic mAbs are mostly produced from mammalian cells. These biological products are heterogeneous due to post-translational modifications (Figure 1). Additional modifications such as oxidation can be introduced during the manufacturing process. A comprehensive characterization of mAb purity, aggregates, and variants is required for the final biopharmaceutical product approval and subsequent manufacturing processes. There is a growing trend to obtain intact mass information as well as the glycan profile in the QC of mAbs using reverse phase chromatography coupled with high resolution mass spectrometry detection. LC/MS analysis of mAb fragments such as light chain (LC), heavy chain (HC), Fc, Fab, scFc and F(ab')2, can accurately reveal the location, as well as nature, of the modification. Moreover, in most QC environments, LC/UV analysis of mAb fragments has been established as a high throughput assay.The reversed phase column (MAbPac RP) used in the fast LC/MS assays is based on wide-pore 4 μmpolymer particles that are stable at extreme pH (0–14) and high temperature (up to 110 °C). The wide-pore size of polymeric particles (1,500 Å) enables efficient separation of large protein molecules with low carry-over. This study focuses on two application areas: 1) monitoring scFc fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation of HC.

Figure 3. LC/UV analysis of mAb fragments containing lysine charge variants.

Figure 4. LC/MS analysis of mAb fragments containing lysine charge variants.

mAb Oxidation Variant AnalysisMet oxidation is one of the critical quality attributes required to be closely monitored. The two Met residues in the CH2-CH3 domain interface of recombinant humanized and fully human IgG1 antibodies were found susceptible to oxidation [1]. It is desirable to monitor the progress of the Met oxidation without complete digestion of mAb. Infliximab was treated with H2O2, resulting in the oxidation of LC and HC. Figure 7 shows the separation of scFc, LC, and Fd’ fragments as well as its oxidized forms on a MAbPac RP column. Figure 8 shows a lesser separation of oxidized fragments from the unmodified fragments when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 9 shows the mass spectra of oxidized scFc at +10 charge state. Figure 10 shows the mass spectra of oxidized LC and Fd’ at +10 charge state. Deconvoluted spectra of scFc and LC show the mass difference between the oxidized form and the unmodified fragment is 16 Da, corresponding to the residue mass of an oxygen (Figure 11 and Figure 12).

CONCLUSIONS mAb fragments scFc, LC, and Fd’ are baseline separated on a 3.0 × 50 mm MAbPac RP column.

Spatial separation of lysine variant and oxidation variant from the unmodified scFc is achieved.

LC/MS analysis of the scFc, LC, and Fd’ fragments successfully pinpoints the location of lysine and oxidation modifications.

A workflow has been developed to successfully remove N-glycan from mAb in 15 min. TrastuzumabHC and its deglycosylated form are baseline separated on a longer (2.1 × 100 mm) MAbPac RP column.

REFERENCES1. Liu H., Gaza-Bulseco G., and Zhou L. Mass Spectrometry Analysis of Photo-InducedMethionine

Oxidation of a Recombinant Human Monoclonal Antibody, J. Am. Soc. Mass. Spectrom., 2009, 20, 525–528.

TRADEMARKS/LICENSING© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

mAb Deglycosylation AnalysisA fast workflow has been developed to remove N-linked glycans in 15 min. Figure 13 shows the separation of LC and HC of trastuzumab before and after deglycosylation. The deglycosylated HC elutes later (retention time at 16.238 min) than the glycosylation HC (retention time at 15.821 min). Figure 14 shows the mass spectra of glycosylated HC and deglycosylated HC. Deconvoluted spectrum of deglycosylated HC shows a single polypeptide of 48,157 Da (Figure 15). The deconvoluted result agrees well with the calculated MW based on the sequence of trastuzumab HC.

Instrument Conditions mAb and mAb FragmentsMass range m/z 1,000–4,000

Spray voltage 3.9 kV

Sheath gas 45 arb. units

Auxiliary gas 15 arb. units

Capillary temperature 320 °CS-lens level 55

In-source CID 40 eV

Microscans 10

AGC target 3 × 106

Maximum IT 200 ms

Resolving power 17,500

Probe temperature 300 °C

Figure 1. Structure of IgG and typical forms of heterogeneity.

Figure 2. mAb reduction and IdeS digestion flowchart.

Column: MAbPac RP, 4 µmFormat: 3.0 × 50 mmMobile phase A: H2O/FA/TFA (99.88 : 0.1 : 0.02

v/v/v)Mobile phase B: MeCN/ H2O/FA/TFA (90 : 9.88 :

0.1 : 0.02 v/v/v/v)Gradient:

Time (min) %A %B0.0 80 201.0 80 2011.0 55 4512.0 55 4514.0 80 2015.0 80 20

Temperature: 80 ºCFlow rate: 0.5 mL/minInj. volume: 2 µL MS Detection: positive-ion mode Mass Spec: Q Exactive™ PlusSample: Infliximab+IdeS+DTT

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

7.89

8.33

7.006.93

scFc

scFc- Lys

LC

Fd´

Figure 5. Mass spectra of scFc, LC, and Fd’ fragments.

+10scFc-Lys

scFc

LC

Fd’

+10

+10

+10

2400 2420 2440 2460 2480 2500 2520 2540 2560 2580 2600 2620 2640 2660 2680 2700m/z

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

0

20

40

60

80

100 2533.75

2549.79

2520.77 2566.462498.572520.91

2536.95

2498.38 2553.472516.75

2485.85 2653.622604.75

2609.532467.57 2600.16 2618.282424.23

2565.30

2569.092561.132443.44 2459.95

Figure 6. Deconvoluted spectra of infliximab scFc fragments containing lysine charge variants.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.588

25489.688

25199.288 25285.08825099.088 25123.688 25651.888

25199.42125361.521

25327.521 25524.02125221.62125157.42124995.621 25383.921

Mass

Lysine

Figure 7. LC/UV analysis of mAb fragments containing oxidation variants.

Figure 8. LC/MS analysis of mAb fragments containing oxidation variants.

Figure 9. Mass spectra of scFc fragments. Figure 10. Mass spectra of LC and Fd’ fragments.

