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Etanercept case study
Ph. Eur. Training Session on Biologicals Workshop 1 (Biotherapeutic products)
Dr Mihaela Buda
European Pharmacopoeia Department European Directorate for the Quality of Medicines & HealthCare
Mihaela BUDA©2017 EDQM, Council of Europe. All rights reserved. 1
Outline of presentation
2 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
Monograph specifications
Reference standard(s) for physico-chemical testing
Reference preparation for bioassay calibration
Etanercept ‒ Monograph elaboration
3 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
Expert group composed of regulators from Ph. Eur. Member states
Single-source approach (collaboration with Innovator)
Public consultation Pharmeuropa
28.2
(Q2/2016)
Monograph adoption Ph. Eur.
Commission Nov. 2016
P4 procedure (P4Bio pilot
phase)
934 amino acids: 467 amino acids/monomer (C2224H3472N618O701S36); 235 amino acids form the extracellular portion of sTNFR2, the remaining 232 amino acids form the Fc domain of human IgG1
S-S bridges
Glycosylation (covers 1/3 of the whole molecular weight):
N-glycosylation in the Fc’s CH2 region,
N-linked and O-linked glycosylation in sTNFR2 region
Calculated mass: approx. 150 kDa
4 Mihaela BUDA ©2016 EDQM, Council of Europe. All rights reserved.
Etanercept
• Dimeric fusion protein consisting of two soluble p75 TNFR molecules (sTNFR2) fused to the Fc fragment of human IgG1. The Fc component of etanercept contains the CH2 domain, the CH3 domain and the hinge region, but not the CH1 domain of IgG1.
• Produced in mammalian cells by recombinant DNA technology
5 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
Etanercept (2895) monograph
DEFINITION
…..
glycosylated protein with multiple N- and O-linked glycosylation sites;
full occupancy of N-linked glycans at N149, N171 and N317;
Content (milligrams of protein per
millilitre): as approved by the competent authority.
Potency: 1.0 × 106 to 2.9 × 106 IU
per milligram of protein. C2224H3472N618O701S36 (monomer)
Mr (monomer without glycosylation) approx. 51 200
ASSAY
Protein (UV determination) Potency (apoptosis assay)
6 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
Etanercept (2895) monograph
Infliximab B Infliximab A
IDENTIFICATION
A. Assay/Potency B. Peptide mapping
PRODUCTION
Host-cell-derived proteins Host-cell and vector-derived DNA N-Glycan analysis
TESTS
pH Sialic acid
Related proteins (HIC) Impurities with molecular
masses greater than that of etanercept (SEC)
Impurities with molecular masses differing than that of
etanercept (SDS-PAGE)
ASSAY
Protein (UV determination) Potency
METHOD. Ph. Eur. General chapter Peptide mapping (2.2.55)
SYSTEM SUITABILITY. Reference solution (‘Etanercept CRS´)
ACCEPTANCE CRITERIA
Specific procedure, detailed instructions:
• Selective cleavage of the peptide bonds:
‒ Reduction and alkylation; ‒ Digestion
• Chromatographic separation: Liquid chromatography (2.2.29)
Etanercept (2895) monograph
IDENTIFICATION. B. Peptide mapping
7 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
The chromatogram obtained with the reference solution is qualitatively similar to the chromatogram supplied with ‘etanercept CRS’.
The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution.
Etanercept (2895) monograph
IDENTIFICATION. B. Peptide mapping
8 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
the purpose of the test is not to the identification of the peptide fragments and complete elucidation of the primary structure, but to establish identity and provide direct evidence of the sequence based on a specific fingerprint.
13 peaks/peptide fragments (marker peaks) present in each chromatogram
9 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
PRODUCTION section
Infliximab B Infliximab A
PRODUCTION
Host-cell-derived proteins Host-cell and vector-derived DNA N-Glycan analysis
Statements under the heading Production draw attention to particular aspects of the manufacturing process but are not necessarily comprehensive. They constitute mandatory requirements for manufacturers, unless otherwise stated.
They may relate, for example, to source materials; to the manufacturing process itself and its validation and control; to in-process testing; or to testing that is to be carried out by the manufacturer on the final article, either on selected batches or on each batch prior to release. These statements cannot necessarily be verified on a sample of the final article by an independent analyst. The competent authority may establish that the instructions have been followed, for example, by examination of data received from the manufacturer, by inspection of manufacture or by testing appropriate samples.
Ph. Eur. General Notices Etanercept monograph
10 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
PRODUCTION ‒ Glycosylation
O-Glycosylation:
Part of extended characterisation
All O-linked oligosaccharides expected to be either singly or doubly sialylated.
Determination of total sialic acid (TESTS) directly related to the amount of O-glycosylation.
