Selective Oxidation of Methionine and Tryptophan for ... AWS
Selective Oxidation of Methionine and Tryptophan for Biologics Comparability Studies May 2nd 2017 Jorge Alexander Pavon
Accelerated and Forced Degradation Studies Accelerated StabilityStability and Forced Degradation Studies for Biologics Development for Biologics Development
Formulation Development Manufacturing Process Development
Research
Development
Phase 1
Phase 2
Phase 3
Commercialization
2
Development Stages No given candidate is perfect, primary and secondary post-translational modifications (PTMs) liabilities are always present Examples: • Deamidation, isomerization, formation of succinimide in CDR • Tryptophan oxidation (CDR) • Free Cysteine • Methionine oxidation (CDR, Fc CH2 and CH3) • Glycosylation sites in CDR • Deamidation of glutamine • Lysine glycation (non-conserved) • (Asp-Pro) clipping
Current and Future Issues in the Manufacturing and Development of Monoclonal Antibodies Advanced Drug Delivery Reviews 2006, 58: 707– 722 Developability Assessment During the Selection of Novel Therapeutic Antibodies. J. Pharm Sci. 2015 104:1885-98
3
Post-translational modifications and Forced Degradation: • PTMs = Critical Quality Attributes (CQAs), if the impact on protein structure leads to effects on function, biological response, safety and efficacy. • Forced degradation can be utilized to rapidly assess potential CQAs during development stages, assessment of stability indicating analytical methods and in later phases comparability assessments.
PTMs = Oxidized Variants = CQAs • Met oxidation can decrease bioactivity and stability and affect serum half-life, can also affect potency • Trp oxidation in the CDR in most cases has been shown to correlate with activity loss • Oxidized Variants can occur during purification, formulation, storage or any step of process development
Characterization of Therapeutic Antibodies and Related Products. Anal. Chem. 2013, 85: 715– 736 Slide 4 www.aaps.org/nationalbiotech
Outline • Selection of Reagents for Trp and Met oxidation • Role of solvent exposure • Selective oxidation of IgG1 and IgG4: case studies • Computational modeling: Trp liabilities and solvent accessibility
5
CDR liabilities: Tryptophan and Methionine in Antibody Fab Domains Exposed Trp residues are more common in antibody CDRs and more solvent accessible than Met
Jarasch A., Koll H. et al., (2015) Developability Assessment During the Selection of Novel Therapeutic Antibodies. J Pharm Sci. 104(6):1885-98
6
Fab (CDR) and Fc (CH2 and CH3) Liabilities in mAbs mAb-A Trp(1a)XXXXTrp(2a) CDR3 (HC) mAb-B Trp(1b)XXXXXXTrp(2b) CDR3 (HC) mAb-D
Trp(1d)XXXXXXXXTrp(2d) CDR3 (HC)
mAb-C Trp(1c) CDR1 (HC) (underlined part of CDR3) The Fc fragment of IgG1 and IgG4 molecules contains two conserved Met residues at positions 252 CH2 domain and 428 CH3 domain, (EU numbering) that are highly susceptible to oxidation.
7
Reactivity of Tryptophan Residues in Proteins (mAbs) Reactivity of residues to photo oxidation is related to surface exposure photosensitization and/or the reaction of Reactive Oxygen Species (ROS).