+10

+10

+10

+10

Oxidized scFc

scFc

Oxidized scFc-lys

Twice OxidizedscFc-lys

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002537.20

2553.15

2577.242524.142427.30 2644.16 2662.24

2535.26

2551.472522.72

2512.79 2561.72

2522.46

2533.56

2538.84

2501.92 2566.022520.86

2537.17

2498.18 2553.14 2583.71

+10

+10

+10

+10

Oxidized Fd’

Fd’

LC

Oxidized LC

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002606.55

2613.102641.212466.04 2600.002439.26

2604.71

2611.792467.92 2598.41 2645.882566.80

2572.822444.57 2558.23

2565.17

2571.362677.81

Figure 11. Deconvoluted spectra of infliximab scFc fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.61425215.214

25489.91425377.814

25345.31425011.514 25237.114 25651.81425285.81425172.814 25447.41425124.414 25508.714

25199.66625361.666

25237.66624971.266 25156.766 25523.76625400.76625318.866 25482.16625013.466 25126.966 25643.966

Mass

O

Figure 12. Deconvoluted spectra of LC fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

23450.164

23406.964

23434.234

23472.53423417.334

Mass

O

Figure 13. LC/UV analysis of trastuzumabLC and HC fragments before and after deglycosylation.

Figure 14. Mass spectra of trastuzumab HC before and after deglycosylation.

control

+PNGaseF

1320 1340 1360 1380 1400 1420m/z

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

1332.66

1373.04 1406.691336.951368.69

1341.15 1415.691364.69 1387.731377.35

1330.651421.74

1404.72

1358.701319.69 1347.72

1366.45 1405.481329.65

1369.721326.68 1418.721402.75

1387.721358.681348.711336.731315.68

1413.73

Figure 15. Deconvoluted spectra of trastuzumab HC before and after deglycosylation.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

49157.554

49138.354 49321.654

50763.886

50454.786

Mass

Deglycosylated HC

HC

Table 1. MS conditions.

Middle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationShanhua Lin1, Zoltan Szabo1, Yury Agroskin1, Terry Zhang2, Jonathan Josephs2, and Xiaodong Liu1, 1Thermo Fisher Scientific, Sunnyvale; 2Thermo Fisher Scientific, San Jose

RESULTSmAb Charge Variant Analysis

A workflow to generate smaller mAb fragments scFc, LC, and Fd’s is shown in Figure 2. Each of these fragments has approximate 25 KDa molecular weight. LC and Fd’ are single polypeptide chain. scFccontains the N-glycan modification site and therefore more heterogenerous. mAb charge variants often include C-terminal lysine variant. Figure 3 shows the baseline separation of scFc, LC, and Fd’ of infliximab on a MAbPac RP column. In addition, there is sufficient spatial separation of lysine containing scFc fragment from the unmodified scFc when using mobile phase containing 0.1% TFA ion-pairing reagent. Figure 4 shows a lesser separation of lysine modified scFc and unmodified scFc when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 5 shows the mass spectra of scFc and scFc-Lys at +10 charge state. Deconvoluted spectra show the mass difference between scFc-Lys and scFc is 128 Da, corresponding to the residue mass of a lysine (Figure 6).

MATERIALS AND METHODSChemicals and ReagentsFabRICATOR™ (IdeS) protease was purchased from Genovis (Lund, Sweden). PNGase F was purchased from Prozyme (Hayward, CA) .Sample Preparation

Reduction: reduction of inter-chain disulfides in a mAb (4 mg/mL) was achieved by incubation of mAbwith 20 mM DTT at 37 °C for 30 min.IdeS Digestion: 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.Deglycosylation: glycoprotein solutions were prepared at 10 mg/mL concentration in PBS buffer. Prior to deglycosylation, buffer containing HEPES at pH 7.9, reductant (TCEP) and detergent (proprietary) were added to the glycoprotein solutions. This was followed by heat denaturation at 95 °C for 5 min. PNGaseF was added to the mixture followed by incubation at 50 ⁰C for 15 min. Deglycosylated proteins were injected onto Thermo Scientific™ MAbPac™ RP analytical columns without further sample cleanup.Oxidation: dilute the mAb solution (5 mg/mL) in half with the 2X oxidation buffer (360 mM sodium chloride, 10 mM sodium acetate, pH 5.0). Then add H2O2 to a final concentration of 0.01% (v/v) and incubate the sample for 24h at room temperature.High Performance Liquid Chromatography

Thermo Scientific™ Dionex™ UltiMate™ 3000 BioRS system was used for mAb fragment separation. The column used was the MAbPac RP analytical column (3.0 x 50 mm, P/N 088645; 2.1 x 100 mm, P/N 088647). The following mobile phase A and B are used for LC/UV experiment: H2O/TFA (99.9 : 0.1 v/v) and MeCN/ H2O/TFA (90 : 9.9 :0.1 v/v/v). The following mobile phase A and B are used for LC/MS experiment: H2O/FA/TFA (99.88 : 0.1 : 0.02 v/v/v) and MeCN/ H2O/FA/TFA (90 : 9.88 : 0.1 : 0.02 v/v/v/v).

Mass Spectrometry Conditions

The Thermo Scientific™ Q Exactive™ Plus Orbitrap™ mass spectrometer was used for this study. Intact mAb or mAb fragments were analyzed by ESI-MS. H-ESI II probe was used. The resolution was set at 17.5 k (FWHM) at m/z 200, see Table 1.

Data Analysis

Full MS spectra of intact mAbs and mAb fragments were analyzed using Thermo Scientific™ Protein Deconvolution™ software 4.0 that utilizes the ReSpect algorithm for molecular mass determination.

ABSTRACTWith the recent growth of the biopharmaceutical industry, sensitive and fast methods are required to monitor microheterogeneity and PTMs during all stages of process development to guarantee product safety and efficacy. Therapeutic mAbs, such as rituximab, trastuzumab, infliximab, and bevacizumab, are mostly produced from mammalian cells. These biological products are heterogeneous, containing multiple charge variants and glycosylation forms. Additional modifications such as oxidation can be introduced during the manufacturing process. In the current study, mAbs are broken down into several large fragments using reduction reagent and IdeS enzyme. A fast LC/MS separation method is employed for the following two applications: 1) monitoring mAb fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation.