TNF-ɑ receptor
Fc
Monograph specifications:
During the course of product development, it must be demonstrated that the manufacturing process consistently produces a product with the expected O-glycan occupancy using a suitably qualified assay.
11 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
PRODUCTION ‒ Glycosylation
N-Glycan analysis
METHOD
• Ph. Eur. general chapter Glycan analysis of glycoproteins (2.2.59)
‒ Release of glycans
‒ Labelling of released glycans
‒ LC analysis (fluorescence detection)
• Specific procedure ‘given as an example’
• Identification of peaks (2 groups): based on chromatogram in the monograph.
Neutral N-glycans
Sialylated N-glycans
12 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
PRODUCTION ‒ Glycosylation
N-Glycan analysis ‒ monograph specifications:
SYSTEM SUITABILITY: Reference solution (a) (‘etanercept CRS’)
ACCEPTANCE CRITERIA: Reference solution (b) (in-house reference preparation)
‒ RS chromatogram is qualitatively similar to the chromatogram supplied with ‘etanercept CRS’;
‒ peaks 1 to 9 are clearly visible.
‒ the chromatogram obtained with the test solution consistent with the chromatogram obtained with reference solution (b);
‒ requirements for ‘retention times’ and ‘additional peaks’;
Limits: % neutral and sialylated N-glycans: ‘as authorised by the competent authority’.
Due to complexity and the link between DS quality and manufacturing process, tests that measure process dependent heterogeneity are mainly seen as a demonstration of production consistency.
These tests cannot be included in the TESTS section of the monograph as a direct transfer of the lot-release specifications set.
Glycan analysis is included in the PRODUCTION section (Ph. Eur. General Notices), as it cannot be performed by an independent analyst:
the glycan profile depends on the manufacturing process;
the test prescribe the use of an in-house reference preparation and this material is available only to the manufacturer;
the user needs acceptance criteria in form of numerical limits, which are not prescribed in the monograph;
given the variability of the glycan profile associated with process changes, acceptance criteria in form of “one-fit-all” numerical limits may not be suitable and have to be set by the manufacturer in agreement with the competent authority
13 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
Etanercept (2895) monograph
PRODUCTION. Glycosylation
• Ph. Eur. General chapter Glycan analysis of glycoproteins (2.2.59)
• Specific method for release of SA, labelling of released SA and LC analysis
• SST: visual comparison of chromatograms, repeatability (‘etanercept CRS’), linearity
of standard curve (NANA)
Sialic acid
• Ph. Eur. General chapter Liquid chromatography (2.2.29) ‒ HIC, specific chromatographic conditions
• Relative retentions (for information only)
• SST: visual comparison of chromatograms, criteria related to peak separation (p/v) (‘etanercept CRS’)
Related proteins
• Ph. Eur. General chapter Liquid chromatography (2.2.29) ‒ SEC, specific chromatographic conditions
• Relative retentions (for information only)
• SST: visual comparison of chromatograms, criteria related to column performance (N), peak separation (Rs) (‘etanercept CRS’)
Impurities with molecular masses greater than that of etanercept
Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved. 14
Etanercept (2895) monograph ‒ TESTS
sialic acid (SA)
peak 2
aggregates
HMW species monomer
Limit: 8-19 moles sialic acid/mole of etanercept
p/v p/v
peak 1 peak 3
Limits: peak 1: ≤ 5%; peak 3: ≤ 28%; Σ all peaks other than peak 2: ≤ 30% Rs
Limit: Σ all peaks eluted before main peak: ≤ 8%
ASSAY
Protein (UV determination) Potency (apoptosis assay)
15 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
Etanercept (2895) monograph
Infliximab B Infliximab A
IDENTIFICATION
A. Assay/Potency B. Peptide mapping
PRODUCTION
Host-cell-derived proteins Host-cell and vector-derived DNA N-Glycan analysis
TESTS
pH Sialic acid
Related proteins (HIC) Impurities with molecular
masses greater than that of etanercept (SEC)
Impurities with molecular masses differing than that of
etanercept (SDS-PAGE)
ASSAY
Protein (UV determination) Potency
The following procedure has been found suitable: apoptosis-based assay based on ability of etanercept to induce apoptosis in histiocytic lymphoma cell-line U937 via caspase activation.
Reference solution: ‘etanercept BRP’
Preparation of assay medium, test/ref., TNF-ɑ working solutions: indications given as an example.
Result: estimated potency: 80-140% relative to the reference solution; confidence limits (P = 0.95): 80-125% of the estimated potency.
