The Impact of Forced Degradation Mechanisms of
The Impact of Forced Degradation Mechanisms of Proteins on the Development of Biosimilars 13 November 2017
Olivier Mozziconacci
Slide 1
#AAPS2017
Session Description and Objectives What to expect during this presentation:
Our objectives:
• A focus on the most relevant degradation products which allow for the differentiation of example of biosimilars
• Overview of protein degradation mechanisms • Why does chemistry matter?
• Assembling the data to get conclusive information
• How do we expand this topic from proteins to biopolymers?
• Applying these techniques to biopolymers
Slide 2
#AAPS2017
Definitions of a biosimilar from regulatory agencies
Agency
Naming
Definition
FDA, USA
Follow-on Biologic or Biosimilar
biological product that is highly similar to a U.S.-licensed […] for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product
Biosimilar
A biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product […] in the EEA. Similarity to the reference medicinal product in terms of quality characteristics, biological activity, safety and efficacy based on a comprehensive comparability exercise needs to be established
Biologic Product
biologic medicine with known biologic activity that contains no new molecules, already licensed in Brazil and that has gone through all the production steps (including formulation, vialing, freeze drying, labeling, packaging, storage, quality control and biologic product lot release)
EMA, EU
ANVISA, Brazil
Tsuruta, L. R., et al. (2015). "Biosimilars advancements: Moving on to the future." Biotechnology Progress 31(5): 1139-1149.
Slide 3
#AAPS2017
FDA guideline in Quality considerations in demonstrating biosimilarity of a therapeutic protein product to a reference product – Section I. Stability
[…] accelerated and stress stability studies, as well as forced degradation studies, should be used to establish degradation profiles and to provide a direct comparison of the proposed product with the reference product.
These comparative studies should be conducted under multiple stress conditions (e.g., high temperature, freeze thaw, light exposure, and agitation) that can cause incremental product degradation over a defined time period. Results of these studies may reveal product differences that warrant additional evaluations and also identify conditions under which additional controls should be employed in manufacturing and storage. […] Sufficient real time, real condition stability data from the proposed product should be provided to support the proposed shelf life.
Slide 4
#AAPS2017
Protein comparability assessments Chemical stability profiling
Physical stability profiling
Mass spectrometry
Ion Mobility Mass spectrometry
Primary structure ?
Alsenaidy, M. A., et al. (2014). "Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles." Front Pharmacol 5: 39.
Slide 5
#AAPS2017
Deamidation reaction Asparagine
Succinimide intermediate
Aspartic acid
Geiger, T. & Clarke, S. J. Biol. Chem. 262, (1987) 785-794
Consequences of a deamidation reaction: i) adds a negative charge, iii) racemizes the protein
Slide 6
#AAPS2017
The formation of diketopiperazine
Aspartame (aspartyl-phenylalanine methyl ester)
Loss of a molecule of methanol S.S. Leung, D.J.W. Grant, Solid state stability studies of model dipeptides: Aspartame and aspartylphenylalanine, J. Pharm. Sci. 86 (1997) 64–71
Slide 7
#AAPS2017
The formation of diketopiperazine Arginine
Proline
Substance P Pathway #1 U. Kertscher, M. Bienert, E. Krause, N.F. Sepetov, B. Mehlis, Spontaneous chemical degradation of substance P in the solid phase and in solution, Int. J. Pept. Protein Res. 41 (1993) 207–211
cyclo(Lys-Pro) Pathway #2 Slide 8
#AAPS2017
Primary and secondary structures influence the rate of the reactions
Gly-Pro
Phe-Pro
C. Goolcharran, R.T. Borchardt, Kinetic of dikitopiperazine formation using model peptides, J. Pharm. Sci. 87 (1998) 283–288
Slide 9
#AAPS2017
Biosimilars evolution
mAbs Comparative analysis of glycans: A key point in determining the degree of similarity in biosimilars
Highly glycosylated protein: Erythropoietin
Recombinant mammalian proteins Less complex molecules produced in microorganisms
Slide 10
#AAPS2017
A first step toward the relation between conformation and chemical degradation: The case of a stability study High Mannose (10-12 mannoses + 2 Glc-Nac)
GlcNAc (1 N-acetylGlucosamine)
Man5 (5 mannoses)
N297Q Fc mutant (no glycan)
Fc protein
Variable Glycans
Trp277 -> hydroxyglycine (stability study) Deamidation Asn315 (stability study)
Mozziconacci, O., et al. (2016). "Comparative Evaluation of the Chemical Stability of 4 WellDefined Immunoglobulin G1-Fc Glycoforms." J. Pharm. Sci. 105(2): 575-587.
