ANALYSIS OF THE FINAL PRODUCT

(QC TEST)
ANALYSIS OF THE FINAL PRODUCT
All pharmaceutical finished products undergo rigorous QC
testing, in order to confirm their conformance to pre-
determined specifications.

The routine analysis of complex biopharmaceutical product

are-

i) Potency testing→ ensuring that the drug will be

efficacious when administered to the patient

ii) Safety testing→ entails analysis of product for the

presence of various potential contaminants
Product potency
Conform to final product potency specifications.

Such specifications are usually expressed in term s of

‘units of activity’ per vial of product

A number of different approaches may be undertaken

to determine product potency. Such as-
-bioassay
-cytopathic effect inhibition assay
-Immunoassay
Bioassays

A known quantity of the substance applied to a biological

system (can be whole animals, specific organs or tissue
types, or individual mammalian cells in culture.)

The response of biological system is measured

quantitatively, allowing an activity value to be assigned to
the substance being assayed.

Exp.→ bioassay of erythropoietin (EPO) involves a mouse-

based bioassay
Cytopathic effect inhibition assays
Assay based upon the ability of many interferons to render animal cells resistant to
viral attack

interferon is incubated with cells sensitive to destruction by a specific virus.

That virus is then subsequently added,

the percentage of cells that survive thereafter is proportional to the levels of interferon
present in the assay sample.

Addition of the dye such as neutral red (Viable cells can assimilate/absorb these dyes)

Quantify the percentage of cell survived (dye assimilated) by spectrophotometer.

drawbacks→
i) Lack of precision
ii) Time
iii) Cost
Immunoassay
Monoclonal or polyclonal antibody preparations is to detect and quantify the
product.

The specificity of antibody–antigen interaction ensures good assay precision.

The use of conjugated radiolabels (radioimmunoassay; RIA) or enzymes

(enzyme immunoassay: EIA) to allow detection of antigen–antibody binding
renders such assays very sensitive.

Disadvantages→
i) It is not correlate directly with biological activity.
ii) Relatively minor modifications of the protein fail to bind antibody

For such reasons , conduct during downstream processing and on at very

least the final product is necessary to prove that potency falls within
specification
Determination of protein concentration

Three technique is used-

i) measuring absorbency at 280 nm

ii) dye-binding procedure
iii) copper-based reagent containing bicinchonic acid
Measuring absorbency at 280 nm
This approach is based on the fact that the side-chains of tyrosine and
tryptophan absorb at this wave length.

The method is popular, fast , easy to perform and non-destructive to the

sample.

Drawback-
Identical concentrations of different proteins will yield different absorbance
values if their contents of tyrosine and tryptophan vary to any significant
extent .

For these reasons-

this method is rarely used to determine the protein concentration.
 but it is routinely used during downstream processing to detect protein
elution off chromatographic columns , and hence track the purification
process .
dye-binding procedure
Various dyes are known to bind quantitatively to proteins,
resulting in an alteration of the characteristic absorption
spectrum of the dye.

For example, Coomassie brilliant blue G-250 becomes

protonated in phosphoric acid, and provide absorbance
maximum at 450 nm

When Coomassie brilliant blue G-250 bind to a protein (via

ionic interactions) provide absorbance at 595 nm

 Quantification of proteins in this case can thus be undertaken

by measuring absorbance at 595 nm.
copper-based reagent containing bicinchonic acid

Incubation of this reagent with protein sample, the

copper is reduced

In the reduced state it react s with bicinchonic acid,

yielding a purple colour which absorbs maximally at
562 nm
Detection of protein-based product impurities

For the assessment of final product purity.

SDS–P AGE (run under reducing conditions)

The reducing agent β-marceptoethanol or dithiothreitol (DTT)

disrupts inter-chain (and intra-chain) disulphide linkages.

 Individual polypeptides held together will thus separate from

each other on the basis of their molecular mass

The presence of additional bands represent protein

contaminants.
SDS–P AGE
Detection of protein-based product impurities

If additional band is found further characterization

may include by Western blot analysis.

This involves eluting the protein bands from the

electrophoretic gel onto a nitrocellulose filter.

The filter can then be probed using antibodies raised

against the product.

Binding of the antibody to the ‘contaminant’ bands

suggests that they are variants of the product.
2-dimensional (2-D) electrophoretic
 Contaminants of the same molecular mass as the product will go undetected, as
they will co-migrate with target protein.

2-dimensional (2-D) electrophoretic analysis would overcome this eventuality in

most instances.

The isoelectric point (pI, pH(I), IEP)→

The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no
net electrical charge or is electrically neutral in the statistical mean.

The isoelectric point (pI) of a protein is defined as the pH at which the net
charge of a protein molecule is zero.

