The use of bioassays for establishing the biological potency

of biologicals Robin Thorpe PhD.,FRCPath.

Head, Biotherapeutics Group, NIBSC, UK. email: Robin.Thorpe@nibsc.org

Measuring potency of Biotherapeutics

• Measuring the potency of Biologicals

normally requires the use of a bioassay

• This applies to Biotherapeutics as these

are a sub-group of Biologicals

Bioassay

• A quantitative procedure for measuring

the potency of a biological substance

• Can be carried out in vivo or in vitro

• A functional response is measured and

analysed to provide a statistically

qualified potency estimate

Bioassay

• Bioassays involve the response of a living system to a biological substance

• This distinguishes them in an important way from physico-chemical methods and procedures which simply involve binding, e.g. immunoassays and receptor binding assays

• This affects the results obtained and their significance

Bioassay

In vivo - Whole animal assays

In vitro - Isolated organ assays

- primary cultured cell assays

- Continuously growing cell line

assays

Bioassay

Appropriately designed bioassays reflect

(assess):

(i) ligand-receptor binding

(ii) signal transduction processes

(iii) final (observed) biological effects

Use of bioassays

Substance Bioassays

Viral Vaccines

Bacterial Vaccines

Neutralization of Infectivity

Tumour “Vaccines” Killing of tumour cells

Coagulation Factors Clotting Assays

Fibrinolytics Clot Lysis

(Enzyme Assays)

Immunoglobulins Neutralisation of relevant micro-organisms.

Binding to appropriate antigens

Endocrine hormones In Vivo & In Vitro assays assessing the biological effect of hormone

Cytokines Bioassays which detect biological effects of cytokines (various)

In vivo and in vitro assays for biological

therapeutics Protein In vivo Assay Biological Response

Qualified

In vitro Assay Biological Response Qualified

GM-CSF Bone marrow stimulation Number of leukocytes Stimulation of cell lines: TF-1, MO7e, AML-193

Cellular proliferation

TNF Necrosis of tumours Number of tumours Cytotoxicity on cell lines: L929, KYM-D4

Cell survival

Erythropoietin Stimulate erythrocytes in hypobaric rats

Number of erythrocytes Stimulation of cell line TF-1 Cellular proliferation

Epidermal Growth Factor

Maturation of mouse newborn

Time to eye opening Stimulation of cell lines: 3T3, Balb/MK, 4MBr-5

Cell proliferation

Interferon-alpha Treatment of virally infected rats

Elevation of IFN inducible enzymes or MHC antigens

Antiviral activity on cell lines, e.g.: HEp2, WISH, MDBK

Cell survival

Interleukin-1 Pyrogenicity in rabbits Temperature Stimulation of cell lines: D10, NOB-1, Monomac

Cellular proliferation Production of cytokines

Bone Morphogenetic Protein-2

Bone callous formation at injection site

Size of callous Stimulation of cell line W20 Production of alkaline phosphatase

Growth Hormone Growth of rats Weight Stimulation of cell lines: NB2, 3T3

Cellular proliferation differentiation

Heparin Antithrombotic activity in rabbits

Size of jugular vein thrombi Anticoagulant activity Inhibition of activated clotting factors

Influenza Vaccine Mouse immunogenicity test Levels of anti-flu antibodies Single radial difussion Area of precipitation

Follicle Stimulating Hormone

Steelman-Pohley test on immature female rats

Ovarian weight Stimulation of sertoli cells or granulosa cells

Aromatisation of androgens, levels of cAMP

Choice of assay

• Assay needs to be selected for each biological

• Assay may use primary cells or cell-lines

• Cell-lines -

– continuously growing

– factor dependent, cloned cell-lines

– transfected with an appropriate receptor

• Select a cell-line which yields a functional end-point

response. These can be –

– Early – phosphorylation of intracellular substrates

– Late – cytokine secretion, proliferation, cytotoxic effects etc

– Early/Late (depending on kinetics) – changes in mRNA expression; PCR readout, reporter gene based assa

When are bioassays useful?

During pre-product development:

Research and R & D activities

During product development:

Characterization of biological activity(s) of product(s)

Stability studies

Dosing

Formulation studies

Post-product development:

Batch-to-batch consistency

Stability

Bioassays

• The responses of samples and standards in

the assay must be parallel

• If not, the assay is responding differently to

the molecular species or forms in the

samples/standards

Dose-response plots

Standardization of bioassays

requires biological potency standards

- calibrated in units (e.g. IU)

Standardization of bioassays

Bioassays - Units

• International Units - relate to

potency of ampoule of I.S. contents

Often arbitrary units

Use of one standard world-wide

facilitates comparability of assay

results

Bioassays - Standards

Biological estimates of IL-4

(nominal ng/ampoule)

Same standard ‘In-house’ standards

Bioassay-derived potency data must

be accompanied by a valid statement

of the uncertainty associated with the

measurement

Bioassays

Bioassays must be carefully designed

to minimize and allow assessment of

errors

Bioassays

Problems with bioassays

1. Can be non-specific - respond to several substances, culture additives, LPS etc.

2. Can be variable, tedious and time-consuming (and difficult)

3. Can require long-term maintenance of (factor-dependant) cell lines

4. Can be ‘inhibited’ by a variety of specific and non-specific molecules

5. Cannot distinguish between molecular ‘forms’ of some molecules, e.g. IL-1α and IL-1ß; TNF-α and TNF-ß

Measuring potency of Biotherapeutics

Need to carry out a validated bioassay

for each batch. This must be analysed

to provide:

(i) A mean potency estimate

(ii) Fiducial limits of error for the batch

(usually equivalent to confidence, i.e.