INTRODUCTIONThe monoclonal antibody (mAb) therapeutics market is growing at a rapid rate owing to increasing demand for targeted treatments. Therapeutic mAbs are mostly produced from mammalian cells. These biological products are heterogeneous due to post-translational modifications (Figure 1). Additional modifications such as oxidation can be introduced during the manufacturing process. A comprehensive characterization of mAb purity, aggregates, and variants is required for the final biopharmaceutical product approval and subsequent manufacturing processes. There is a growing trend to obtain intact mass information as well as the glycan profile in the QC of mAbs using reverse phase chromatography coupled with high resolution mass spectrometry detection. LC/MS analysis of mAb fragments such as light chain (LC), heavy chain (HC), Fc, Fab, scFc and F(ab')2, can accurately reveal the location, as well as nature, of the modification. Moreover, in most QC environments, LC/UV analysis of mAb fragments has been established as a high throughput assay.The reversed phase column (MAbPac RP) used in the fast LC/MS assays is based on wide-pore 4 μmpolymer particles that are stable at extreme pH (0–14) and high temperature (up to 110 °C). The wide-pore size of polymeric particles (1,500 Å) enables efficient separation of large protein molecules with low carry-over. This study focuses on two application areas: 1) monitoring scFc fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation of HC.

Figure 3. LC/UV analysis of mAb fragments containing lysine charge variants.

Figure 4. LC/MS analysis of mAb fragments containing lysine charge variants.

mAb Oxidation Variant AnalysisMet oxidation is one of the critical quality attributes required to be closely monitored. The two Met residues in the CH2-CH3 domain interface of recombinant humanized and fully human IgG1 antibodies were found susceptible to oxidation [1]. It is desirable to monitor the progress of the Met oxidation without complete digestion of mAb. Infliximab was treated with H2O2, resulting in the oxidation of LC and HC. Figure 7 shows the separation of scFc, LC, and Fd’ fragments as well as its oxidized forms on a MAbPac RP column. Figure 8 shows a lesser separation of oxidized fragments from the unmodified fragments when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 9 shows the mass spectra of oxidized scFc at +10 charge state. Figure 10 shows the mass spectra of oxidized LC and Fd’ at +10 charge state. Deconvoluted spectra of scFc and LC show the mass difference between the oxidized form and the unmodified fragment is 16 Da, corresponding to the residue mass of an oxygen (Figure 11 and Figure 12).

CONCLUSIONS mAb fragments scFc, LC, and Fd’ are baseline separated on a 3.0 × 50 mm MAbPac RP column.

Spatial separation of lysine variant and oxidation variant from the unmodified scFc is achieved.

LC/MS analysis of the scFc, LC, and Fd’ fragments successfully pinpoints the location of lysine and oxidation modifications.

A workflow has been developed to successfully remove N-glycan from mAb in 15 min. TrastuzumabHC and its deglycosylated form are baseline separated on a longer (2.1 × 100 mm) MAbPac RP column.

REFERENCES1. Liu H., Gaza-Bulseco G., and Zhou L. Mass Spectrometry Analysis of Photo-InducedMethionine

Oxidation of a Recombinant Human Monoclonal Antibody, J. Am. Soc. Mass. Spectrom., 2009, 20, 525–528.

TRADEMARKS/LICENSING© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

mAb Deglycosylation AnalysisA fast workflow has been developed to remove N-linked glycans in 15 min. Figure 13 shows the separation of LC and HC of trastuzumab before and after deglycosylation. The deglycosylated HC elutes later (retention time at 16.238 min) than the glycosylation HC (retention time at 15.821 min). Figure 14 shows the mass spectra of glycosylated HC and deglycosylated HC. Deconvoluted spectrum of deglycosylated HC shows a single polypeptide of 48,157 Da (Figure 15). The deconvoluted result agrees well with the calculated MW based on the sequence of trastuzumab HC.

Instrument Conditions mAb and mAb FragmentsMass range m/z 1,000–4,000

Spray voltage 3.9 kV

Sheath gas 45 arb. units

Auxiliary gas 15 arb. units

Capillary temperature 320 °CS-lens level 55

In-source CID 40 eV

Microscans 10

AGC target 3 × 106

Maximum IT 200 ms

Resolving power 17,500

Probe temperature 300 °C

Figure 1. Structure of IgG and typical forms of heterogeneity.

Figure 2. mAb reduction and IdeS digestion flowchart.

Column: MAbPac RP, 4 µmFormat: 3.0 × 50 mmMobile phase A: H2O/FA/TFA (99.88 : 0.1 : 0.02

v/v/v)Mobile phase B: MeCN/ H2O/FA/TFA (90 : 9.88 :

0.1 : 0.02 v/v/v/v)Gradient:

Time (min) %A %B0.0 80 201.0 80 2011.0 55 4512.0 55 4514.0 80 2015.0 80 20

Temperature: 80 ºCFlow rate: 0.5 mL/minInj. volume: 2 µL MS Detection: positive-ion mode Mass Spec: Q Exactive™ PlusSample: Infliximab+IdeS+DTT

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

7.89

8.33

7.006.93

scFc

scFc- Lys

LC

Fd´

Figure 5. Mass spectra of scFc, LC, and Fd’ fragments.

+10scFc-Lys

scFc

LC

Fd’

+10

+10

+10

2400 2420 2440 2460 2480 2500 2520 2540 2560 2580 2600 2620 2640 2660 2680 2700m/z

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

0

20

40

60

80

100 2533.75

2549.79

2520.77 2566.462498.572520.91

2536.95

2498.38 2553.472516.75

2485.85 2653.622604.75

2609.532467.57 2600.16 2618.282424.23

2565.30

2569.092561.132443.44 2459.95

Figure 6. Deconvoluted spectra of infliximab scFc fragments containing lysine charge variants.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.588

25489.688

25199.288 25285.08825099.088 25123.688 25651.888

25199.42125361.521

25327.521 25524.02125221.62125157.42124995.621 25383.921

Mass

Lysine

Figure 7. LC/UV analysis of mAb fragments containing oxidation variants.

Figure 8. LC/MS analysis of mAb fragments containing oxidation variants.

Figure 9. Mass spectra of scFc fragments. Figure 10. Mass spectra of LC and Fd’ fragments.