Sreedhara A., et al., Mol. Pharmaceutics 10 (2013): 278-288 Duenas et al. Pharm. Res. 18 ﴾2001﴿: 1455-1460. Lam et al. Pharm. Res. (2011):2543–2555 Kerwin Bruce et al. Jour. Pharma. Sciences 6 (2007):1468-1479 8
Favored Reagents and Source of ROS Oxidation of Met
methionine
Oxidation of trp: 2,2'-Azobis-2-Methyl-Propanimidamide, Dihydrochloride (AAPH)
met sulfoxide (+16)
Junyan A. JI, et al., . (2009) Methionine, Tryptophan, and Histidine Oxidation in a Model Protein, PTH: Mechanisms and Stabilization. Jour. of Pharma. Sciences, (98) 4485-4500 CasbeerErik et al., (2013) Kinetics and Mechanism of Oxidation of Tryptophan by Ferrate(VI) Environ. Sci. Technol. (47) 4572−4580 Thomas H, Andres et. al (2013) Tryptophan oxidation photosensitized by pterin) Free Radical Biologyand Medicine (63) 467–475 S. Jovanović, I. Čudina, Lj. Josimović., (1977) Gamma radiolysis of oxygenated aqueous solutions of tryptophan. Radiation Physics and Chemistry (22) 765–770
9
Case Study 1: mAb-A (IgG1) - Trp(1a) (HC CDR3) Met (Fc CH2) and Trp1a are Primarily Oxidized by AAPH (60 fold excess 2.0 mM ) Reduced Peptide Mapping (RPM) percent oxidation Stress condition
Modification
Heavy Chain
Met CH2 Trp1a
control
2hr AAPH abundance (%) 10.5 10.8
7.2 2.7
6hr AAPH 39.4 25.9
Trp1a unmodified peptide
Extracted Ion Chromatogram (EIC) Trp oxidation products
Trp1a control 6 hrs AAPH
or
Met CH2 Control 6 hrs AAPH
Methionine sulfoxide peptide
Met CH2 unmodified peptide
10
Mixed Mode Analysis of AAPH Oxidation of an IgG1 (pre-peaks oxidized variants)
20 mM Citrate Buffer neat
Molar Mass vs. time
time (40ºC) main peak purity % 6 hrs 86.7 2 mM AAPH 2 hrs 61.2 2 mM AAPH 6 hrs 29.9
Control sample main peak percent not affected by 6 hrs 40ºC incubation
aTIGIT#2[Aug 18th] LS
1.0x10 1.0x10 1.0x10
Molar Mass (g/mol)
mAb-A (5 mg/mL) Control 2 mM AAPH 2 mM AAPH
1.0x10 1.0x10
8
MW ~ 144 kDa (monomeric)
7
6
5
4
1000.0
Sepax Technologies: Zenix column SEC-300 PBS + 600 mM NaCL
100.0 10.0 1.0 15.0
20.0
25.0 time (min)
30.0
35.0
11
Mixed Mode Analysis of AAPH Oxidation: Effect of (Met and Trp) on chromatography profile
mAU
200.00 100.00
mAb-A
Main peak% 86.7
(pre-peaks oxidized variants)
Control 40ºC 6 hrs
mAU
0.00
50.00
mAb-A
2 mM AAPH 5.0 mM met 40ºC 2 hrs
mAb-A
2 mM AAPH 5.0 mM met 40ºC 6 hrs
41.0
mAb-A
2 mM AAPH 2.5 mM trp 40ºC 2 hrs
82.5
mAb-A
2 mM AAPH 2.5 mM trp 40ºC 6 hrs
77.5
mAb-A
2 mM AAPH 5 mM trp 40ºC 6 hrs
72.4
0.00
mAU
60.00 40.00 20.00 0.00
mAU
100.00 50.00 0.00
mAU
100.00 50.00 0.00
mAU
100.00
86.0
50.00 0.00 0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
Minutes
12
Reduced Peptide Mapping Analysis of AAPH Oxidation in the presence of Met and Trp Selective protection suggest AAPH utilizes different mechanisms for met and trp oxidation » Free –Met suppresses Fc Met oxidation » Free-Trp suppresses Trp1a oxidation (RPM) % oxidation
stress condition chain
control modification Met CH2 Trp1a
HC
7.2 2.7
2hr AAPH
10.5 10.8
6hr AAPH
5mM Met 2hr
39.4 25.9
Trp1a AAPH 6 hrs 5 mM met Trp oxidation products
5mM Met 6hr
abundance (%) 6.7 7.5 9.5 24.4
2.5mM Trp 2hr
