Development of the 1st International

Standard for Pegylated Granulocyte

Colony Stimulating Factor (PEG-G-CSF).

Meenu Wadhwa

WHO Reference Standards

for Biologicals

• Analyte often undefined – exerts biological/immunological effect/response

• No established primary method

• A “physical” standard ie ampoule content defines the unit of measurement (potency)

• Assigned a value in arbitrary units (IU)

• Primarily intended for calibration of bioassays by assessing potency of test sample vs reference standard

• Representative of the diverse cohort of products (WHO guidelines)

Role in development of internationally agreed system for measurement

of activity of a biological

WHO International Standard:

Material of Higher Order

WHO Biological International Standards

and Reference Preparations

• Collaborative study: multi-centre; multi-methods

• Value assignment based on consensus results

• Participants comments and approvals on analysis of

results and recommendations

• Review and approval by the Expert Committee on

Biological Standardisation (ECBS). For some materials,

review and approval from appropriate expert/

professional organisations eg International Society for

Thrombosis and Haemostasis (ISTH) prior to ECBS.

• Serve as ‘primary’ standard for calibration of secondary

standards/in-house standards etc

Intended for calibration of potency assays to

achieve consistency and harmonization

Attributes of Primary Reference

Materials

• Definition of unit - Arbitrary unit, relates to effect/response of

the biological

• Similar characteristics and behaviour as test samples - Like

against like

• Minimise assay bias - Multiple method assignment

• Stable

• Long-term use

• Depends on batch size and demand; assuming no

significant degradation

• Replacement standard - Continuity of unit

• Calibrate against preceding standard and value assign

WHO International Standardscode

Erythropoietin, Human Recombinant (3rd WHO IS) 11/170

Follicle Stimulating Hormone, Human, Recombinant for bioassay (1st

WHO IS)

92/642

Follicle Stimulating Hormone and Luteinizing Hormone, Human Urinary,

for bioassay (5th WHO IS)

10/286

Granulocyte Colony Stimulating Factor rDNA (2nd WHO IS) 09/136

Granulocyte Macrophage Colony Stimulating Factor rDNA (1st WHO IS) 88/646

Growth Hormone, Human, Pituitary (1st WHO IS) 80/505

Heparin, Low Molecular Weight (2nd WHO IS) 01/608

Insulin, Human (1st WHO International Standard) 83/500

Interferon alpha 2a rDNA (2nd WHO IS) 95/650

Interferon alpha 2b rDNA (2nd WHO IS) 95/566

Interferon beta rDNA (3rd WHO IS) 00/572

Interferon beta Ser17 rDNA (NIBSC Reference Reagent) 00/574

Interleukin-2 rDNA (2nd WHO IS) 86/504

Interleukin-11 rDNA (WHO Reference Reagent) 92/788

Luteinizing Hormone, Human, recombinant (1st WHO IS) 96/602

Parathyroid Hormone, Human, recombinant (1st WHO IS) 95/646

ICH guideline Q6B:

“The results of biological assay should be expressed in units of activity calibrated

against an international or national reference standard. Where no such

reference standard exists, a characterised in-house reference material

should be established and assay results reported as in-house

units.”

Establishment of NIBSC/WHO

bioassay standards for traditional

biologicals (e.g IFN’s, EPO) is a

well established process.

It presents significant problems

for “next-generation” molecules

Next generation molecules

• Proteins with chemical modifications e.g., polysialylation,

PEGylation, fusion to Ig domains or serum proteins (albumin)

• Likely to be used in the same clinical context and indication as the

parent

• Single manufacturer; labelled in mass (in ‘mg’)

• Unlike the parent, which is likely to have a potency based on the

use of the IS for biological activity, there are no IS for these

products..

• Intentionally modified, unique so cannot be standardised in a

similar way as the parent

Establishment of 1st IS for Peg-G-CSF

Pegylated G-CSF

• 1 licensed product – NEULASTA (INN - Peg-Filgrastim)

• G-CSF (parent protein)

• 20 Kd PEG, Linear form, coupling chemistry known

• Site - N-terminal Methionine of G-CSF.