Accordingly, proteins are positively charged at a pH below their pI and

negatively charged at a pH above their pI
2-dimensional (2-D) electrophoretic
proteins
are
positively
charged
at a pH
below
their pI
and
negativel
y charged
at a pH
above
their pI
2-dimensional (2-D) electrophoretic
entails setting up a pH gradient along the length of an
electrophoretic gel.
 Applied proteins will migrate under the influence of
an electric field, until they reach a point in the gel at
which the pH equals the protein’s isoelectric point (pI;
the pH at which the protein exhibits no overall net
charge — only species with a ne t charge will move
under the influence of an electric field).
IEF thus separates proteins on the basis of charge
characteristics.
2-dimensional (2-D) electrophoretic
Capillary electrophoresis

Separation is based upon different rates of protein migration in

an electric field.

This separation occurs within a capillary tube have a diameter of

20–50 mm and be up to 1 m long

The dimensions of this system yield greatly increased surface area:

volume ratio (when compared to slab gels).

Capillary tube speed up the migration rate of proteins.

 Sample analysis can be undertaken in 15–30 min, and on-line

detect ion at the end of the column allows automatic detection
and quantification of eluting bands.
Capillary electrophoresis
Capillary electrophoresis
High-pressure liquid chromatography (HPLC )

The separation principle of HPLC is based on the

distribution of the analyte (sample) between a
mobile phase (eluent) and a stationary phase
(packing material of the column).

 Depending on the chemical structure of the analyte,

the molecules are retarded while passing the
stationary phase.
High-pressure liquid chromatography (HPLC )

The separation
principle of HPLC is
based on the
distribution of the
analyte (sample)
between a mobile
phase (eluent) and a
stationary phase
(packing material of
the column).

 Depending on the
chemical structure of
the analyte, the
molecules are retarded
while passing the
stationary phase.
High-pressure liquid chromatography (HPLC )

Two typesps-

Reverse-phase HPLC (RP-HPLC)→

Widely used for analysis
separates proteins on the basis of differences in their
surface hydrophobicity.

Size exclusion HP LC (SE-H PLC) →

Limited used
separates proteins on the basis of size and
shape.
Reverse-phase HPLC (RP-HPLC)
The stationary phase in the HPLC column normally consists of
silica or a polymeric support to which hydrophobic arms have
been attached.

It is powerful analytical technique, capable of separating very

similar molecules, displaying minor differences in
hydrophobicity.

Single amino acid substitution or the removal of a single amino

acid from the end of a polypeptide chain can be detected by RP -
HPLC.

 In most instances modifications such as deamidation will also

cause peak shifts and thus may be used to detect impurities
Immunological approaches to detect contaminants
The strategy is an immunoassay termed the ‘blank run approach’.

Construct a host cell identical in all respects to the natural producer cell, except gene of interest.
↓
Then subjected to upstream processing
↓
Cellular extracts are subsequently subjected to the normal product purification process
↓
This produces an array of proteins which could co-purify with the final product.
↓
These proteins are used to immunize horses, goats or other suitable animals.
↓
Produce Polyclonal antibody
↓
Purification of the antibodies allows their incorporation in radioimmunoassay or enzyme-based
immunoassay systems
↓
Labeled antibodies will be used as probe.
↓
Such multi-antigen assay systems will detect the sum
total of host cell-derived impurities present in the product
Peptide mapping
Point mutations in the product’s gene, leading to an altered
primary structure (i.e. amino acid sequence). Thats why it is done

Protein sample is treated with specific reagent that hydrolysis

peptide bonds at specific points and generates peptide fragments.

Reagent should generate smaller peptide fragment as large

fragment is difficult to detect single amino acid alteration.

Exp. Of Reagent:
cyanogen bromide→ it cleaves the peptide bond on the
carboxyl side of methionine residues
V8 protease→ along with trypsin
Peptide mapping
Expose of the protein product to a reagent which hydrolyze peptide bonds at
specific points and generates a series of peptide fragments.
↓
Fragments are separated from each other one- or two-dimensional electrophoresis
and, RP-HPLC.
↓
A standardized sample of the protein product, yield a characteristic peptide
fingerprint, or map,
↓
The peptide maps obtained with each batch of product can subsequently be
compared.
↓
If the peptides generated are relatively short, a change in a single amino acid
residue is likely to alter the peptide’s physicochemical properties sufficiently to
alter its position within the peptide map
Peptide mapping

In this way single (or multiple) amino acid substitutions,

deletions, insertions or modifications can usually be
detected.
N-terminal sequencing
Sequencing of first 20–30 amino acid residues is a popular
quality control test as it:

-positively identifies the protein

-confirms (or otherwise) the accuracy of the amino

acid sequence of at least the N-terminus of the protein

-readily identifies the presence of modified forms (one or

more amino acids are missing from the N-terminus)

N-terminal sequencing is normally undertaken by Edman

degradation
N-terminal sequencing

The phenylthiohydantoin derivative can then be isolated and its

constituent amino acids identified by comparison to
phenylthiohydantion derivatives of standard amino acid solutions.
The shorter peptide is then subjected to a second round of treatment
Analysis of secondary and tertiary structure

protein’s 3-D conformation may be studied by-

-X-ray crystallography or
-NMR spectroscopy,
-More recently proton-NMR has also been applied.