+10

+10

+10

+10

Oxidized scFc

scFc

Oxidized scFc-lys

Twice OxidizedscFc-lys

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002537.20

2553.15

2577.242524.142427.30 2644.16 2662.24

2535.26

2551.472522.72

2512.79 2561.72

2522.46

2533.56

2538.84

2501.92 2566.022520.86

2537.17

2498.18 2553.14 2583.71

+10

+10

+10

+10

Oxidized Fd’

Fd’

LC

Oxidized LC

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002606.55

2613.102641.212466.04 2600.002439.26

2604.71

2611.792467.92 2598.41 2645.882566.80

2572.822444.57 2558.23

2565.17

2571.362677.81

Figure 11. Deconvoluted spectra of infliximab scFc fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.61425215.214

25489.91425377.814

25345.31425011.514 25237.114 25651.81425285.81425172.814 25447.41425124.414 25508.714

25199.66625361.666

25237.66624971.266 25156.766 25523.76625400.76625318.866 25482.16625013.466 25126.966 25643.966

Mass

O

Figure 12. Deconvoluted spectra of LC fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

23450.164

23406.964

23434.234

23472.53423417.334

Mass

O

Figure 13. LC/UV analysis of trastuzumabLC and HC fragments before and after deglycosylation.

Figure 14. Mass spectra of trastuzumab HC before and after deglycosylation.

control

+PNGaseF

1320 1340 1360 1380 1400 1420m/z

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

1332.66

1373.04 1406.691336.951368.69

1341.15 1415.691364.69 1387.731377.35

1330.651421.74

1404.72

1358.701319.69 1347.72

1366.45 1405.481329.65

1369.721326.68 1418.721402.75

1387.721358.681348.711336.731315.68

1413.73

Figure 15. Deconvoluted spectra of trastuzumab HC before and after deglycosylation.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

49157.554

49138.354 49321.654

50763.886

50454.786

Mass

Deglycosylated HC

HC

Table 1. MS conditions.

Middle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationShanhua Lin1, Zoltan Szabo1, Yury Agroskin1, Terry Zhang2, Jonathan Josephs2, and Xiaodong Liu1, 1Thermo Fisher Scientific, Sunnyvale; 2Thermo Fisher Scientific, San Jose

RESULTSmAb Charge Variant Analysis

A workflow to generate smaller mAb fragments scFc, LC, and Fd’s is shown in Figure 2. Each of these fragments has approximate 25 KDa molecular weight. LC and Fd’ are single polypeptide chain. scFccontains the N-glycan modification site and therefore more heterogenerous. mAb charge variants often include C-terminal lysine variant. Figure 3 shows the baseline separation of scFc, LC, and Fd’ of infliximab on a MAbPac RP column. In addition, there is sufficient spatial separation of lysine containing scFc fragment from the unmodified scFc when using mobile phase containing 0.1% TFA ion-pairing reagent. Figure 4 shows a lesser separation of lysine modified scFc and unmodified scFc when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 5 shows the mass spectra of scFc and scFc-Lys at +10 charge state. Deconvoluted spectra show the mass difference between scFc-Lys and scFc is 128 Da, corresponding to the residue mass of a lysine (Figure 6).

MATERIALS AND METHODSChemicals and ReagentsFabRICATOR™ (IdeS) protease was purchased from Genovis (Lund, Sweden). PNGase F was purchased from Prozyme (Hayward, CA) .Sample Preparation

Reduction: reduction of inter-chain disulfides in a mAb (4 mg/mL) was achieved by incubation of mAbwith 20 mM DTT at 37 °C for 30 min.IdeS Digestion: 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.Deglycosylation: glycoprotein solutions were prepared at 10 mg/mL concentration in PBS buffer. Prior to deglycosylation, buffer containing HEPES at pH 7.9, reductant (TCEP) and detergent (proprietary) were added to the glycoprotein solutions. This was followed by heat denaturation at 95 °C for 5 min. PNGaseF was added to the mixture followed by incubation at 50 ⁰C for 15 min. Deglycosylated proteins were injected onto Thermo Scientific™ MAbPac™ RP analytical columns without further sample cleanup.Oxidation: dilute the mAb solution (5 mg/mL) in half with the 2X oxidation buffer (360 mM sodium chloride, 10 mM sodium acetate, pH 5.0). Then add H2O2 to a final concentration of 0.01% (v/v) and incubate the sample for 24h at room temperature.High Performance Liquid Chromatography

Thermo Scientific™ Dionex™ UltiMate™ 3000 BioRS system was used for mAb fragment separation. The column used was the MAbPac RP analytical column (3.0 x 50 mm, P/N 088645; 2.1 x 100 mm, P/N 088647). The following mobile phase A and B are used for LC/UV experiment: H2O/TFA (99.9 : 0.1 v/v) and MeCN/ H2O/TFA (90 : 9.9 :0.1 v/v/v). The following mobile phase A and B are used for LC/MS experiment: H2O/FA/TFA (99.88 : 0.1 : 0.02 v/v/v) and MeCN/ H2O/FA/TFA (90 : 9.88 : 0.1 : 0.02 v/v/v/v).

Mass Spectrometry Conditions

The Thermo Scientific™ Q Exactive™ Plus Orbitrap™ mass spectrometer was used for this study. Intact mAb or mAb fragments were analyzed by ESI-MS. H-ESI II probe was used. The resolution was set at 17.5 k (FWHM) at m/z 200, see Table 1.

Data Analysis

Full MS spectra of intact mAbs and mAb fragments were analyzed using Thermo Scientific™ Protein Deconvolution™ software 4.0 that utilizes the ReSpect algorithm for molecular mass determination.

ABSTRACTWith the recent growth of the biopharmaceutical industry, sensitive and fast methods are required to monitor microheterogeneity and PTMs during all stages of process development to guarantee product safety and efficacy. Therapeutic mAbs, such as rituximab, trastuzumab, infliximab, and bevacizumab, are mostly produced from mammalian cells. These biological products are heterogeneous, containing multiple charge variants and glycosylation forms. Additional modifications such as oxidation can be introduced during the manufacturing process. In the current study, mAbs are broken down into several large fragments using reduction reagent and IdeS enzyme. A fast LC/MS separation method is employed for the following two applications: 1) monitoring mAb fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation.