• Labelling in ‘mass’ units – PFS containing 6 mg (0.6 mls of 10 mg/ml)

• Patent expiry imminent

• In clinical development worldwide:

• Copy’ products including biosimilars – similar size peg, targeting same site

and using same chemistry

• Novel products – peg of different size, form, targeting different site &

possibly using different chemistry (1 approved in 2013 – lipegfilgrastim)

Pegylated G-CSF

Company name, Country Product name Stage of development

Apotex, Canada - Submitted for approval in the US in

December 2014

Dr Reddy’s Laboratories,

India*

Peg-grafeel ‘Similar biologic’ approved in India in May

2010

Intas Biopharmaceuticals,

India*

Neupeg ‘Similar biologic’ approved in India in

August 2007

Gennova

Biopharmaceuticals

(Emcure), India*

Pegex ‘Similar biologic’ approved in India in

January 2010

Lupin, India* Peg-filgrastim ‘Similar biologic’ approved in India in

September 2013

Merck, USA MK-6302 Reported to be in clinical development as of

2011 for neutropenia caused by cancer

chemotherapy

Sandoz, Switzerland LA-EP2006 Two global phase III trials (PROTECT 1 &

2) completed. Trials will be used to support

registration in the European Union and the

US

Biosimilars and non-originator biologicals of pegfilgrastim approved or in development

Reprint with permission. GaBI Online – Generics and Biosimilars Initiative. Biosimilars of pegfilgrastim [www.gabionline.net]. Mol, Belgium:

Pro Pharma Communications International. Available from: www.gabionline.net/Biosimilars/General/Biosimilars-of-pegfilgrastim

• In 2013 - worldwide

sales of US$5.8 billion

• Patent expiry

• Oct 2015 (USA)

• Aug 2017(EU)

Rationale for PEG-G-CSF IS

• As a biological, there is an absolute requirement to demonstrate

biological activity in a validated bioassay.

• An international standard is necessary.

• Some manufacturers using the WHO 2nd IS for G-CSF for measuring the

activity of their PEG-products but the suitability of use of this IS had not

been formally established. Concern regarding the suitability of the G-CSF

IS and demand for a new IS

• Complexities – Pegylated products (size, form, site, coupling chemistries)

PEGylation can variably reduce potency in vitro while increasing activity half-life

in vivo.

• Feasibility Study - Evaluated different Peg-G-CSF preparations (copy/biosimilar)

with the aim of developing a standard for biological activity of pegylated G-CSF

PEG-G-CSF

• Towards a Standard - Should we consider developing a standard?

– Successful pilot fill - suitable formulation identified and stability assessed at 3

months.

– Two suitable candidate rDNA preparations of PEGylated G-CSF were lyophilized

with the intention of assessing these relative to the WHO 2nd IS for G-CSF (09/136)

in a multi-centre international collaborative study

• If the standard is developed, how should the unitage be assigned?

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

1 10 100 1000

cpm

IU/ml

09/136A Batch 1A Batch 2C Batch 1

PEG-G-CSF

• Source/Type Of Material

Two candidates lyophilized; both pegylated at the same site, same size

PEG, same pegylation chemistry.

B and C – coded duplicates

Ampoule

code

Fill

date

Study

code

No Of

Ampoule

s in Stock

Protein Protein

(Predicted

Mass - mg)

Expression

System

Excipients

12/222 1/11/12 B, C 4,700~ PEG-G-CSF 1 E.coli Trehalose

Tween -20

Phenylalanine

Arginine

Human Serum

Albumin

12/188 13/9/12 A 4,700~ PEG-G-CSF 1 E.coli

09/136 2/07/09 2nd IS

G-CSF

3,400~ G-CSF 1 E.coli

Ist IS

PEG-G-CSF• Collaborative study (launched November 2012)

– 23 participants (3 control, 1 pharmacopoeia, 16 manufacturers’ and 2 CRO

laboratories) from 10 countries submitted data

– China, Korea, India, Germany, Croatia, UK, Switzerland, USA, Uruguay,

Argentina

• Aim - Characterize a candidate WHO 1st IS for the bioassay of human

PEG-G-CSF and assign a unitage for in vitro biological activity.