Slide 11
#AAPS2017
Effect of the size of the glycans on the deamidation of Asn 315 during stability study
12 weeks
Mozziconacci, O., et al. (2016). "Comparative Evaluation of the Chemical Stability of 4 WellDefined Immunoglobulin G1-Fc Glycoforms." J. Pharm. Sci. 105(2): 575-587.
Slide 12
#AAPS2017
Change of conformation for pharmacokinetic enhancement
L-Trp
D-Trp
modified to enhance lasting effect
Octreotide
Somatostatin
NOT photostable !
Photostable ! Mozziconacci, O. and C. Schöneich (2014). "Effect of conformation on the photodegradation of Trp- and cystine-containing cyclic peptides: Octreotide and somatostatin." Molecular Pharmaceutics 11(10): 3537-3546.
Slide 13
#AAPS2017
Mechanism
e-
Slide 14
#AAPS2017
Mass spectrometry analysis of the translocation of methyl-indole
Slide 15
#AAPS2017
Effect of the size of the glycans on the transformation of Trp277 into hydroxyglycine
5 weeks Trp277 -> Hydroxyglycine
Formation hydroxyglycine
Further degradation of hydroxyglycine
Mozziconacci, O., et al. (2016). "Comparative Evaluation of the Chemical Stability of 4 WellDefined Immunoglobulin G1-Fc Glycoforms." J. Pharm. Sci. 105(2): 575-587.
Slide 16
#AAPS2017
Are Asn315 and Trp277 useful for a comparability study during a forced stress degradation? Stability
Relevant for stability studies
Trp277
hydroxyglycine
Asn315
Relevant for forced stress studies
Forced degradation fragmentation
deamidation
Trp313
Translocation of methyl-indole
Met428
Methionine sulfoxide
Slide 17
#AAPS2017
4 biosimilars, 4 forced stress studies, 3 modification targets
Man5
Glc NAc
Man12
N297 Q
Met428
control
UV
metal
Ccentered radical
H2O2 Asn315
STRESSES Trp313
Slide 18
#AAPS2017
Deamidation of Asn 315 is not discriminant during forced stress studies Man5
Man12
Glc NAc
N297 Q
Met428
Asn315
Trp313
Slide 19
#AAPS2017
Rate of degradation of tryptic peptides T3, T4, and T9 is inversely proportional to the size of the glycan
T4
T3
T9
Fc-High-Mannose Fc-Man5 Fc-GlcNAc Fc—N297q Slide 20
Rate degradation
Size glycan
Fc-Glycan
Rate of degradation of T3, T4, and T9 tryptic peptides 1075 ions/h 1080 ions/h 1585 ions/h 2800 ions/h #AAPS2017
Beyond proteins, the challenging analysis of complex drug mixtures. The example of crofelemer.
Hewarathna, A., Mozziconacci, O., et al. (2017). "Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs." J. Pharm. Sci.
Slide 21
#AAPS2017
Let’s first separate low and high molecular weight molecules Thiolysis reveals higher content of gallocatechine than catechine
Total Crofelemer
Light fraction Crofelemer
Monomer (%)
25oC
Time (days)
gallocatechine
Heavy fraction Crofelemer
40oC
25oC
Time (days)
Time (days)
Gallocatechine Catechine Epi-Catechine Epi-Gallocatechine
catechine
Hewarathna, A., Mozziconacci, O., et al. (2017). "Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs." J. Pharm. Sci.
Slide 22
#AAPS2017
Mutual Information Scores (MIS) to identify differences in datasets
~ 800,000 data points m/z 607.12, time 2.7 min
Hewarathna, A., Mozziconacci, O., et al. (2017). "Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs." J. Pharm. Sci.
Slide 23
#AAPS2017
What is the structure of the discriminant oxidation product?
Original product of oxidation
Combination of ion-source fragmentation and thiolysis reaction
Slide 24
#AAPS2017
Acknowledgments • Department of Pharmaceutical Chemistry at The University of Kansas • • • • • • • •
Pr. Christian Schöneich Pr. Thomas Tolbert Pr. David Volkin Pr. M. Laird Forrest Pr. C. Russell Middaugh Dr. Sangeeta B Joshi Dr. Asha Hewarathna Dr. Eric J. Deeds
Slide 25
#AAPS2017
Questions
Slide 26
#AAPS2017
Olivier Mozziconacci
Slide 1
#AAPS2017
Session Description and Objectives What to expect during this presentation:
Our objectives:
• A focus on the most relevant degradation products which allow for the differentiation of example of biosimilars
• Overview of protein degradation mechanisms • Why does chemistry matter?