2.5mM Trp 6hr
13.1 2.8
27.5 3.8
Trp1a unmodified peptide
EIC AAPH 6 hrs 2.5 mM trp
13
Case Study 2: mAb-B (IgG4) - Trp(1b) (HC CDR3) Reversed-phase HPLC with limited proteolysis: AAPH Oxidation of an IgG4 (36 fold AAPH up to 24 hours)
Fc
25.0 mg/mL Control 40ºC 24 hrs
Fab *
25.0 mg/mL 6 mM AAPH 2 hrs *
*
25.0 mg/mL 6 mM AAPH 6 hrs * 0.00
12.00
24.00
36.00
48.00
60.00
Minutes
Example chromatograph: method under development/optimization
14
Reduced Peptide Mapping Met (Fc CH2) and Trp1b are Primarily Oxidized by AAPH: Partial protection by (Met and Trp) = percent oxidation RPM stress condition chain HC
EIC
control modification Met CH2 Trp1b
6hr AAPH
3.1 2.4
24hr AAPH
21.6 46.6
5mM Met 6hr
5mM Met 24hr
abundance (%) 5.6 12.7 43.0 91.5
66.6 90.7
5mM Trp 6hr
22.1 9.0
5mM Trp 24hr
50.0 19.5
15
Trp1b AAPH 24h
Trp1b unmodified peptide
+4Da
Trp1b AAPH 24h+ 5 mM Met
+16Da
Met/Trp1b +32/16Da
+16Da +32Da +16Da
+32Da
Trp1b unmodified peptide
Extracted Ion Chromatogram
+16Da
Trp1b unmodified peptide
Trp1b AAPH 24h + 5 mM Trp +16Da likely Met
For mAb-B: partial protection is observed, Higher oxidation levels for Trp1b vs Met CH2
Proposed Mechanism for Selective Oxidation by AAPH - Involvement of different reactive intermediates originating from same species
free trp Inhibits reaction of alkyl peroxide with CDR3 Trp
free met inhibits AAPH peroxide reaction with Fc Met
16
Selective Oxidation of Tryptophan and Methionine
Junyan A. JI, boyan Zhang, Wilson Cheng Y., John Wang. (2009) Methionine, Tryptophan, and Histidine Oxidation in a Model Protein, PTH: Mechanisms and Stabilization. Journal of Pharmaceutical Sciences, (98) 4485-4500 Folzer E., Diepold K., Bomans K., Koulov AV, et al., (2015) Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule. Journal of Pharmaceutical Sciences, 104(9)2824-2831
17
Case Study 3: mAb-C (IgG1) – Trp1c (HC CDR1) Oxidation with AAPH: yields Fc Met oxidation, only (36 fold excess for up to 24 hours)
RPM stress condition chain HC
W
control modification Met CH2 Trp1c
3.4 Not detected
6hr AAPH abundance (%) 29.3 Not detected
24hr AAPH 88.1 Not detected M M
Role of solvent exposure and Trp oxidation
18
Computational Modeling of fragment antigen-binding (Fab) of mAb-A, mAb-B • Trp1a and Trp1b are surface exposed as measured by accessible surface area, calculated using a homology model • Other Trp residues in Fv domain are much less surface exposed, as indicated by a small accessible surface area Selective Residues
H3
mAb (chain) mAb-A LC mAb-A HC mAb-A HC mAb-B HC mAb-B HC mAb-B HC
Residue TRP TRP TRP TRP TRP TRP
UID A 1a 2a 1b 2b B
Trp1a
ASA(A^2) 10.96 120.05 10.25 85.25 30.75 21.15
H3
(%) 3.52 38.60 3.30 27.41 9.88 6.80 Trp1b
LC HC
LC
HC
Computational Modeling (Fab) of mAb-C • Trp1c displays good level of exposure to solvent, but showed no oxidation H2
H1
All Trp residues in mAb-C Fab
Trp1c
H3 LC HC
mAb-C LC LC HC HC HC HC HC
Residue TRP TRP TRP TRP TRP TRP TRP
UID A B 1c C D E F
(A^2) 0 7.30 53.63 0 0 27.97 1.74
(%) 0 2.35 17.24 0 0 8.99 0.56