Ph. Eur. General chapter Total protein (2.5.33, Method 1); UV absorbance at 280 nm
Reference solution: ‘etanercept CRS’
Protein concentration calculated taking into account the assigned content of ‘etanercept CRS’
Protein (UV determination)
16 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
Etanercept (2895) monograph ‒ ASSAY
Potency (apoptosis assay)
Outline of presentation
17 Mihaela BUDA ©2017 EDQM, Council of Europe. All rights reserved.
Monograph specifications
Reference standard(s) for physico-chemical testing
Reference preparation for bioassay calibration
Etanercept Case Study
Reference standard for physico-chemical testing
Dr Sylvie JORAJURIA Head of the Biology Section – Laboratory Department
EDQM – Council of Europe
1
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
European Pharmacopoeia Training Session on Biologicals
Workshop 1 (Biotherapeutic products)
Etanercept monograph
2
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
7 tests involve use of a CRS
4 monograph sections
2 types of purposes
1 CRS: Main substance
N-glycan analysis Production
Qualitative:
- for method evaluation -> System suitability
- for sample
compliance testing ->Results
Etanercept CRS
Peptide mapping Identification
Sialic acid
Tests
Related proteins (HIC)
Impurities with molecular masses greater than that of etanercept (SEC)
Impurities with molecular masses differing from that of etanercept (SDS-PAGE)
Protein content Assay Quantitative
3
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
System suitabilty?
Etanercept CRS
Yes
Protein content
Related proteins
Sialic acid Peptide mapping
Impurities (SDS-PAGE)
Aggregates (SEC)
N-glycan
Qualitative use
Quantitative use
Results? Chromato in leaflet
Resolution, p/v?
Resolution? Chromato in leaflet
Chromato in leaflet
Assigned content?
Yes No
No
Yes
Yes
Yes No
No Yes Yes
Yes
Yes
No
Mapping of Etanercept CRS use
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
System suitabilty?
Etanercept CRS
Yes
N-glycan
Qualitative use
Results? Chromato in leaflet
Resolution?
No
No
Yes
System suitability: The chromatogram obtained with etanercept CRS is qualitatively similar to the chromatogram supplied with etanercept CRS
CRS is used to assess of the validity of the analytical setting
5
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
System suitabilty?
Etanercept CRS
Yes
Aggregates (SEC)
Qualitative use
Results? Chromato in leaflet
Resolution?
No
Yes
Yes
System suitability: • The chromatogram obtained with
etanercept CRS is qualitatively similar to the chromatogram supplied with etanercept CRS
• resolution: minimum 1.7 between the peaks due to the high molecular weight species and etanercept monomer
CRS is used to assess of the validity of the analytical setting
6
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
System suitabilty?
Etanercept CRS
Yes
Impurities (SDS-PAGE)
Qualitative use
Results? Resolution,
p/v?
Chromato in leaflet
Yes
No
No
System suitability: The bands in the electropherogram obtained with etanercept CRS are clearly visible Results:
the electropherogram obtained with the test solution is similar to the electropherogram obtained with etanercept CRS
CRS is used as a benchmark to produce batch data
CRS is used to assess of the validity of the analytical setting
7
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
System suitabilty?
Etanercept CRS
Yes
Peptide mapping
Qualitative use
Results? Resolution,
p/v?
Chromato in leaflet
Yes
No
Yes
Sialic acid
Yes
Peptide mapping System suitability: the chromatogram obtained with etanercept CRS is qualitatively similar to the chromatogram supplied with etanercept CRS Results: The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with etanercept CRS
Sialic acid System suitability: The sialic acid peak in the chromatogram obtained with etanercept CRS is visible and is similar to the corresponding peak in the chromatogram supplied with etanercept CRS Results: The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with etanercept CRS
8
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
System suitabilty?
Etanercept CRS
Yes
Related proteins
Qualitative use
Results? Resolution,
p/v?
Chromato in leaflet
Yes
Yes
Yes
System suitability: • the chromatogram
obtained with etanercept CRS is similar to the chromatogram supplied with etanercept CRS
• peak-to-valley
ratio: ≥ 1.1 between peak1 and peak2; ≥ 1.9 between peak 3 and peak 2
Results: No additional peaks are observed in the chromatogram obtained with the test solution in comparison with the chromatogram obtained with etanercept CRS
9
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
Etanercept CRS
Protein content
Qualitative use
Quantitative use
Assigned content?
Yes
No
Calculate the protein concentration of etanercept taking into account the assigned content of etanercept CRS
Any value assigned to a reference standard is valid for the intended use and not necessarily for other uses [Ph.Eur. Chapter 5.12.]
10
Sylvie Jorajuria©2017 EDQM, Council of Europe. All rights reserved.
System suitabilty?
Etanercept CRS
Yes
Protein content
Related proteins
Sialic acid Peptide mapping
Impurities (SDS-PAGE)
Aggregates (SEC)
N-glycan
Qualitative use
Quantitative use
Results? Chromato in leaflet
Resolution, p/v?
Resolution? Chromato in leaflet
Chromato in leaflet
Assigned content?
