Download SI and non-SI units for Biological quantities

SI and non-SI units for Biological quantities
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 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
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
Measurement of quantities in medicine
Therapeutic intervention
Most drugs (eg aspirin) are dosed in
units of the SI (mg)
A significant minority, the
“biologicals”, are in arbitrary units
Diagnosis
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?
Biological assays and standardization in
medicine: Historical background
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
HPLC of therapeutic insulin
1925
1995
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
Even now it would be impossible to
assign any kind of value in SI to the 1925
insulin.
however:
How did they do it then so successfully?
The Insulin Story: accurate quantification of
an unknown quantity
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?
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
SH
TL
TH
Assay 1
SH = 20/24
SL = 8/24
TH = 21/24
TL = 6/24
Standard low dose (30mU/ml)
Standard high dose (60mU/ml)
Test low dose ?
Test high dose ? (2 x TL)
30
Standard
20
10
Test
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)
Convulsion-inducing activity compared
to reference standard
A comparative mouse convulsion insulin assay (N = 24)
SL
SH
TL
TH
Assay 1
SH = 20/24
SL = 8/24
TH = 21/24
TL = 6/24
Standard low dose (30mU/ml)
Standard high dose (60mU/ml)
Test low dose ?
Test high dose ? (2 x TL)
30
Assay 2
SH = 8/24
SL = 1/24
Standard
20
Th = 9/24
Tl = 2/24
10
30
20
Standard
Test
10
Test
0
0
30
60
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)
Combination:
Assay 2: 111.3% (74.0-181)
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 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
“Biologicals” and Biological Reference materials:
the current portfolio
WHO Biological reference materials
Established by NIBSC (>400)
Include:
Examples
Clotting factors
Blood Coagulation factor VIII
Thrombolytics
Tissue plasminogen activator
Hormones
Follicle stimulating hormone
Cytokines and growth factors
Interferon
Enzymes
Thrombin
Vaccines
Whole cell Pertussis vaccine
Micobiological antigens
Hepatitis B antigen
Toxins
Botulinum toxin
Antisera and immunoglobulins
Anti-HPV-16 Ab
Genomic DNA, cDNA and RNA
HIV clade 1 genetic reference panel
“Biologicals” and Biological Reference materials:
the current portfolio
WHO Biological reference materials
Established by NIBSC
Number > 400, including:
Examples of quantitative measurement
methods (bioassay)
Clotting factors
Thrombolytics
Clot lysis in vitro
Hormones
Cytokines and growth factors
Stimulation of cell proliferation in vitro
Enzymes
Vaccines
Protection of test animals against microbiological challenge
Micobiological antigens
Toxins
Antisera and immunoglobulins
Genomic DNA, cDNA and RNA
Animal or cell death
Assignment of quantities to biological medicines:
An old problem re-discovered
Chemical drugs
Where the dose or clinical effect
can be related to a fundamental
physical constant, measuring
quantity or effect
Biological drugs
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 21st century?
It is true that our understanding of what meets
the definition of a “biological is changing
m.w
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
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
180
333
371
376
5808
22K
28K
~30.6K
~65K
67K
150K
~280K
>100kb DNA
1950
2007
2010
Non-biological
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
Is this definition still valid?
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.
The short answer is yes!
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
Clotting factor VIII
2361 amino acids
268kD (protein)
Glycosylated
Insulin
51 amino acids,
5.8kD
Growth hormone
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
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
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
biantennary
Sialic acid
Sulphate
Galactose
GalNac
Mannose
N-acetyl glucosamine
triantennary
tetraantennary
Asparagine
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 -
In vivo biological activity
200
Mol protein is not a
measurement that reflects the
biological activity of the
molecule
150
100
50
0
200
250
300
Terminal glycosylatyion (Z)
350
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 vivo activity
In vivo biological activity
In vitro biological activity
In vitro activity (receptor binding)
400
300
200
100
0
200
250
300
Terminal glycosylatyion (Z)
350
200
150
100
50
0
200
250
300
Terminal glycosylatyion (Z)
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
350
Assignment of quantities to biological medicines:
An old problem re-discovered
Activity-determining factors
In vitro
In vivo
Amount of substance
Amount of substance
Receptor affinity
Receptor affinity
Signal Transduction in
responsive cells
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
Where the dose or clinical effect
can be related to a fundamental
physical constant, measuring
quantity or effect
Biological drugs
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!
Thank you for your attention