SI and non-SI units for Biological · PDF fileSI and non-SI units for Biological quantities Adrian F. Bristow ... – The unit of insulin can only be described in terms of a reference

National Institute for Biological Standards and Control

Assuring the quality of biological medicines

SI and non-SI units for Biological quantities

Adrian F. Bristow

National Institute for Biological Standards and control

(Health Protection Agency)

Assignment of quantities to biological medicines:

An old problem re-discovered



On March 13th 2006, 6 healthy male volunteers were given low doses of

TGN1412, an experimental monoclonal antibody which was under clinical

trial as a potential immune-activating agent, through agonistic

(stimulatory) interaction with the CD28 T-cell receptor.

All developed rapid, life-threatening adverse reactions, leading to multiple

organ dysfunction. This was later confirmed to arise from a toxic

“cytokine storm”. Thanks to rapid intervention the men all survived, but

at least some will have persistent, irreversible sequelae.

The dose (mg/kg) used was 500 times lower than the dose that had been

safe in monkeys









Why have I told you this?

TGN1412 is a super-agonist for the CD28 T-cell receptor.

It is meant to stimulate release of T-cell cytokines

It could be argued that qualitatively the response was not unexpected.

What was unexpected was the magnitude of the response. What the

clinical trial lacked was any reference framework for relating the dose

quantity (mg) to the magnitude of the response in humans.

In medicine, numbers, quantities and units matter.

They make the difference between lack of efficacy, effective therapy and

over-dose

They make the difference between mis-diagnosis and accurate diagnosis

.



Measurement of quantities in medicine

Therapeutic intervention Diagnosis

Most drugs (eg aspirin) are dosed in

units of the SI (mg)

A significant minority, the

“biologicals”, are in arbitrary units

Several hundred quantities are

measured in the diagnosis of disease

About 20% are measured in units of the

SI (mg or mol)

The majority are “biologicals”, and not

traced to the SI



What, in this context, is a “biological” medicine ?

and

Why is it an old problem re-discovered?

Consider insulin:

A story of successful quantification of an unknown quantity

Biological assays and standardization in

medicine: Historical background

Diabetes Voice (Kambaskovic, D., June

2002 Volume 47 Issue 2) describes two diabetics with remarkably long life spans

Hazel Davies, who had lived for 80 years on insulin therapy after being diagnosed in 1921, and Roy Cross, who lived to be >100 after being diagnosed in 1938

This suggests that insulin was a well-controlled drug

however:

Even now it would be impossible to assign any kind of value in SI to the 1925 insulin.

How did they do it then so successfully?

HPLC of therapeutic insulin

1925 1995

Even with today’s technology, the 1920’s preparation would be too

impure to assay by an HPLC method

(Insulin structure determined by Sanger in the 1950’s)

Paradox:

Insulin is a drug whose dose needs to be precisely controlled +/- 10%

This was clearly achieved (Hazel Davies lived to be >100)

How was this possible?

The Insulin Story: accurate quantification of an unknown quantity

How do you measure, to within 10%, the potency of insulin in 1925?

You have to assign a precise figure to the biological activity of insulin

preparations, even when you don’t know what insulin is

•What do you know?

– It lowers blood sugar

•Can you measure blood sugar?

– Yes but not easily

•What can you measure?

– You can count!

- Mouse convulsion insulin assay:

Injecting fasting mice with insulin will cause some to go into hypoglycaemic

convulsions. The number that do appears to be related to the amount of

insulin you inject

Convulsion-inducing activity compared

to reference standard

A comparative mouse convulsion insulin assay (N = 24)

SL Standard low dose (30mU/ml)

SH Standard high dose (60mU/ml)

TL Test low dose ?

TH Test high dose ? (2 x TL)

Assay 1

SH = 20/24

SL = 8/24

TH = 21/24

TL = 6/24

30 60

30

20

10

0

Standard

Test

Statistical analysis is based on calculations of a common slope, then chi-squared for deviations for parallelism and linearity

Assay 1: 97.0% (75-126)

Convulsion-inducing activity compared

to reference standard

A comparative mouse convulsion insulin assay (N = 24)

SL Standard low dose (30mU/ml)

SH Standard high dose (60mU/ml)

TL Test low dose ?

