Ph. Eur. Training Session on Biologicals Workshop 1 ... · Ph. Eur. Training Session on Biologicals...

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


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


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


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)



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)


IDENTIFICATION. B. Peptide mapping


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.


IDENTIFICATION. B. Peptide mapping


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


PRODUCTION section


PRODUCTION


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


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.



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



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



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)




IDENTIFICATION

A. Assay/Potency B. Peptide mapping

PRODUCTION


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)


Etanercept (2895) monograph ‒ ASSAY

Potency (apoptosis assay)

Outline of presentation


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


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


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


System suitabilty?

Etanercept CRS

Yes

N-glycan

Qualitative use


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


System suitabilty?

Etanercept CRS

Yes

Aggregates (SEC)

Qualitative use


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


6


System suitabilty?

Etanercept CRS

Yes


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


7


System suitabilty?

Etanercept CRS

Yes

Peptide mapping

Qualitative use


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


System suitabilty?

Etanercept CRS

Yes

Related proteins

Qualitative use


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


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


System suitabilty?

Etanercept CRS

Yes

Protein content

Related proteins

Sialic acid Peptide mapping


Aggregates (SEC)

N-glycan

Qualitative use

Quantitative use


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


WHO/BSP Study materials


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


a a

BSP/WHO study summary results

(relative potency estimates/A)


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



Box-plot summary of individual assay potency estimates relative to

sample A



Methods

Equivalence between methods except for ELISA (2 labs only, results not included)

Apoptosis (U937)

Cytotoxivity (L929 or other)

Reporter gene assay


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)


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


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


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