INTRODUCTIONThe monoclonal antibody (mAb) therapeutics market is growing at a rapid rate owing to increasing demand for targeted treatments. Therapeutic mAbs are mostly produced from mammalian cells. These biological products are heterogeneous due to post-translational modifications (Figure 1). Additional modifications such as oxidation can be introduced during the manufacturing process. A comprehensive characterization of mAb purity, aggregates, and variants is required for the final biopharmaceutical product approval and subsequent manufacturing processes. There is a growing trend to obtain intact mass information as well as the glycan profile in the QC of mAbs using reverse phase chromatography coupled with high resolution mass spectrometry detection. LC/MS analysis of mAb fragments such as light chain (LC), heavy chain (HC), Fc, Fab, scFc and F(ab')2, can accurately reveal the location, as well as nature, of the modification. Moreover, in most QC environments, LC/UV analysis of mAb fragments has been established as a high throughput assay.The reversed phase column (MAbPac RP) used in the fast LC/MS assays is based on wide-pore 4 μmpolymer particles that are stable at extreme pH (0–14) and high temperature (up to 110 °C). The wide-pore size of polymeric particles (1,500 Å) enables efficient separation of large protein molecules with low carry-over. This study focuses on two application areas: 1) monitoring scFc fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation of HC.

Figure 3. LC/UV analysis of mAb fragments containing lysine charge variants.

Figure 4. LC/MS analysis of mAb fragments containing lysine charge variants.

mAb Oxidation Variant AnalysisMet oxidation is one of the critical quality attributes required to be closely monitored. The two Met residues in the CH2-CH3 domain interface of recombinant humanized and fully human IgG1 antibodies were found susceptible to oxidation [1]. It is desirable to monitor the progress of the Met oxidation without complete digestion of mAb. Infliximab was treated with H2O2, resulting in the oxidation of LC and HC. Figure 7 shows the separation of scFc, LC, and Fd’ fragments as well as its oxidized forms on a MAbPac RP column. Figure 8 shows a lesser separation of oxidized fragments from the unmodified fragments when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 9 shows the mass spectra of oxidized scFc at +10 charge state. Figure 10 shows the mass spectra of oxidized LC and Fd’ at +10 charge state. Deconvoluted spectra of scFc and LC show the mass difference between the oxidized form and the unmodified fragment is 16 Da, corresponding to the residue mass of an oxygen (Figure 11 and Figure 12).

CONCLUSIONS mAb fragments scFc, LC, and Fd’ are baseline separated on a 3.0 × 50 mm MAbPac RP column.

Spatial separation of lysine variant and oxidation variant from the unmodified scFc is achieved.

LC/MS analysis of the scFc, LC, and Fd’ fragments successfully pinpoints the location of lysine and oxidation modifications.

A workflow has been developed to successfully remove N-glycan from mAb in 15 min. TrastuzumabHC and its deglycosylated form are baseline separated on a longer (2.1 × 100 mm) MAbPac RP column.

REFERENCES1. Liu H., Gaza-Bulseco G., and Zhou L. Mass Spectrometry Analysis of Photo-InducedMethionine

Oxidation of a Recombinant Human Monoclonal Antibody, J. Am. Soc. Mass. Spectrom., 2009, 20, 525–528.

TRADEMARKS/LICENSING© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

mAb Deglycosylation AnalysisA fast workflow has been developed to remove N-linked glycans in 15 min. Figure 13 shows the separation of LC and HC of trastuzumab before and after deglycosylation. The deglycosylated HC elutes later (retention time at 16.238 min) than the glycosylation HC (retention time at 15.821 min). Figure 14 shows the mass spectra of glycosylated HC and deglycosylated HC. Deconvoluted spectrum of deglycosylated HC shows a single polypeptide of 48,157 Da (Figure 15). The deconvoluted result agrees well with the calculated MW based on the sequence of trastuzumab HC.

Instrument Conditions mAb and mAb FragmentsMass range m/z 1,000–4,000

Spray voltage 3.9 kV

Sheath gas 45 arb. units

Auxiliary gas 15 arb. units

Capillary temperature 320 °CS-lens level 55

In-source CID 40 eV

Microscans 10

AGC target 3 × 106

Maximum IT 200 ms

Resolving power 17,500

Probe temperature 300 °C

Figure 1. Structure of IgG and typical forms of heterogeneity.

Figure 2. mAb reduction and IdeS digestion flowchart.

Column: MAbPac RP, 4 µmFormat: 3.0 × 50 mmMobile phase A: H2O/FA/TFA (99.88 : 0.1 : 0.02

v/v/v)Mobile phase B: MeCN/ H2O/FA/TFA (90 : 9.88 :

0.1 : 0.02 v/v/v/v)Gradient:

Time (min) %A %B0.0 80 201.0 80 2011.0 55 4512.0 55 4514.0 80 2015.0 80 20

Temperature: 80 ºCFlow rate: 0.5 mL/minInj. volume: 2 µL MS Detection: positive-ion mode Mass Spec: Q Exactive™ PlusSample: Infliximab+IdeS+DTT

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

7.89

8.33

7.006.93

scFc

scFc- Lys

LC

Fd´

Figure 5. Mass spectra of scFc, LC, and Fd’ fragments.

+10scFc-Lys

scFc

LC

Fd’

+10

+10

+10

2400 2420 2440 2460 2480 2500 2520 2540 2560 2580 2600 2620 2640 2660 2680 2700m/z

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

0

20

40

60

80

100 2533.75

2549.79

2520.77 2566.462498.572520.91

2536.95

2498.38 2553.472516.75

2485.85 2653.622604.75

2609.532467.57 2600.16 2618.282424.23

2565.30

2569.092561.132443.44 2459.95

Figure 6. Deconvoluted spectra of infliximab scFc fragments containing lysine charge variants.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.588

25489.688

25199.288 25285.08825099.088 25123.688 25651.888

25199.42125361.521

25327.521 25524.02125221.62125157.42124995.621 25383.921

Mass

Lysine

Figure 7. LC/UV analysis of mAb fragments containing oxidation variants.

Figure 8. LC/MS analysis of mAb fragments containing oxidation variants.