Routine application of such techniques to

biopharmaceutical manufacture is impractical from
both a technical and economic standpoint
Endotoxin and other pyrogenic contaminants
Pyrogens are substances which, when they enter the blood stream,
influence hypothalamic
regulation of body temperature, usually resulting in fever.

In severe cases results in patient death.

Pyrogens represent a diverse group of substances, including various

chemicals, particulate matter and endotoxin (lipopolysaccharide)

LPS→ a molecule derived from the outer membrane

of Gram-negative bacteria

Entry of endotoxin → stimulates the production of interleukin 1 by

macrophages →initiates the fever response
Endotoxin and other pyrogenic contaminants
Endotoxin and other pyrogenic contaminants
Contamination of the final product with endotoxin is
more difficult to control because:

i) Product source is also a source of endotoxin.

eg→ Gram-negative bacterial systems

ii) rigorous implementation of GMP

iii) the heat-stability exhibited by endotoxin.

eg→ autoclaving of process equipment will not
destroy endotoxin present on such equipment
Pyrogen detection
Pyrogens may be detected in parenteral preparations
(or other substances) by a number of methods.
Two such methods are widely employed in the
pharmaceutical industry-

i) rabbit pyrogen test

ii) Limulus amoebocyte lysate (LAL) test
Rabbit pyrogen test
Administration of the product to a group of healthy rabbits, with subsequent
monitoring of rabbit temperature using rectal probes.

Increased rabbit temperature above a certain point suggests the presence of

pyrogenic substances.

The basic rabbit method, as outlined in the European Pharmacopoeia,

If increased temperature is-

less than 1.15 8C→ product may be consider

greater than 2.65 8C→ product has failed

Between 1.15 8C to 2.65 8C→ the result is considered inconclusive, and the
test must be repeated using a further batch of animals
Rabbit pyrogen test
Disadvantages:

i) expensive (there is a requirement for animals, animal

facilities and animal technicians)

ii) excitation/poor handling of the rabbits can affect the

results obtained, usually prompting a false-positive result

iii) sub-clinical infection/poor overall animal health can

also lead to false-positive results

iv) use of different rabbit colonies/breeds can yield variable

results.
Limulus amoebocyte lysate (LAL) test
Detection the presence of DNA

Presence of DNA in biopharmaceuticals focuses

presence of active oncogenes in the genome of several
producer cell types

Uptake and expression of such DNA in human cells

could lead to cancer.

DNA hybridization studies (e.g. the ‘dot-blot’ assay)

utilizing radio labelled DNA probes allows detection
of DNA contaminants in the product
Detection the presence of DNA
DNA in product can be achieved by ethanol precipitation
↓
The isolated DNA is then applied onto nitrocellulose filter paper and heated the
filter at 80 8C under vacuum.
↓
This promotes DNA denaturation, yielding single strands, DNA on the filter
↓
total DNA derived from the cells in which the product is produced is then
radiolabelled with 32P and heated at 90 C for denaturation
↓
incubated with the baked filter for several hours at 40 8C.
↓
Labelled DNA will re-anneal with any complementary DNA strands
immobilized on the filter.
↓
After the filter is washed (to remove non-specifically bound radiolabelled probe) it
is subjected to autoradiography, which allows detection of any bound probe.
 
Viral assays
Newly evolved viral strains, or uncharacterized/unknown
viral is difficult/ not possible

This fact underlines the importance of including at least

one step in downstream processing which is likely to
indiscriminately inactivate or remove viruses from the
product.

Current viral assays fall into one of three categories:

i) immunoassays;
ii) assays based on viral DNA probes;
iii) bioassays.
Viral assays (Immunoassays)
A sample of live or attenuated virus, or a purified
component of the viral caspid, can be injected into animals
to stimulate polyclonal antibody production (or to facilitate
monoclonal antibody production by hybridom a
technology).

Harvested antibodies are then employed to develop

specific immunoassays which can be used to routinely
screen test samples for the presence of that specific virus
Viral assays (based on viral DNA probes and Bioassays)

Based on viral DNA probes

These can be used to screen the biopharmaceutical
product for the presence of viral DNA. The assay
strategy is similar to the dot-blot assays used to detect
host cell derive d DNA contaminants

Bioassays
Entails incubation of the final product with cell lines
sensitive to a range of viruses. The cell s are
subsequently monitored for cytopathic effects or other
obvious signs of viral infection.