• Solvent accessibility is required for oxidation • However, exposure to solvent does not necessarily equate to higher oxidation levels (Trp1a and Trp1b) • Other structural elements likely play a role
Computational modeling of the fragment antigen-binding (Fab) mAb-D (selective oxidation data not available) •
The structural mode predicts two Trp residues, Trp1d and Trp1d(framework), are solvent exposed
•
Subsequent preliminary data suggest (light stress) both residues are oxidized
mAb D LC LC LC HC HC HC HC HC HC HC
Residue TRP TRP TRP TRP TRP TRP TRP TRP TRP TRP
UID A B C
Trp1d(framework) D E F Trp1d
Trp(2d) G
(A^2) 0 30.67 73.12 118.26 0 0 0 243.93 15.2 82.85
(%) 0 9.86 23.51 38.02 0 0 0 78.42 4.89 26.64
Conclusions Selective oxidation can be achieved by utilizing free Met or Trp in the stress conditions. This observation strongly suggests the presence of two different reactive intermediates generated from the AAPH alkylperoxide radical. The selective oxidation system coupled with RPM-mass spectrometry and analytical chromatography methods can be used to identify and monitor individually potential CQAs in therapeutic monoclonal antibodies. Surface-exposed tryptophan residues in the CDR is a prerequisite for the reactivity of this amino acid with reactive oxygen species Computational modeling, coupled to historical data can be a powerful tool in studying PCQAs in therapeutic monoclonal antibodies.
Questions and Contact Information Jorge Alexander Pavon PhD Associate Principal Scientist Forced Degradation & Impurity Profiling 2015 Galloping Hill Road K-15 B428C Kenilworth, NJ 07033 (908)740-6886 [email protected]
Slide 23 www.aaps.org/nationalbiotech
Acknowledgements
Yan-Hui Liu Li Xiao Danielle Aldredge Alex Fridman Eugene Dank Umesh Kishnani Jia Zha Peter Salmon
24
Accelerated and Forced Degradation Studies Accelerated StabilityStability and Forced Degradation Studies for Biologics Development for Biologics Development
Formulation Development Manufacturing Process Development
Research
Development
Phase 1
Phase 2
Phase 3
Commercialization
2
Development Stages No given candidate is perfect, primary and secondary post-translational modifications (PTMs) liabilities are always present Examples: • Deamidation, isomerization, formation of succinimide in CDR • Tryptophan oxidation (CDR) • Free Cysteine • Methionine oxidation (CDR, Fc CH2 and CH3) • Glycosylation sites in CDR • Deamidation of glutamine • Lysine glycation (non-conserved) • (Asp-Pro) clipping
Current and Future Issues in the Manufacturing and Development of Monoclonal Antibodies Advanced Drug Delivery Reviews 2006, 58: 707– 722 Developability Assessment During the Selection of Novel Therapeutic Antibodies. J. Pharm Sci. 2015 104:1885-98
3
Post-translational modifications and Forced Degradation: • PTMs = Critical Quality Attributes (CQAs), if the impact on protein structure leads to effects on function, biological response, safety and efficacy. • Forced degradation can be utilized to rapidly assess potential CQAs during development stages, assessment of stability indicating analytical methods and in later phases comparability assessments.