Figure 9. Mass spectra of scFc fragments. Figure 10. Mass spectra of LC and Fd’ fragments.

+10

+10

+10

+10

Oxidized scFc

scFc

Oxidized scFc-lys

Twice OxidizedscFc-lys

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002537.20

2553.15

2577.242524.142427.30 2644.16 2662.24

2535.26

2551.472522.72

2512.79 2561.72

2522.46

2533.56

2538.84

2501.92 2566.022520.86

2537.17

2498.18 2553.14 2583.71

+10

+10

+10

+10

Oxidized Fd’

Fd’

LC

Oxidized LC

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002606.55

2613.102641.212466.04 2600.002439.26

2604.71

2611.792467.92 2598.41 2645.882566.80

2572.822444.57 2558.23

2565.17

2571.362677.81

Figure 11. Deconvoluted spectra of infliximab scFc fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.61425215.214

25489.91425377.814

25345.31425011.514 25237.114 25651.81425285.81425172.814 25447.41425124.414 25508.714

25199.66625361.666

25237.66624971.266 25156.766 25523.76625400.76625318.866 25482.16625013.466 25126.966 25643.966

Mass

O

Figure 12. Deconvoluted spectra of LC fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

23450.164

23406.964

23434.234

23472.53423417.334

Mass

O

Figure 13. LC/UV analysis of trastuzumabLC and HC fragments before and after deglycosylation.

Figure 14. Mass spectra of trastuzumab HC before and after deglycosylation.

control

+PNGaseF

1320 1340 1360 1380 1400 1420m/z

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

1332.66

1373.04 1406.691336.951368.69

1341.15 1415.691364.69 1387.731377.35

1330.651421.74

1404.72

1358.701319.69 1347.72

1366.45 1405.481329.65

1369.721326.68 1418.721402.75

1387.721358.681348.711336.731315.68

1413.73

Figure 15. Deconvoluted spectra of trastuzumab HC before and after deglycosylation.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

49157.554

49138.354 49321.654

50763.886

50454.786

Mass

Deglycosylated HC

HC

Table 1. MS conditions.

Middle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationShanhua Lin1, Zoltan Szabo1, Yury Agroskin1, Terry Zhang2, Jonathan Josephs2, and Xiaodong Liu1, 1Thermo Fisher Scientific, Sunnyvale; 2Thermo Fisher Scientific, San Jose

RESULTSmAb Charge Variant Analysis

A workflow to generate smaller mAb fragments scFc, LC, and Fd’s is shown in Figure 2. Each of these fragments has approximate 25 KDa molecular weight. LC and Fd’ are single polypeptide chain. scFccontains the N-glycan modification site and therefore more heterogenerous. mAb charge variants often include C-terminal lysine variant. Figure 3 shows the baseline separation of scFc, LC, and Fd’ of infliximab on a MAbPac RP column. In addition, there is sufficient spatial separation of lysine containing scFc fragment from the unmodified scFc when using mobile phase containing 0.1% TFA ion-pairing reagent. Figure 4 shows a lesser separation of lysine modified scFc and unmodified scFc when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 5 shows the mass spectra of scFc and scFc-Lys at +10 charge state. Deconvoluted spectra show the mass difference between scFc-Lys and scFc is 128 Da, corresponding to the residue mass of a lysine (Figure 6).

MATERIALS AND METHODSChemicals and ReagentsFabRICATOR™ (IdeS) protease was purchased from Genovis (Lund, Sweden). PNGase F was purchased from Prozyme (Hayward, CA) .Sample Preparation

Reduction: reduction of inter-chain disulfides in a mAb (4 mg/mL) was achieved by incubation of mAbwith 20 mM DTT at 37 °C for 30 min.IdeS Digestion: 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.Deglycosylation: glycoprotein solutions were prepared at 10 mg/mL concentration in PBS buffer. Prior to deglycosylation, buffer containing HEPES at pH 7.9, reductant (TCEP) and detergent (proprietary) were added to the glycoprotein solutions. This was followed by heat denaturation at 95 °C for 5 min. PNGaseF was added to the mixture followed by incubation at 50 ⁰C for 15 min. Deglycosylated proteins were injected onto Thermo Scientific™ MAbPac™ RP analytical columns without further sample cleanup.Oxidation: dilute the mAb solution (5 mg/mL) in half with the 2X oxidation buffer (360 mM sodium chloride, 10 mM sodium acetate, pH 5.0). Then add H2O2 to a final concentration of 0.01% (v/v) and incubate the sample for 24h at room temperature.High Performance Liquid Chromatography

Thermo Scientific™ Dionex™ UltiMate™ 3000 BioRS system was used for mAb fragment separation. The column used was the MAbPac RP analytical column (3.0 x 50 mm, P/N 088645; 2.1 x 100 mm, P/N 088647). The following mobile phase A and B are used for LC/UV experiment: H2O/TFA (99.9 : 0.1 v/v) and MeCN/ H2O/TFA (90 : 9.9 :0.1 v/v/v). The following mobile phase A and B are used for LC/MS experiment: H2O/FA/TFA (99.88 : 0.1 : 0.02 v/v/v) and MeCN/ H2O/FA/TFA (90 : 9.88 : 0.1 : 0.02 v/v/v/v).

Mass Spectrometry Conditions

The Thermo Scientific™ Q Exactive™ Plus Orbitrap™ mass spectrometer was used for this study. Intact mAb or mAb fragments were analyzed by ESI-MS. H-ESI II probe was used. The resolution was set at 17.5 k (FWHM) at m/z 200, see Table 1.

Data Analysis

Full MS spectra of intact mAbs and mAb fragments were analyzed using Thermo Scientific™ Protein Deconvolution™ software 4.0 that utilizes the ReSpect algorithm for molecular mass determination.

ABSTRACTWith the recent growth of the biopharmaceutical industry, sensitive and fast methods are required to monitor microheterogeneity and PTMs during all stages of process development to guarantee product safety and efficacy. Therapeutic mAbs, such as rituximab, trastuzumab, infliximab, and bevacizumab, are mostly produced from mammalian cells. These biological products are heterogeneous, containing multiple charge variants and glycosylation forms. Additional modifications such as oxidation can be introduced during the manufacturing process. In the current study, mAbs are broken down into several large fragments using reduction reagent and IdeS enzyme. A fast LC/MS separation method is employed for the following two applications: 1) monitoring mAb fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation.