95% limits)

Measuring potency of Biotherapeutics

The mean potency estimate should lie

within specified limits - these limits

should be derived from results of mean

estimate of potency for different batches

and limit the batch-to-batch variation in

potency

Measuring potency of Biotherapeutics

The fiducial limits of error are derived

from the individual potency estimates

(from which the means are derived) -

they are a limit to the assay variability

Example of how statistical limits are

applied to biological assays

Measuring potency of Biotherapeutics

Stated potency

Potency on label. Backed by mean potency

estimate (i.e. this is within limits) + fiducial

limits of error (these are acceptable)

Usually inappropriate to label vials with

individual batch mean potency estimate value

New bioassays in use

Recent bioassays include :

• Kinase receptor activation assays (KIRA) in which

ligand receptor interaction triggers phosphorylation

of a protein

• Bioassays using receptor transfected cells

• Reporter gene assays in which ligand-induced

promoter genes are linked to easily detectable

‘tags’ e.g. enzymes

• Quantitative PCR based assays

N

Cyt 1R Cyt 2R

does not respond to cyt 3 N

Cyt 1R

Cyt 3R

Responds to Cyt 3

TRANSFECTION

Cyt 3R

Principle

Transfection of cells with cytokine receptors

Transfection of non-responsive cells with

cytokine receptors

CT.h4S

mIL-2R mIL-4R

CTLL-2

mIL-2R mIL-4R

32D/Mpl+ 32D

mIL-3R mIL-3R

responds to huTPO

hu TPO R

Ba8.1cl BAF3

mIL-3R mIL-3R

responds to hu IL-10

hu IL-10R

grows in mIL-3

(a) human IL-4R

Transfection

with hu IL-4R

Transfection

with hu TPO R

Transfection

with hu IL-10R

(b) human TPO

(c) human IL-10

m/hu IL-4R

responds to hu IL-4

grows in mIL-3

grows in hu IL-2

A549 Luciferase Reporter Gene Assay

.

0

1000

2000

3000

4000

5000

6000

7000

0.1 1 10 100 1000 10000

IFN ALPHA (pg/ml)

CP

S

IFN ALPHA PRODUCT

MODIFIED IFN ALPHA PRODUCT

IFN-a2

IL-28

IL-29

Dose responses for IFN-α and IFN-l

preparations ( A) and 2 IFN-α preparations

(B) from a reporter gene assay using a cell

line transfected with the promoter of the

MxA gene fused with luciferase. The MxA

gene codes for a type 1 interferon-inducible

protein involved in antiviral activity.

A

B

qPCR assay protocol

Incubate cells in 96-well plate with doses

of test substance and standard

Lyse cells and isolate good quality RNA

Reverse transcribe RNA into cDNA

Measure mRNA expression using real-time qPCR

Data analysis

20 minutes – 1 hour

Approx 1hr

Approx 1hr

Approx 2hr

Liquid-handling robots

Qiacube RNA isolation

VEGF bioassays – IL-8 mRNA in HUVECs

0.0 1.0 2.0 3.9 7.8 15.6 31.3 62.5 125.0 250.0

VEGF (ng/mL)

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

ala

marB

lue r

eduction (

A570nm

-A600nm

)

62.531.315.67.83.92.01.00.50.20.0

VEGF (ng/mL)

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

Tis

sue f

acto

r (U

/mL)

0 0.25 0.5 1 2 4 8 16 32 64 1250

3

6

9

12

15

18

VEGF (ng/mL)

rela

tive IL8 e

xpre

ssio

n

IL-8 mRNA - qPCR

0 10 20 40 80 160 320 640 12800.0

2.5

5.0

7.5

10.0

12.5

15.0

VEGF mAb 293(ng/ml)

VEGF (7.8ng/ml)

rela

tive IL8 e

xpre

ssio

n0 7.8 31.2 62.5 125 250 500 1000

0

2

4

6

8

10

12

14

VEGF sR1(ng/ml)

VEGF (7.8ng/ml)

rela

tive IL

8 e

xp

ressio

n

64hr

24hr

4.5hr

Conclusions

• Bioassays provide crucial data at all

stages of product development

• Bioassay data is essential as part of

product characterization and

complements information obtained

using non-bioassay procedures

Acknowledgements

• Meenu Wadhwa

• Chris Burns

• Jane Robinson

• Paula Dilger

• Chris Bird

• Tony Meager

• Guenther Adolf

• Tony Mire-Sluis