TH Test high dose ? (2 x TL)

Assay 1

SH = 20/24

SL = 8/24

TH = 21/24

TL = 6/24

30 60

30

20

10

0

Standard

Test

Statistical analysis is based on calculations of a common slope, then chi-squared for deviations for parallelism and linearity

30 60

30

20

10

0

Standard

Test

Assay 2

SH = 8/24

SL = 1/24

Th = 9/24

Tl = 2/24

Sir Henry Dale established that a unit of insulin can only effectively be described in

terms of a reference material, not in terms of an absolute response

Assay 1: 97.0% (75-126) Assay 2: 111.3% (74.0-181)

Combination: 100.75% (80.2 – 126)

The Insulin Story exemplifies all aspects of a

“biological” as the term is used in medicine:

• The medicinal substance was isolated from a biological source

(extracts of animal pancreata – bovine, porcine)

• Accurate quantification of the active principle was achieved by

quantifying its activity in a test measuring function rather than

quantity of substance

– i.e. what it does, rather than what it is

• Quantification is dependent on the science of biological

standardization

– The unit of insulin can only be described in terms of a reference material,

not in terms of an absolute response (eg. number of convulsions)

– Statistical combination of independent assays can produce precise

numbers from an inherently imprecise and irreproducible method

Clotting factors

Thrombolytics

Hormones

Cytokines and growth factors

Enzymes

Vaccines

Micobiological antigens

Toxins

Antisera and immunoglobulins

Genomic DNA, cDNA and RNA

WHO Biological reference materials

Established by NIBSC (>400)

Include:

“Biologicals” and Biological Reference materials:

the current portfolio

Examples

Blood Coagulation factor VIII

Tissue plasminogen activator

Follicle stimulating hormone

Interferon

Thrombin

Whole cell Pertussis vaccine

Hepatitis B antigen

Botulinum toxin

Anti-HPV-16 Ab

HIV clade 1 genetic reference panel

Clotting factors

Thrombolytics

Hormones

Cytokines and growth factors

Enzymes

Vaccines

Micobiological antigens

Toxins

Antisera and immunoglobulins

Genomic DNA, cDNA and RNA

WHO Biological reference materials

Established by NIBSC

Number > 400, including:

Stimulation of cell proliferation in vitro

Protection of test animals against microbiological challenge

Clot lysis in vitro

Animal or cell death

Examples of quantitative measurement

methods (bioassay)

“Biologicals” and Biological Reference materials:

the current portfolio



Chemical drugs Biological drugs

Where the dose or clinical effect

can be related to a fundamental

physical constant, measuring

quantity or effect

Where the dose or clinical effect can only be

related to the response in a complex

measurement system such as an animal or a

living cell, and can be traced only to the

effect produced by a reference material

(bioassay)

This complexity is irreducible!

A “biological” analytes is considered by WHO as one

“…of biological origin,

which cannot be characterized adequately by chemical

and/or physical means alone…”

(WHO, Tech. Rep. Ser. 800, 1990, 181-213).

This is a practical definition, relating the structural complexity of the

material being standardised to the current utility of analytical methods.



Does this paradigm really still reflect analytical reality in the

second decade of the 21st century?

It is true that our understanding of what meets

the definition of a “biological is changing

• Aspirin 180

• Adrenaline 333

• Ampicillin 371

• Oestradiol 376

• Insulin 5808

• Growth hormone 22K

• Rec Hep-B vaccine 28K

• Erythropoietin ~30.6K

• Tissue plasminogen activator ~65K

• Albumin 67K

• Anti-TNF receptor MAb 150K

• Clotting factor VIII ~280K

• Viral gene delivery vectors >100kb DNA

• Meningococcus vaccine

• Stem cells

• Organs

1950 2007m.w 1950 2010

Biological

Non-biological



Although the last twenty years has seen a progressive move towards

physcichemical analytical methods for many smaller proteins, the

“new” medicine, based on biotechnology encompasses therapeutic

interventions such as engineered antibodies, complex glycoproteins,

gene-therapy delivery vectors, stem cells, and engineered vaccines.

These are comfortably below the “biologicals” cut-off line and their

measurement remains dependant on complex biological systems.

A “biological” analytes is considered by WHO as one

“…of biological origin,

which cannot be characterized adequately by chemical

and/or physical means alone…”

(WHO, Tech. Rep. Ser. 800, 1990, 181-213).

This is a practical definition, relating the structural complexity of the

material being standardised to the current utility of analytical methods.



Is this definition still valid?

The short answer is yes!