INTRODUCTIONThe monoclonal antibody (mAb) therapeutics market is growing at a rapid rate owing to increasing demand for targeted treatments. Therapeutic mAbs are mostly produced from mammalian cells. These biological products are heterogeneous due to post-translational modifications (Figure 1). Additional modifications such as oxidation can be introduced during the manufacturing process. A comprehensive characterization of mAb purity, aggregates, and variants is required for the final biopharmaceutical product approval and subsequent manufacturing processes. There is a growing trend to obtain intact mass information as well as the glycan profile in the QC of mAbs using reverse phase chromatography coupled with high resolution mass spectrometry detection. LC/MS analysis of mAb fragments such as light chain (LC), heavy chain (HC), Fc, Fab, scFc and F(ab')2, can accurately reveal the location, as well as nature, of the modification. Moreover, in most QC environments, LC/UV analysis of mAb fragments has been established as a high throughput assay.The reversed phase column (MAbPac RP) used in the fast LC/MS assays is based on wide-pore 4 μmpolymer particles that are stable at extreme pH (0–14) and high temperature (up to 110 °C). The wide-pore size of polymeric particles (1,500 Å) enables efficient separation of large protein molecules with low carry-over. This study focuses on two application areas: 1) monitoring scFc fragments containing charge variants and oxidation variants; 2) confirming complete deglycosylation of HC.

Figure 3. LC/UV analysis of mAb fragments containing lysine charge variants.

Figure 4. LC/MS analysis of mAb fragments containing lysine charge variants.

mAb Oxidation Variant AnalysisMet oxidation is one of the critical quality attributes required to be closely monitored. The two Met residues in the CH2-CH3 domain interface of recombinant humanized and fully human IgG1 antibodies were found susceptible to oxidation [1]. It is desirable to monitor the progress of the Met oxidation without complete digestion of mAb. Infliximab was treated with H2O2, resulting in the oxidation of LC and HC. Figure 7 shows the separation of scFc, LC, and Fd’ fragments as well as its oxidized forms on a MAbPac RP column. Figure 8 shows a lesser separation of oxidized fragments from the unmodified fragments when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 9 shows the mass spectra of oxidized scFc at +10 charge state. Figure 10 shows the mass spectra of oxidized LC and Fd’ at +10 charge state. Deconvoluted spectra of scFc and LC show the mass difference between the oxidized form and the unmodified fragment is 16 Da, corresponding to the residue mass of an oxygen (Figure 11 and Figure 12).

CONCLUSIONS mAb fragments scFc, LC, and Fd’ are baseline separated on a 3.0 × 50 mm MAbPac RP column.

Spatial separation of lysine variant and oxidation variant from the unmodified scFc is achieved.

LC/MS analysis of the scFc, LC, and Fd’ fragments successfully pinpoints the location of lysine and oxidation modifications.

A workflow has been developed to successfully remove N-glycan from mAb in 15 min. TrastuzumabHC and its deglycosylated form are baseline separated on a longer (2.1 × 100 mm) MAbPac RP column.

REFERENCES1. Liu H., Gaza-Bulseco G., and Zhou L. Mass Spectrometry Analysis of Photo-InducedMethionine

Oxidation of a Recombinant Human Monoclonal Antibody, J. Am. Soc. Mass. Spectrom., 2009, 20, 525–528.

TRADEMARKS/LICENSING© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

mAb Deglycosylation AnalysisA fast workflow has been developed to remove N-linked glycans in 15 min. Figure 13 shows the separation of LC and HC of trastuzumab before and after deglycosylation. The deglycosylated HC elutes later (retention time at 16.238 min) than the glycosylation HC (retention time at 15.821 min). Figure 14 shows the mass spectra of glycosylated HC and deglycosylated HC. Deconvoluted spectrum of deglycosylated HC shows a single polypeptide of 48,157 Da (Figure 15). The deconvoluted result agrees well with the calculated MW based on the sequence of trastuzumab HC.

Instrument Conditions mAb and mAb FragmentsMass range m/z 1,000–4,000

Spray voltage 3.9 kV

Sheath gas 45 arb. units

Auxiliary gas 15 arb. units

Capillary temperature 320 °CS-lens level 55

In-source CID 40 eV

Microscans 10

AGC target 3 × 106

Maximum IT 200 ms

Resolving power 17,500

Probe temperature 300 °C

Figure 1. Structure of IgG and typical forms of heterogeneity.

Figure 2. mAb reduction and IdeS digestion flowchart.

Column: MAbPac RP, 4 µmFormat: 3.0 × 50 mmMobile phase A: H2O/FA/TFA (99.88 : 0.1 : 0.02

v/v/v)Mobile phase B: MeCN/ H2O/FA/TFA (90 : 9.88 :

0.1 : 0.02 v/v/v/v)Gradient:

Time (min) %A %B0.0 80 201.0 80 2011.0 55 4512.0 55 4514.0 80 2015.0 80 20

Temperature: 80 ºCFlow rate: 0.5 mL/minInj. volume: 2 µL MS Detection: positive-ion mode Mass Spec: Q Exactive™ PlusSample: Infliximab+IdeS+DTT

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

7.89

8.33

7.006.93

scFc

scFc- Lys

LC

Fd´

Figure 5. Mass spectra of scFc, LC, and Fd’ fragments.

+10scFc-Lys

scFc

LC

Fd’

+10

+10

+10

2400 2420 2440 2460 2480 2500 2520 2540 2560 2580 2600 2620 2640 2660 2680 2700m/z

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

0

20

40

60

80

100 2533.75

2549.79

2520.77 2566.462498.572520.91

2536.95

2498.38 2553.472516.75

2485.85 2653.622604.75

2609.532467.57 2600.16 2618.282424.23

2565.30

2569.092561.132443.44 2459.95

Figure 6. Deconvoluted spectra of infliximab scFc fragments containing lysine charge variants.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.588

25489.688

25199.288 25285.08825099.088 25123.688 25651.888

25199.42125361.521

25327.521 25524.02125221.62125157.42124995.621 25383.921

Mass

Lysine

Figure 7. LC/UV analysis of mAb fragments containing oxidation variants.