– Assess the suitability of the current G-CSF IS or alternatively a human PEG-

G-CSF candidate as the 1st IS for the bioactivity of human PEG-G-CSF in

various bioassays.

– Assess the activity of the PEG-G-CSF preparations in different bioassays

and calibrate the candidate IS against the 2nd IS for G-CSF if possible.

– Compare the PEG-G-CSF preparations with characterised 'in-house'

laboratory standards of PEG-G-CSF where available.

PEG-G-CSF Standard

• In the Study protocol, participating manufacturers were encouraged to

• Include their own PEG-G-CSF and G-CSF protein (material for PEG

conjugation) in the bioassays.

• Provide data on in-house PEG-G-CSF standards (many)

• Provide information on their own product (many)

– Size of Peg, Form e.g., mono/di, Type of peg e.g., Linear/branched

– Site of Pegylation, Coupling Chemistry & reagents used

• Requested information provided (many)

Assigning the biological unit to

Pegylated G-CSF ?

• How should the unitage be assigned to the standard?

Option 1 - Should it be traceable to the IS for G-CSF?

Option 2 - Should it be assigned independent units?

Option 3 - Assign independent units and indicate relationship with G-CSF IS

• How would the assigned unitage relate to labelling of product (i.e. clinical dose

or units for expressing the potency)?

• Risk : if modified products are considered equivalent to the parent protein when

intended for same indication and given the same unitage as the parent

• Not intended for dosing

• Retrospective standardisation of the innovator product & consequences for

product labelling???

• Not intended for product labelling

Strategy for assigning unitageOpti

on

Question Answer Pros Cons

1 Should the

unitage be

traceable

to the IS

for G-CSF?

Possible

2nd IS for G-CSF

included

Traceability to

be determined

by statistical

analysis of

data. If assays

valid relative to

the IS, units can

be traceable to

G-CSF IS.

Traceable to G-CSF IS

Align with other products if

this approach has been used

Likely to encourage

developers of novel PEG-G-

CSF products to consider

calibration of in-house

reference standards using the

WHO 2nd IS for G-CSF, if

possible

Provides objectivity for

independent testing

Difficult to ensure

similar relationship

between the two

standards and

between PEG-G-

CSF products and

G-CSF IS

Risk of

discontinuity when

G-CSF IS is

replaced

Strategy for assigning unitage

Optio

n Question

Answer Pros Cons

2 Should

the

standard

be

assigned

independ

ent units?

Will be

determined by

statistical

analysis of study

data. If data

relative to G-CSF

IS inappropriate

and gives

statistically

invalid estimates,

independent

units likely to be

assigned.

Usual and Easy approach

No impact in case of

replacement of current G-CSF

IS

Risk of

disconnection with

novel PEG-G-CSF &

other modified G-

CSF products

Potential for

confusion for users

Strategy for assigning unitage

Opti

on

Question Answer Pros Cons

3 Assign

independent

units and

indicate

relationship

with G-CSF

IS

Possible -

Study

includes 2nd IS

for G-CSF.

Will be

determined by

statistical

analysis of

study data as

described

above.

Ideal approach - provides an

independent unitage as well as a

relationship with G-CSF IS (and

consequently a link with the parent

molecule).

May be suitable for novel

PEGylated G-CSF products

Provides a basis for linking novel

PEG-G-CSF and other modified G-

CSF products to the parent

molecule

PEG-G-CSF Study Assays

Several proliferation assays using

• different cell-lines and different readouts mainly colorimetric

although fluorescence and luminescence also used

• NFS-60 –

• murine myelogenous leukemic cell-line induced with the Cas-Br-

MuLV wild mouse ecotropic retrovirus

• 9 labs, MTS/ Cell titer Glo(4 labs), Alamar Blue (1),MTT (4)

• M-NFS-60 –

• variant of NFS-60 produced by culturing in M-CSF

• 12 labs, MTS/ Cell titer Glo(9 labs), Alamar Blue (2),WST-1 (1)

• G-NFS-60 –

• variant of NFS-60 produced by culturing in G-CSF

• 1 lab; thymidine incorporation

Reporter gene assay (proprietary) – 1 lab; luminescence

PEG-G-CSF Study Results

Better parallelism when sample A

used as the standard

2423222120191816151413121110090807060504030201

2.5

2

1.5

1

0.67

0.5

0.4

Lab

Ratio

0.8

1.25

A:CS

With use of G-CSF IS (09/136) as

standard,

• response showed more non–

parallelism. Similar data with B and C.