Description of the amount of material in terms of basic physical constant is

complicated by:

Extreme complexity

Heterogeniety



Insulin

51 amino acids,

5.8kD

Growth hormone

191 amino acids

22kD

Clotting factor VIII

2361 amino acids

268kD (protein)

Glycosylated

1 Complexity

Consider clotting factor VIII, a large (ish) glycoprotein used

to treat haemophilia



Peptide mapping is a front-line technique for analysing proteins

Somatropin

Tryptic peptides:

3-10 14

11-20 5

>20 1

Factor VIII

Tryptic peptides:

3-10 108

11-20 53

>20 28

Irresolvable in one dimension



A single subunit globular glycoprotein, 165 amino acids

3 N-linked and 1 O-linked glycosylation sites

Mr Approx 30,600 (ie 35% CHO)

Kidney-derived hormone maintaining erythrocyte maturation in vivo

Used in treatment of anaemia associated with renal failure and other conditions

Erythropoietin: a therapeutic glycoprotein

Sialic acid

SulphateGalactose

GalNac

N-acetyl glucosamine

Mannose

Asparagine

Glycosylation: N-linked glycan structures

biantennary

triantennary

tetraantennary

Glycosylation: a non-template-directed process which

produces heterogeneous products

Charge based fractionation of

pharmaceutical erythropoietin

products

2 Heterogeneity



For heterogeneous glycoproteins, quantification by mass (mg) is

meaningless – the structures all have different molecular weights

Quantification in mol protein is feasible, but -

200 250 300 350

0

50

100

150

200

In v

ivo

bio

log

ica

l a

cti

vit

y

Terminal glycosylatyion (Z)

Mol protein is not a

measurement that reflects the

biological activity of the

molecule



Complexity of vaccines

Proteomics-based identification of antigenically-active components present in OMV

Vaccines against Meningococcal group B disease

-At least 30 different antigens can be identified

in this vaccine

-The relationship between antigen quantity and

protective immunity is not understood

-Protective immunity can only be measured in a

(biological) challenge protection assay



So description of quantity in terms of basic physical constants is

both difficult and misleading.

What about description of function in terms of basic physcal

constants?

Most pharmacologically active biologicals exert their actions by

interacting with a receptor

Can this be an analytical target for expressing function in terms

of physical constants?



Analysis of receptor binding by

surface plasmon resonanceInteraction of active bio-molecules with their

receptors can be measured and described in

physico-chemical terms by techniques such

as surface plasmon resonance

Receptor binding is a good analogue of

biological activity in cell bioassays in vitro

What’s the problem?

http://www.ncbi.nlm.nih.gov/books/NBK20740/figure/A266/?report=objectonly



200 250 300 350

0

50

100

150

200

In v

ivo

bio

log

ica

l a

cti

vit

y


In v

itro

bio

log

ica

l a

cti

vit

y


200 250 300 350

0

100

200

300

400

Erythropoietin: relationships between glycosylation and biological activity

In vitro activity (receptor binding) In vivo activity

Factors affecting the ability of erythropoietin to bind to its receptor are not

the same as those affecting its ability to stimulate red blood cell formation in

a whole organism



Activity-determining factors

In vitro In vivo

Amount of substance

Receptor affinity

Signal Transduction in

responsive cells

Amount of substance

Receptor affinity

Signal Transduction in

responsive cells

Access to target tissue

Plasma half life



-Insulin analogues with up to 10-fold higher receptor affinity, or with similarly

reduced receptor affinity have been produced (eg B-10 Asp insulin)

-The receptor-binding properties of such analogues are reflected in their in vivo

potency

-It is not reflected in the in vivo potency, which is usually similar to the parent

molecule

-Similarly, insulin analogues with reduced receptor binding do not show reduced

activity in vivo

-??



Ribel et al(1990) Diabetes 39 10333-1039

Maintenance of the biological blood glucose-lowering activity action

of insulin in vivo depends on the circulating plasma levels

The main route of clearance of insulin in vivo is through its receptor

Increasing the affinity for the receptor increases the rate of removal

from the plasma compartment and vice versa

Changes in receptor affinity are not reflected in changes in in vivo

activity (and therefore in clinical efficacy)



Chemical drugs Biological drugs

Where the dose or clinical effect

can be related to a fundamental

physical constant, measuring

quantity or effect

Where the dose or clinical effect can only be

related to the response in a complex

measurement system such as an animal or a

living cell, and can be traced only to the

effect produced by a reference material

(bioassay)

This complexity is irreducible!



-There is a need for robust and accurate quantitation in all areas of

medicine, including biological medicines

-Although some progress has been made in applying measurement

science to bio-molecules, much of this area of medicine remains

beyond its scope present capacities

-The use of complex measurement systems based on biological

responses remains embedded in the approach to controlling this type

of medicine

-A reductionist approach to measurement in this field, breaking down

the system into measurable components, seems unpromising –

biological systems are not the sum of their parts

-Developing a metrology for complex systems seems overdue!

Thank you for your attention

Documents

SI and non-SI units for Biological · PDF fileSI and non-SI units for Biological quantities Adrian F. Bristow ... – The unit of insulin can only be described in terms of a reference