Adrian F. Bristow
National Institute for Biological Standards and control
(Health Protection Agency)
National Institute for Biological Standards and Control
Assuring the quality of biological medicines

Assignment of quantities to biological medicines:
An old problem re-discovered
Assignment of quantities to biological medicines:
An old problem re-discovered

Assignment of quantities to biological medicines:
An old problem re-discovered
On March 13 th 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

Assignment of quantities to biological medicines:
An old problem re-discovered

Assignment of quantities to biological medicines:
An old problem re-discovered

Assignment of quantities to biological medicines:
An old problem re-discovered
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

Assignment of quantities to biological medicines:
An old problem re-discovered
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 .

Assignment of quantities to biological medicines:
An old problem re-discovered
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

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 wellcontrolled drug however:
HPLC of therapeutic insulin
1925 1995
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?

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?

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

A comparative mouse convulsion insulin assay (N = 24)
SL
SH
TL
TH
Standard low dose (30mU/ml)
Standard high dose (60mU/ml)
Test low dose ?
Test high dose ? (2 x TL)
Assay 1
SH = 20/24
SL = 8/24
TH = 21/24
TL = 6/24
30
20
Standard
10
0
30
Test
60
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)

A comparative mouse convulsion insulin assay (N = 24)
SL
SH
TL
TH
Standard low dose (30mU/ml)
Standard high dose (60mU/ml)
Test low dose ?
Test high dose ? (2 x TL)
Assay 1
SH = 20/24
SL = 8/24
TH = 21/24
TL = 6/24
30
20
Standard
Assay 2
SH = 8/24
SL = 1/24
Th = 9/24
Tl = 2/24
30
20
Standard
Test
10 10
0
30
Test
60
0
30 60
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) Assay 2: 111.3% (74.0-181)
Combination: 100.75% (80.2
– 126)
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

• 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

“Biologicals” and Biological Reference materials: the current portfolio
WHO Biological reference materials
Established by NIBSC (>400)
Include:
Clotting factors
Thrombolytics
Hormones
Cytokines and growth factors
Enzymes
Vaccines
Micobiological antigens
Toxins
Antisera and immunoglobulins
Genomic DNA, cDNA and RNA
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

“Biologicals” and Biological Reference materials: the current portfolio
WHO Biological reference materials
Established by NIBSC
Number > 400, including:
Clotting factors
Thrombolytics
Hormones
Cytokines and growth factors
Enzymes
Vaccines
Micobiological antigens
Toxins
Antisera and immunoglobulins
Genomic DNA, cDNA and RNA
Examples of quantitative measurement methods (bioassay)
Clot lysis in vitro
Stimulation of cell proliferation in vitro
Protection of test animals against microbiological challenge
Animal or cell death

Assignment of quantities to biological medicines:
An old problem re-discovered
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.

Assignment of quantities to biological medicines:
An old problem re-discovered
Does this paradigm really still reflect analytical reality in the second decade of the 21 st century?

It is true that our understanding of what meets the definition of a “biological is changing
• Aspirin
• Adrenaline
• Ampicillin
• Oestradiol
• Insulin
• Growth hormone
• Rec Hep-B vaccine
• Erythropoietin
• Tissue plasminogen activator
• Albumin
• Anti-TNF receptor MAb
• Clotting factor VIII
• Viral gene delivery vectors
• Meningococcus vaccine
• Stem cells
• Organs m.w
Non-biological
180
333
371
376
5808
22K
28K
~30.6K
~65K
67K
150K
~280K
>100kb DNA
Biological

Assignment of quantities to biological medicines:
An old problem re-discovered
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.

Assignment of quantities to biological medicines:
An old problem re-discovered
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.

Assignment of quantities to biological medicines:
An old problem re-discovered
Description of the amount of material in terms of basic physical constant is complicated by:
Extreme complexity
Heterogeniety

Assignment of quantities to biological medicines:
An old problem re-discovered
1 Complexity
Consider clotting factor VIII, a large (ish) glycoprotein used to treat haemophilia
2361 amino acids
268kD (protein)
Glycosylated
51 amino acids,
5.8kD
191 amino acids
22kD

Assignment of quantities to biological medicines:
An old problem re-discovered
Peptide mapping is a front-line technique for analysing proteins
Tryptic peptides:
3-10 14
11-20
>20
5
1
Tryptic peptides:
3-10 108
11-20
>20
53
28
Irresolvable in one dimension

Assignment of quantities to biological medicines:
An old problem re-discovered
Erythropoietin: a therapeutic glycoprotein
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

2 Heterogeneity
Glycosylation: a non-template-directed process which produces heterogeneous products
Glycosylation: N-linked glycan structures
Sialic acid
Sulphate
Galactose
GalNac
Mannose
N-acetyl glucosamine
Asparagine biantennary triantennary tetraantennary
Charge based fractionation of pharmaceutical erythropoietin products

Assignment of quantities to biological medicines:
An old problem re-discovered
For heterogeneous glycoproteins, quantification by mass (mg) is meaningless – the structures all have different molecular weights
Quantification in mol protein is feasible, but -
200
150
100
50
0
200 250 300
Terminal glycosylatyion (Z)
350
Mol protein is not a measurement that reflects the biological activity of the molecule

Assignment of quantities to biological medicines:
An old problem re-discovered
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

Assignment of quantities to biological medicines:
An old problem re-discovered
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?

Assignment of quantities to biological medicines:
An old problem re-discovered
Analysis of receptor binding by surface plasmon resonance
Interaction 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?

Assignment of quantities to biological medicines:
An old problem re-discovered
Erythropoietin: relationships between glycosylation and biological activity
In vitro activity (receptor binding) In vivo activity
400 200
150 300
100
200
50
100
0
200 250 300
Terminal glycosylatyion (Z)
350
0
200 250 300
Terminal glycosylatyion (Z)
350
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

Assignment of quantities to biological medicines:
An old problem re-discovered
Activity-determining factors
In vitro
Amount of substance
Receptor affinity
Signal Transduction in responsive cells
In vivo
Amount of substance
Receptor affinity
Signal Transduction in responsive cells
Access to target tissue
Plasma half life

Assignment of quantities to biological medicines:
An old problem re-discovered
-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
-??

Assignment of quantities to biological medicines:
An old problem re-discovered
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)
Ribel et al(1990) Diabetes 39 10333-1039

Assignment of quantities to biological medicines:
An old problem re-discovered
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!

Assignment of quantities to biological medicines:
An old problem re-discovered
-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!