• steeper slopes for samples A, B and C

evident in some laboratories

Slope ratios for B relative to A

Slope ratios for A relative to G-CSF IS Acceptable parallelism was seen between

all study samples as indicated by the slope

ratios in a majority of laboratories.

Comparison of potency estimates

relative to the 1st G-CSF IS

Peg-G

Vs 1st G-CSF IS Vs 1st G-CSF IS1

Potency estimates

(95% CL) IU/ampoule

Inter-lab

GCV

Potency estimates

(95% CL) IU/ampoule

Inter-lab

GCV

A 0.49 (0.38 – 0.62) 76.0 % 0.35 (0.30 – 0.40) 30.2%

B 0.48 (0.36 – 0.63) 92.6% 0.32 (0.27 – 0.38) 34.8%

C 0.48 (0.37 – 0.63) 83.3% 0.34 (0.29 – 0.40) 34.8%

• 1NFS-60 assays unable to discriminate between G-CSF and Peg-G-CSF.

Data from NFS-60 assays excluded. B & C are coded duplicates

• In most labs, PEG-G-CSF samples lower in potency relative to G-CSF

IS but high inter-laboratory variability seen

Potencies of A,B&C relative to 09/136

Data from NFS-60 assays

LabSample A Sample B Sample C

GM GCV GM GCV GM GCV

2 0.66 14.3 0.71 15.9 0.71 10.4

4 0.33 8.0 0.32 9.9 0.32 8.4

5 1.00 14.4 0.96 10.4 0.95 7.0

7 0.85 22.9 0.90 22.9 0.95 .

8 1.39 19.4 1.28 9.1 1.27 20.9

16 0.36 40.9 0.45 36.0 0.36 44.5

18 1.09 25.2 1.02 37.8 1.00 33.8

21 1.08 63.4 1.30 67.6 1.40 44.3

22 1.33 . 2.15 . 1.44 .

Potency

Estimate Range 0.33-1.39 0.32-2.15 0.32-1.44

Potencies of A,B&C relative to 09/136

Data from MNFS-60 assays

LabSample A Sample B Sample C

GM GCV GM GCV GM GCV

1 0.24 10.0 0.23 5.0 0.23 17.4

9 0.38 27.5 0.31 27.0 0.39 20.4

10 0.26 14.0 0.25 9.8 0.24 13.6

11 0.36 14.4 0.34 18.6 0.35 11.9

12 0.40 10.5 0.38 7.8 0.38 12.8

13 0.27 11.2 0.25 17.2 0.26 13.1

14 0.37 16.6 0.40 21.9 0.39 13.8

15 0.24 7.3 0.22 9.6 0.23 7.9

19 0.41 12.3 0.34 35.4 0.29 3.0

20 0.30 18.7 0.26 23.9 0.28 23.3

23 0.35 44.6 0.26 24.9 0.36 46.3

24 0.58 19.0 0.61 18.9 0.59 15.5

Potency

Estimate Range 0.24-0.58 0.22-0.61 0.23-0.59

Proliferation 0.39 0.36 0.38

Reporter Gene 0.55 0.53 0.61

Comparison of potency estimates

relative to a PEG-G-CSF sample

Peg-G

Vs A Vs A*

Potency estimates

(95% CL) IU/ampoule

Inter-lab

GCV

Potency estimates

(95% CL) IU/ampoule

Inter-lab

GCV

B 0.99 (0.95 – 1.04) 10.1% 0.97 (0.94 – 1.00) 5.7%

C 1.02 (0.99 – 1.06) 8.7% 1.01 (0.98 – 1.04) 5.7%

• B and C are coded duplicates so mean potency is 1.01

• All assays provided comparable data. 1Potencies if data from NFS-60

assays excluded.