Yes No
No
Yes
Yes
Yes No
No Yes Yes
Yes
Yes
No
_________________
Etanercept BRP
BSP138
M-E. Behr-Gross, DBO
BSP project to establish Etanercept BRP
BSP138
Project leader Dr. Meenu Wadhwa (NIBSC, UK)
Principle: joint WHO & EDQM (P4BIO/BSP) project
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved. 2
BRP establishment study context
WHO study AIMS
Assess suitability of ampouled preparations of human TNF Receptor type II Fusion protein (TNFR II-Fc, Etanercept) to serve as 1st WHO IS for the bioassay of human TNFR II-Fc by assaying their biological activity in a range of bioassays.
Determine relative activity of the ampouled preparations in different assays in current use for these materials and concentrations of TNFR II-Fc required to neutralise specific amounts of TNF- α IS.
Compare the ampouled preparations with characterised 'in-house' laboratory standards where these are available
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
BSP138 Study aims
BSP138 was a pilot project run under the joint aegis of EDQM programs P4 BIO and BSP and also as part of a WHO IS establishment study
• to provide additional information on the proposed bioassay that will be included in the Ph. Eur. monograph
• to establish Etanercept BRP batch 1 for the Ph.Eur. bioassay
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved. 4
WHO/BSP Study materials and methods
Materials Etanercept candidates WHO IS/BRP: samples from 3 preparations coded by letter A to D containing each 5 micrograms (5μg) TNFRII Fc fusion protein, freeze-dried
TNFa WHO 3rd IS (12/154) 43000 IU of TNF-a/ampoule
Methods Cytotoxicity, apoptosis (cytotoxicity neutralisation TNF sensitive cell based), reporter gene and binding assays
Participants Design 28 laboratories 3 ind. assays/ participants
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
WHO/BSP Study materials
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
Ampoule
code
Fill
date
Study
code
No Of
Ampoules
in Stock
Protein
(Predicted
Mass -
mg)
Protein
Expression
System
Excipients
13/192
10/10/13
A, D
4,500
5
TNFR II-Fc
CHO
1% Sucrose
4% Mannitol
10mM Tris HCL
0.2% Human Serum
Albumin
13/204
24/10/13
B
4,700
5
13/260
20/03/14
C
6,601
5
BSP/WHO study results
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
a a
BSP/WHO study summary results
(relative potency estimates/A)
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
Assay Sample GM 95% Confidence Limits Between-lab GCV (%) n
U937 B 0.93 0.90 0.97 5.5 12
C 0.93 0.91 0.96 4.7 12
D 1.00 0.96 1.04 6.8 12
L929 B 0.93 0.88 0.97 8.0 12
C 0.95 0.89 1.01 10.4 12
D 1.01 0.95 1.08 10.4 12
Other cytotoxicity B 0.93 0.78 1.10 14.5 5
C 0.95 0.87 1.03 7.4 5
D 0.99 0.89 1.09 8.7 5
Reporter Gene B 0.93 . . . 2
C 0.94 . . . 2
D 1.03 . . . 2
Binding B 0.91 . . . 2
C 0.89 . . . 2
D 0.96 . . . 2
Potency (all
assays)
B
C
D
0.93
0.94
1.00
0.90
0.92
0.97
0.95
0.96
1.03
7.8
7.6
8.3
33
33
33
Potency
(excluding binding
assays)
B
C
D
0.93
0.94
1.00
0.90
0.92
0.98
0.96
0.97
1.03
7.9
7.4
8.2
31
31
31
BSP/WHO study results
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
Box-plot summary of individual assay potency estimates relative to
sample A
BSP/WHO study results
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
Methods
Equivalence between methods except for ELISA (2 labs only, results not included)
Apoptosis (U937)
Cytotoxivity (L929 or other)
Reporter gene assay
BSP/WHO study results
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved
Final report submitted to ECBS in October 2015
BSP/WHO conclusions
13/204 was adopted as the first International Standard for TNF receptor Fc fusion protein (Etanercept) with an assigned in vitro bioactivity of 10,000 IU per ampoule
(13/192 could serve as replacement batch)
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
WHO/BSP Study results for proposed Ph. Eur. method
Method Apoptosis assay using the TNF-sensitive U937 line
Principles The assay is based on the inhibitory action of TNFR II-Fc on the biological activity of TNF-a.
Results - Geometric mean potencies relative to A with 95% CL
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.
BSP138 conclusions
The Etanercept candidate batch, established as the first International Standard for Etanercept
(13/204 sample A) will be proposed to Ph. Eur. Commission for adoption as Etanercept BRP batch 1 for use in the assay of Etanercept according to the
prescription of the monograph 2895 with an assigned potency of 10,000 IU per ampoule
Behr-Gross©2017 EDQM, Council of Europe. All rights reserved.