PTMs = Oxidized Variants = CQAs • Met oxidation can decrease bioactivity and stability and affect serum half-life, can also affect potency • Trp oxidation in the CDR in most cases has been shown to correlate with activity loss • Oxidized Variants can occur during purification, formulation, storage or any step of process development
Characterization of Therapeutic Antibodies and Related Products. Anal. Chem. 2013, 85: 715– 736 Slide 4 www.aaps.org/nationalbiotech
Outline • Selection of Reagents for Trp and Met oxidation • Role of solvent exposure • Selective oxidation of IgG1 and IgG4: case studies • Computational modeling: Trp liabilities and solvent accessibility
5
CDR liabilities: Tryptophan and Methionine in Antibody Fab Domains Exposed Trp residues are more common in antibody CDRs and more solvent accessible than Met
Jarasch A., Koll H. et al., (2015) Developability Assessment During the Selection of Novel Therapeutic Antibodies. J Pharm Sci. 104(6):1885-98
6
Fab (CDR) and Fc (CH2 and CH3) Liabilities in mAbs mAb-A Trp(1a)XXXXTrp(2a) CDR3 (HC) mAb-B Trp(1b)XXXXXXTrp(2b) CDR3 (HC) mAb-D
Trp(1d)XXXXXXXXTrp(2d) CDR3 (HC)
mAb-C Trp(1c) CDR1 (HC) (underlined part of CDR3) The Fc fragment of IgG1 and IgG4 molecules contains two conserved Met residues at positions 252 CH2 domain and 428 CH3 domain, (EU numbering) that are highly susceptible to oxidation.
7
Reactivity of Tryptophan Residues in Proteins (mAbs) Reactivity of residues to photo oxidation is related to surface exposure photosensitization and/or the reaction of Reactive Oxygen Species (ROS).
Sreedhara A., et al., Mol. Pharmaceutics 10 (2013): 278-288 Duenas et al. Pharm. Res. 18 ﴾2001﴿: 1455-1460. Lam et al. Pharm. Res. (2011):2543–2555 Kerwin Bruce et al. Jour. Pharma. Sciences 6 (2007):1468-1479 8
Favored Reagents and Source of ROS Oxidation of Met
methionine
Oxidation of trp: 2,2'-Azobis-2-Methyl-Propanimidamide, Dihydrochloride (AAPH)
met sulfoxide (+16)
Junyan A. JI, et al., . (2009) Methionine, Tryptophan, and Histidine Oxidation in a Model Protein, PTH: Mechanisms and Stabilization. Jour. of Pharma. Sciences, (98) 4485-4500 CasbeerErik et al., (2013) Kinetics and Mechanism of Oxidation of Tryptophan by Ferrate(VI) Environ. Sci. Technol. (47) 4572−4580 Thomas H, Andres et. al (2013) Tryptophan oxidation photosensitized by pterin) Free Radical Biologyand Medicine (63) 467–475 S. Jovanović, I. Čudina, Lj. Josimović., (1977) Gamma radiolysis of oxygenated aqueous solutions of tryptophan. Radiation Physics and Chemistry (22) 765–770
9
Case Study 1: mAb-A (IgG1) - Trp(1a) (HC CDR3) Met (Fc CH2) and Trp1a are Primarily Oxidized by AAPH (60 fold excess 2.0 mM ) Reduced Peptide Mapping (RPM) percent oxidation Stress condition
Modification
Heavy Chain
Met CH2 Trp1a
control
2hr AAPH abundance (%) 10.5 10.8
7.2 2.7
6hr AAPH 39.4 25.9
Trp1a unmodified peptide
Extracted Ion Chromatogram (EIC) Trp oxidation products
Trp1a control 6 hrs AAPH
or
Met CH2 Control 6 hrs AAPH
Methionine sulfoxide peptide
Met CH2 unmodified peptide
10
Mixed Mode Analysis of AAPH Oxidation of an IgG1 (pre-peaks oxidized variants)
20 mM Citrate Buffer neat
Molar Mass vs. time