Relative Potencies of A, B &C

B relative to A C relative to AC relative to B

(coded duplicates)

Lab GM GCV GM GCV GM GCV

1 0.99 8.1 0.98 16.7 0.99 14.7

2 1.08 11.7 1.07 14.9 1.00 10.8

3 0.97 8.6 1.10 24.1 1.14 16.5

4 0.97 8.6 0.97 7.3 1.00 8.2

5 0.97 6.1 0.99 7.4 1.03 5.5

6 0.96 13.6 1.00 14.5 1.01 12.9

7 0.99 25.4 1.08 28.7 1.14 38.6

8 0.92 31.8 0.86 21.8 1.09 11.9

9 0.99 26.9 1.07 17.9 0.96 35.8

10 0.98 8.6 0.94 12.7 0.96 11.0

11 1.00 9.9 0.99 12.1 0.99 12.3

12 1.00 11.1 1.00 8.7 1.04 8.7

13 0.92 16.0 0.96 8.7 1.05 12.8

14 1.06 13.4 1.10 8.0 1.04 17.0

15 0.89 5.6 0.96 3.2 1.08 4.2

16 1.24 55.4 1.17 13.3 0.94 39.5

18 0.95 22.6 0.96 14.0 1.05 15.9

19 0.90 37.8 1.07 42.4 1.04 48.8

20 0.87 27.0 0.95 22.8 1.08 27.4

21 0.97 28.4 0.98 16.9 0.94 20.4

22 1.33 33.3 1.27 23.2 1.16 .

23 1.03 9.4 1.00 6.7 1.00 11.2

24 0.99 30.9 1.08 14.8 1.09 26.7

PEG-G-CSF

Mean potencies of samples A, B and C

relative to IS for G-CSF (09/136)

Mean potencies of samples B and C

relative to A

PEG-G-CSF IS

Reason for inability of NFS-60 assays to distinguish between parent and

modified form not clear

• Parental line insensitive relative to the variant cell-lines

• Differences in sourcing and maintenance of NFS-60 cells

High variability in data when G-CSF IS used

• Possibly due to the reduced bioactivity in vitro of the Peg-G-CSF vs

parent molecule

Variability due to use of the insoluble formazan dye MTT in some labs

Better agreement when principle of ‘like vs like’ applies

WHO Tech Report Series, no 932, 2006, Annex 2: Recommendations for the

preparation, characterization and establishment of international and other biological

reference standards - the behaviour of the reference standard should resemble as

closely as possible the behaviour of the test samples in the assay used to test them.

The general principle of “like versus like”

PEG-G-CSF: Assigning a

Unitage

Relative to G-CSF IS -

• In most labs, PEG-G-CSF samples lower in potency, high inter-laboratory

variability seen

• In labs using NFS-60 assays, bias towards high potency estimates

• For expressing unitage for sample A, it seemed reasonable to use a mean

potency value of 0.35 (derived by excluding these assays).

• G-CSF IS (09/136) - assigned unitage of 95,000 IU per ampoule, therefore, a

mean potency estimate for sample A is equivalent to 33,250 IU of G-CSF.

Reasonable to assign a unitage independent of G-CSF IS -

• Sample A or B/C – showed comparable data;

• All assays show similar potency estimates; none excluded

• Sample A (coded 12/188) arbitrarily assigned a value of 10, 000 IU/ampoule.

A strategy for assigning a unitage with three options was formulated at the outset.