• Assembling the data to get conclusive information
• How do we expand this topic from proteins to biopolymers?
• Applying these techniques to biopolymers
Slide 2
#AAPS2017
Definitions of a biosimilar from regulatory agencies
Agency
Naming
Definition
FDA, USA
Follow-on Biologic or Biosimilar
biological product that is highly similar to a U.S.-licensed […] for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product
Biosimilar
A biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product […] in the EEA. Similarity to the reference medicinal product in terms of quality characteristics, biological activity, safety and efficacy based on a comprehensive comparability exercise needs to be established
Biologic Product
biologic medicine with known biologic activity that contains no new molecules, already licensed in Brazil and that has gone through all the production steps (including formulation, vialing, freeze drying, labeling, packaging, storage, quality control and biologic product lot release)
EMA, EU
ANVISA, Brazil
Tsuruta, L. R., et al. (2015). "Biosimilars advancements: Moving on to the future." Biotechnology Progress 31(5): 1139-1149.
Slide 3
#AAPS2017
FDA guideline in Quality considerations in demonstrating biosimilarity of a therapeutic protein product to a reference product – Section I. Stability
[…] accelerated and stress stability studies, as well as forced degradation studies, should be used to establish degradation profiles and to provide a direct comparison of the proposed product with the reference product.
These comparative studies should be conducted under multiple stress conditions (e.g., high temperature, freeze thaw, light exposure, and agitation) that can cause incremental product degradation over a defined time period. Results of these studies may reveal product differences that warrant additional evaluations and also identify conditions under which additional controls should be employed in manufacturing and storage. […] Sufficient real time, real condition stability data from the proposed product should be provided to support the proposed shelf life.
Slide 4
#AAPS2017
Protein comparability assessments Chemical stability profiling
Physical stability profiling
Mass spectrometry
Ion Mobility Mass spectrometry
Primary structure ?
Alsenaidy, M. A., et al. (2014). "Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles." Front Pharmacol 5: 39.
Slide 5
#AAPS2017
Deamidation reaction Asparagine
Succinimide intermediate
Aspartic acid
Geiger, T. & Clarke, S. J. Biol. Chem. 262, (1987) 785-794
Consequences of a deamidation reaction: i) adds a negative charge, iii) racemizes the protein
Slide 6
#AAPS2017
The formation of diketopiperazine
Aspartame (aspartyl-phenylalanine methyl ester)
Loss of a molecule of methanol S.S. Leung, D.J.W. Grant, Solid state stability studies of model dipeptides: Aspartame and aspartylphenylalanine, J. Pharm. Sci. 86 (1997) 64–71
Slide 7
#AAPS2017
The formation of diketopiperazine Arginine
Proline
Substance P Pathway #1 U. Kertscher, M. Bienert, E. Krause, N.F. Sepetov, B. Mehlis, Spontaneous chemical degradation of substance P in the solid phase and in solution, Int. J. Pept. Protein Res. 41 (1993) 207–211
cyclo(Lys-Pro) Pathway #2 Slide 8
#AAPS2017
Primary and secondary structures influence the rate of the reactions
Gly-Pro
Phe-Pro
C. Goolcharran, R.T. Borchardt, Kinetic of dikitopiperazine formation using model peptides, J. Pharm. Sci. 87 (1998) 283–288
Slide 9
#AAPS2017
Biosimilars evolution
mAbs Comparative analysis of glycans: A key point in determining the degree of similarity in biosimilars
Highly glycosylated protein: Erythropoietin
Recombinant mammalian proteins Less complex molecules produced in microorganisms
Slide 10
#AAPS2017
A first step toward the relation between conformation and chemical degradation: The case of a stability study High Mannose (10-12 mannoses + 2 Glc-Nac)
GlcNAc (1 N-acetylGlucosamine)
Man5 (5 mannoses)
N297Q Fc mutant (no glycan)
Fc protein
Variable Glycans
Trp277 -> hydroxyglycine (stability study) Deamidation Asn315 (stability study)
Mozziconacci, O., et al. (2016). "Comparative Evaluation of the Chemical Stability of 4 WellDefined Immunoglobulin G1-Fc Glycoforms." J. Pharm. Sci. 105(2): 575-587.