time (40ºC) main peak purity % 6 hrs 86.7 2 mM AAPH 2 hrs 61.2 2 mM AAPH 6 hrs 29.9
Control sample main peak percent not affected by 6 hrs 40ºC incubation
aTIGIT#2[Aug 18th] LS
1.0x10 1.0x10 1.0x10
Molar Mass (g/mol)
mAb-A (5 mg/mL) Control 2 mM AAPH 2 mM AAPH
1.0x10 1.0x10
8
MW ~ 144 kDa (monomeric)
7
6
5
4
1000.0
Sepax Technologies: Zenix column SEC-300 PBS + 600 mM NaCL
100.0 10.0 1.0 15.0
20.0
25.0 time (min)
30.0
35.0
11
Mixed Mode Analysis of AAPH Oxidation: Effect of (Met and Trp) on chromatography profile
mAU
200.00 100.00
mAb-A
Main peak% 86.7
(pre-peaks oxidized variants)
Control 40ºC 6 hrs
mAU
0.00
50.00
mAb-A
2 mM AAPH 5.0 mM met 40ºC 2 hrs
mAb-A
2 mM AAPH 5.0 mM met 40ºC 6 hrs
41.0
mAb-A
2 mM AAPH 2.5 mM trp 40ºC 2 hrs
82.5
mAb-A
2 mM AAPH 2.5 mM trp 40ºC 6 hrs
77.5
mAb-A
2 mM AAPH 5 mM trp 40ºC 6 hrs
72.4
0.00
mAU
60.00 40.00 20.00 0.00
mAU
100.00 50.00 0.00
mAU
100.00 50.00 0.00
mAU
100.00
86.0
50.00 0.00 0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
Minutes
12
Reduced Peptide Mapping Analysis of AAPH Oxidation in the presence of Met and Trp Selective protection suggest AAPH utilizes different mechanisms for met and trp oxidation » Free –Met suppresses Fc Met oxidation » Free-Trp suppresses Trp1a oxidation (RPM) % oxidation
stress condition chain
control modification Met CH2 Trp1a
HC
7.2 2.7
2hr AAPH
10.5 10.8
6hr AAPH
5mM Met 2hr
39.4 25.9
Trp1a AAPH 6 hrs 5 mM met Trp oxidation products
5mM Met 6hr
abundance (%) 6.7 7.5 9.5 24.4
2.5mM Trp 2hr
2.5mM Trp 6hr
13.1 2.8
27.5 3.8
Trp1a unmodified peptide
EIC AAPH 6 hrs 2.5 mM trp
13
Case Study 2: mAb-B (IgG4) - Trp(1b) (HC CDR3) Reversed-phase HPLC with limited proteolysis: AAPH Oxidation of an IgG4 (36 fold AAPH up to 24 hours)
Fc
25.0 mg/mL Control 40ºC 24 hrs
Fab *
25.0 mg/mL 6 mM AAPH 2 hrs *
*
25.0 mg/mL 6 mM AAPH 6 hrs * 0.00
12.00
24.00
36.00
48.00
60.00
Minutes
Example chromatograph: method under development/optimization
14
Reduced Peptide Mapping Met (Fc CH2) and Trp1b are Primarily Oxidized by AAPH: Partial protection by (Met and Trp) = percent oxidation RPM stress condition chain HC
EIC
control modification Met CH2 Trp1b
6hr AAPH
3.1 2.4
24hr AAPH
21.6 46.6
5mM Met 6hr
5mM Met 24hr
abundance (%) 5.6 12.7 43.0 91.5
66.6 90.7
5mM Trp 6hr
22.1 9.0
5mM Trp 24hr
50.0 19.5
15
Trp1b AAPH 24h
Trp1b unmodified peptide
+4Da
Trp1b AAPH 24h+ 5 mM Met
+16Da
Met/Trp1b +32/16Da
+16Da +32Da +16Da
+32Da
Trp1b unmodified peptide
Extracted Ion Chromatogram
+16Da
Trp1b unmodified peptide
Trp1b AAPH 24h + 5 mM Trp +16Da likely Met
For mAb-B: partial protection is observed, Higher oxidation levels for Trp1b vs Met CH2
Proposed Mechanism for Selective Oxidation by AAPH - Involvement of different reactive intermediates originating from same species
free trp Inhibits reaction of alkyl peroxide with CDR3 Trp
free met inhibits AAPH peroxide reaction with Fc Met
16
Selective Oxidation of Tryptophan and Methionine
Junyan A. JI, boyan Zhang, Wilson Cheng Y., John Wang. (2009) Methionine, Tryptophan, and Histidine Oxidation in a Model Protein, PTH: Mechanisms and Stabilization. Journal of Pharmaceutical Sciences, (98) 4485-4500 Folzer E., Diepold K., Bomans K., Koulov AV, et al., (2015) Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule. Journal of Pharmaceutical Sciences, 104(9)2824-2831