12/188 established as IS with its own unitage and has no formal

relationship with G-CSF IS

PEG-G-CSF

242322212019181716151413121110987654321

2.5

2

1.5

1

0.67

0.5

0.4

Lab

Ratio

1.25

0.8

Copy/Biosim Peg

Unmodified GCSF

Peg

Novel Peg

Code

IH:A23 participants

1 – PEG-G-CSF (no details) - green

3 – PEG-G-CSF (novel - 12,20,30 kD

attached at a different site) - blue

13 – Peg-G-CSF (1 innovator; 11

copy/biosimilar) - black

2 – G-CSF – red

Others – no IH standard

Slope ratios for in-house standards relative to the Sample A

Acceptable parallelism seen with in-house stds representative of PEG-G-CSF preps;

the within lab variability of potency estimates of the in-house preps vs A is also very low

PEG-G-CSF : Stability Studies

Potencies (%) of samples of 12/188 stored at elevated temperatures over 7 months relative

to the -70˚C sample.

Potency is not diminished after 1 week storage at either 4˚C or 20˚C

following reconstitution or after repeated freeze-thaw cycles.

Storage

temperature

Potency relative to -70˚C

GM 95% CI GCV n

-20˚C 1.03 0.98 – 1.09 11.018

+20˚C 1.03 0.98 – 1.09 10.918

+37˚C 0.99 0.95 – 1.03 8.918

+45˚C 1.05 1.01 – 1.09 8.318

No detectable loss of potency detected even at 45˚C; Not possible

to predict yearly loss for this preparation.

In stability studies, 12/188 was found to be stable

Conclusion and Establishment

• Sample A, code 12/188 suitable to serve as the WHO IS for

in vitro bioactivity of PEG-G-CSF preparations

(representative of the approved product, INN PEG-

Filgrastim).

• It was established by the ECBS as the WHO 1st IS for PEG-

G-CSF with an assigned in vitro bioactivity of 10, 000

IU/ampoule independent of the G-CSF IS.

PEG-G-CSF IS

Statement in IFU to reflect use

• Since 12/188 has only been evaluated for use in in vitro bioassays, it

cannot be assumed to be suitable for evaluation in vivo or for

pharmacokinetic studies without suitable validation. Users of this standard

will need to perform validation studies if using the standard for purposes

other than evaluation of in vitro biological activity.

• The use of the standard for calibrating other PEG-G-CSF preparations

(i.e. produced using PEG of different sizes, forms, or targeted to different

sites using alternative coupling chemistries or conjugation procedures)

will need to be validated by the user as this has not been sufficiently

addressed in the study (WHO/BS/2013.2218).

• The IS (coded 12/188) intended as a reference standard for in vitro

bioactivity of biosimilar / copy PEG-G-CSF products only. Novel products

not considered.

Acknowledgements

•Donors of the material

•Participants

•Project team at NIBSC

Reference -

Wadhwa M, Bird C, Dougall T, Rigsby P, Bristow A, Thorpe

R; participants of the study.

J Immunol Methods. 2015;416:17-28

PEG-G-CSF : Stability

Potencies (%) of samples of 12/188 stored at elevated temperatures over 7 months relative to the

-70˚C sample.

Potencies (%) of freeze-thaw samples relative to fresh samples

For Proposed IS, potency is not

diminished after 1 week storage

at either 4˚C or 20˚C following

reconstitution or after repeated

freeze-thaw cycles.

Therefore, it will be a future

requirement to assess the stability

of PEG-G-CSF in the residual

ampoules in storage at elevated

temperatures

Storage

temperature

Potency relative to -70˚C

GM 95% CI GCV n

-20˚C 1.03 0.98 – 1.09 11.018

+20˚C 1.03 0.98 – 1.09 10.918

+37˚C 0.99 0.95 – 1.03 8.918

+45˚C 1.05 1.01 – 1.09 8.318

Preparation Cycles GM 95% CI GCV n

12/188 1 0.87 0.74 - 1.00 21.3 8

2 1.02 0.75 - 1.44 28.5 5

3 0.98 0.78 - 1.29 32.4 8

4 0.96 0.82 - 1.22 18.7 7

12/222 1 0.93 0.85 - 1.02 17.7 16

2 0.96 0.86 - 1.04 21.1 15

3 1.04 0.95 - 1.12 18.5 16

4 1.05 0.97 - 1.10 16.6 17

No detectable loss of potency detected even at 45˚C; Not

possible to predict yearly loss for this preparation.