Figure 8. LC/MS analysis of mAb fragments containing oxidation variants.

Figure 9. Mass spectra of scFc fragments. Figure 10. Mass spectra of LC and Fd’ fragments.

+10

+10

+10

+10

Oxidized scFc

scFc

Oxidized scFc-lys

Twice OxidizedscFc-lys

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002537.20

2553.15

2577.242524.142427.30 2644.16 2662.24

2535.26

2551.472522.72

2512.79 2561.72

2522.46

2533.56

2538.84

2501.92 2566.022520.86

2537.17

2498.18 2553.14 2583.71

+10

+10

+10

+10

Oxidized Fd’

Fd’

LC

Oxidized LC

2400 2450 2500 2550 2600 2650 2700m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce 0

50

1002606.55

2613.102641.212466.04 2600.002439.26

2604.71

2611.792467.92 2598.41 2645.882566.80

2572.822444.57 2558.23

2565.17

2571.362677.81

Figure 11. Deconvoluted spectra of infliximab scFc fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

25327.61425215.214

25489.91425377.814

25345.31425011.514 25237.114 25651.81425285.81425172.814 25447.41425124.414 25508.714

25199.66625361.666

25237.66624971.266 25156.766 25523.76625400.76625318.866 25482.16625013.466 25126.966 25643.966

Mass

O

Figure 12. Deconvoluted spectra of LC fragments containing a single oxidation site.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

23450.164

23406.964

23434.234

23472.53423417.334

Mass

O

Figure 13. LC/UV analysis of trastuzumabLC and HC fragments before and after deglycosylation.

Figure 14. Mass spectra of trastuzumab HC before and after deglycosylation.

control

+PNGaseF

1320 1340 1360 1380 1400 1420m/z

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

1332.66

1373.04 1406.691336.951368.69

1341.15 1415.691364.69 1387.731377.35

1330.651421.74

1404.72

1358.701319.69 1347.72

1366.45 1405.481329.65

1369.721326.68 1418.721402.75

1387.721358.681348.711336.731315.68

1413.73

Figure 15. Deconvoluted spectra of trastuzumab HC before and after deglycosylation.

-100

-50

0

50

100

Rel

a tiv

eIn

ten s

i ty

49157.554

49138.354 49321.654

50763.886

50454.786

Mass

Deglycosylated HC

HC

Table 1. MS conditions.

Middle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationShanhua Lin1, Zoltan Szabo1, Yury Agroskin1, Terry Zhang2, Jonathan Josephs2, and Xiaodong Liu1, 1Thermo Fisher Scientific, Sunnyvale; 2Thermo Fisher Scientific, San Jose

RESULTSmAb Charge Variant Analysis

A workflow to generate smaller mAb fragments scFc, LC, and Fd’s is shown in Figure 2. Each of these fragments has approximate 25 KDa molecular weight. LC and Fd’ are single polypeptide chain. scFccontains the N-glycan modification site and therefore more heterogenerous. mAb charge variants often include C-terminal lysine variant. Figure 3 shows the baseline separation of scFc, LC, and Fd’ of infliximab on a MAbPac RP column. In addition, there is sufficient spatial separation of lysine containing scFc fragment from the unmodified scFc when using mobile phase containing 0.1% TFA ion-pairing reagent. Figure 4 shows a lesser separation of lysine modified scFc and unmodified scFc when using a lower concentration of ion-pairing reagent and a steeper organic gradient. Figure 5 shows the mass spectra of scFc and scFc-Lys at +10 charge state. Deconvoluted spectra show the mass difference between scFc-Lys and scFc is 128 Da, corresponding to the residue mass of a lysine (Figure 6).

MATERIALS AND METHODSChemicals and ReagentsFabRICATOR™ (IdeS) protease was purchased from Genovis (Lund, Sweden). PNGase F was purchased from Prozyme (Hayward, CA) .Sample Preparation

Reduction: reduction of inter-chain disulfides in a mAb (4 mg/mL) was achieved by incubation of mAbwith 20 mM DTT at 37 °C for 30 min.IdeS Digestion: 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.Deglycosylation: glycoprotein solutions were prepared at 10 mg/mL concentration in PBS buffer. Prior to deglycosylation, buffer containing HEPES at pH 7.9, reductant (TCEP) and detergent (proprietary) were added to the glycoprotein solutions. This was followed by heat denaturation at 95 °C for 5 min. PNGaseF was added to the mixture followed by incubation at 50 ⁰C for 15 min. Deglycosylated proteins were injected onto Thermo Scientific™ MAbPac™ RP analytical columns without further sample cleanup.Oxidation: dilute the mAb solution (5 mg/mL) in half with the 2X oxidation buffer (360 mM sodium chloride, 10 mM sodium acetate, pH 5.0). Then add H2O2 to a final concentration of 0.01% (v/v) and incubate the sample for 24h at room temperature.High Performance Liquid Chromatography

Thermo Scientific™ Dionex™ UltiMate™ 3000 BioRS system was used for mAb fragment separation. The column used was the MAbPac RP analytical column (3.0 x 50 mm, P/N 088645; 2.1 x 100 mm, P/N 088647). The following mobile phase A and B are used for LC/UV experiment: H2O/TFA (99.9 : 0.1 v/v) and MeCN/ H2O/TFA (90 : 9.9 :0.1 v/v/v). The following mobile phase A and B are used for LC/MS experiment: H2O/FA/TFA (99.88 : 0.1 : 0.02 v/v/v) and MeCN/ H2O/FA/TFA (90 : 9.88 : 0.1 : 0.02 v/v/v/v).

Mass Spectrometry Conditions

The Thermo Scientific™ Q Exactive™ Plus Orbitrap™ mass spectrometer was used for this study. Intact mAb or mAb fragments were analyzed by ESI-MS. H-ESI II probe was used. The resolution was set at 17.5 k (FWHM) at m/z 200, see Table 1.

Data Analysis

Full MS spectra of intact mAbs and mAb fragments were analyzed using Thermo Scientific™ Protein Deconvolution™ software 4.0 that utilizes the ReSpect algorithm for molecular mass determination.

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