17
Case Study 3: mAb-C (IgG1) – Trp1c (HC CDR1) Oxidation with AAPH: yields Fc Met oxidation, only (36 fold excess for up to 24 hours)
RPM stress condition chain HC
W
control modification Met CH2 Trp1c
3.4 Not detected
6hr AAPH abundance (%) 29.3 Not detected
24hr AAPH 88.1 Not detected M M
Role of solvent exposure and Trp oxidation
18
Computational Modeling of fragment antigen-binding (Fab) of mAb-A, mAb-B • Trp1a and Trp1b are surface exposed as measured by accessible surface area, calculated using a homology model • Other Trp residues in Fv domain are much less surface exposed, as indicated by a small accessible surface area Selective Residues
H3
mAb (chain) mAb-A LC mAb-A HC mAb-A HC mAb-B HC mAb-B HC mAb-B HC
Residue TRP TRP TRP TRP TRP TRP
UID A 1a 2a 1b 2b B
Trp1a
ASA(A^2) 10.96 120.05 10.25 85.25 30.75 21.15
H3
(%) 3.52 38.60 3.30 27.41 9.88 6.80 Trp1b
LC HC
LC
HC
Computational Modeling (Fab) of mAb-C • Trp1c displays good level of exposure to solvent, but showed no oxidation H2
H1
All Trp residues in mAb-C Fab
Trp1c
H3 LC HC
mAb-C LC LC HC HC HC HC HC
Residue TRP TRP TRP TRP TRP TRP TRP
UID A B 1c C D E F
(A^2) 0 7.30 53.63 0 0 27.97 1.74
(%) 0 2.35 17.24 0 0 8.99 0.56
• Solvent accessibility is required for oxidation • However, exposure to solvent does not necessarily equate to higher oxidation levels (Trp1a and Trp1b) • Other structural elements likely play a role
Computational modeling of the fragment antigen-binding (Fab) mAb-D (selective oxidation data not available) •
The structural mode predicts two Trp residues, Trp1d and Trp1d(framework), are solvent exposed
•
Subsequent preliminary data suggest (light stress) both residues are oxidized
mAb D LC LC LC HC HC HC HC HC HC HC
Residue TRP TRP TRP TRP TRP TRP TRP TRP TRP TRP
UID A B C
Trp1d(framework) D E F Trp1d
Trp(2d) G
(A^2) 0 30.67 73.12 118.26 0 0 0 243.93 15.2 82.85
(%) 0 9.86 23.51 38.02 0 0 0 78.42 4.89 26.64
Conclusions Selective oxidation can be achieved by utilizing free Met or Trp in the stress conditions. This observation strongly suggests the presence of two different reactive intermediates generated from the AAPH alkylperoxide radical. The selective oxidation system coupled with RPM-mass spectrometry and analytical chromatography methods can be used to identify and monitor individually potential CQAs in therapeutic monoclonal antibodies. Surface-exposed tryptophan residues in the CDR is a prerequisite for the reactivity of this amino acid with reactive oxygen species Computational modeling, coupled to historical data can be a powerful tool in studying PCQAs in therapeutic monoclonal antibodies.
Questions and Contact Information Jorge Alexander Pavon PhD Associate Principal Scientist Forced Degradation & Impurity Profiling 2015 Galloping Hill Road K-15 B428C Kenilworth, NJ 07033 (908)740-6886 [email protected]
Slide 23 www.aaps.org/nationalbiotech
Acknowledgements
Yan-Hui Liu Li Xiao Danielle Aldredge Alex Fridman Eugene Dank Umesh Kishnani Jia Zha Peter Salmon
24