Slide 11
#AAPS2017
Effect of the size of the glycans on the deamidation of Asn 315 during stability study
12 weeks
Mozziconacci, O., et al. (2016). "Comparative Evaluation of the Chemical Stability of 4 WellDefined Immunoglobulin G1-Fc Glycoforms." J. Pharm. Sci. 105(2): 575-587.
Slide 12
#AAPS2017
Change of conformation for pharmacokinetic enhancement
L-Trp
D-Trp
modified to enhance lasting effect
Octreotide
Somatostatin
NOT photostable !
Photostable ! Mozziconacci, O. and C. Schöneich (2014). "Effect of conformation on the photodegradation of Trp- and cystine-containing cyclic peptides: Octreotide and somatostatin." Molecular Pharmaceutics 11(10): 3537-3546.
Slide 13
#AAPS2017
Mechanism
e-
Slide 14
#AAPS2017
Mass spectrometry analysis of the translocation of methyl-indole
Slide 15
#AAPS2017
Effect of the size of the glycans on the transformation of Trp277 into hydroxyglycine
5 weeks Trp277 -> Hydroxyglycine
Formation hydroxyglycine
Further degradation of hydroxyglycine
Mozziconacci, O., et al. (2016). "Comparative Evaluation of the Chemical Stability of 4 WellDefined Immunoglobulin G1-Fc Glycoforms." J. Pharm. Sci. 105(2): 575-587.
Slide 16
#AAPS2017
Are Asn315 and Trp277 useful for a comparability study during a forced stress degradation? Stability
Relevant for stability studies
Trp277
hydroxyglycine
Asn315
Relevant for forced stress studies
Forced degradation fragmentation
deamidation
Trp313
Translocation of methyl-indole
Met428
Methionine sulfoxide
Slide 17
#AAPS2017
4 biosimilars, 4 forced stress studies, 3 modification targets
Man5
Glc NAc
Man12
N297 Q
Met428
control
UV
metal
Ccentered radical
H2O2 Asn315
STRESSES Trp313
Slide 18
#AAPS2017
Deamidation of Asn 315 is not discriminant during forced stress studies Man5
Man12
Glc NAc
N297 Q
Met428
Asn315
Trp313
Slide 19
#AAPS2017
Rate of degradation of tryptic peptides T3, T4, and T9 is inversely proportional to the size of the glycan
T4
T3
T9
Fc-High-Mannose Fc-Man5 Fc-GlcNAc Fc—N297q Slide 20
Rate degradation
Size glycan
Fc-Glycan
Rate of degradation of T3, T4, and T9 tryptic peptides 1075 ions/h 1080 ions/h 1585 ions/h 2800 ions/h #AAPS2017
Beyond proteins, the challenging analysis of complex drug mixtures. The example of crofelemer.
Hewarathna, A., Mozziconacci, O., et al. (2017). "Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs." J. Pharm. Sci.
Slide 21
#AAPS2017
Let’s first separate low and high molecular weight molecules Thiolysis reveals higher content of gallocatechine than catechine
Total Crofelemer
Light fraction Crofelemer
Monomer (%)
25oC
Time (days)
gallocatechine
Heavy fraction Crofelemer
40oC
25oC
Time (days)
Time (days)
Gallocatechine Catechine Epi-Catechine Epi-Gallocatechine
catechine
Hewarathna, A., Mozziconacci, O., et al. (2017). "Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs." J. Pharm. Sci.
Slide 22
#AAPS2017
Mutual Information Scores (MIS) to identify differences in datasets
~ 800,000 data points m/z 607.12, time 2.7 min
Hewarathna, A., Mozziconacci, O., et al. (2017). "Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs." J. Pharm. Sci.
Slide 23
#AAPS2017
What is the structure of the discriminant oxidation product?
Original product of oxidation
Combination of ion-source fragmentation and thiolysis reaction
Slide 24
#AAPS2017
Acknowledgments • Department of Pharmaceutical Chemistry at The University of Kansas • • • • • • • •
Pr. Christian Schöneich Pr. Thomas Tolbert Pr. David Volkin Pr. M. Laird Forrest Pr. C. Russell Middaugh Dr. Sangeeta B Joshi Dr. Asha Hewarathna Dr. Eric J. Deeds
Slide 25
#AAPS2017
Questions
Slide 26
#AAPS2017
