INTERVIEW QUESTIONS
1. HR (HUMAN RESOURCE) INTERVIEW QUESTIONS
2. QUALITY CONTROL INTERVIEW QUESTIONS ON WET CHEMICAL ANALYSIS
3. QUALITY CONTROL INTERVIEW QUESTIONS ON WET INSTRUMENTAL ANALYSIS
4. QUALITY CONTROL INTERVIEW QUESTIONS ON CHROMATOGRAPHY
5. INTERVIEW QUESTIONS ON SYSTEM SUITABILITY PARAMETERS - HPLC & GC
6. INTERVIEW QUESTIONS ON TROUBLESHOOTING - HPLC & GC
pH
1. pH is a
number which represents conventionally the hydrogen ion concentration of an
aqueous solution.
2. The pH of
a solution can be measured by potentiometric instrument (pH meter) capable of
reproducing pH value to 0.02 pH
units.
3. The
potentiometric determination of pH is made by measuring the potential
difference between two appropriate electrodes immersed in the solution to be
examined.
4. One of
these electrode is sensitive to hydrogen ions (usually glass electrode) and
other is reference electrode (for example a saturated calomel electrode)
5. The pH of
a solution to be examined is related to that of a reference solution (pHs) by
following Nernst equation.
E - Es
pH= pHs - ---------------
K
Where
E = Potential in volts of
cell containing the solution to be examined.
Es= Potential in volts of
cell containing the known pH (pHs)
K = Change in potential per
unit change in pH expressed in volts.
Theoretically K=
(0.05916+.000198(t-25) volts at any temperature t.
Measurement of pH:
pH is defined as -Log [H+].
It is important to be able to measure pH accurately. And also it is important
to know how the instrument measure pH because several factors can cause the
observed value to differ from the
actual pH.
A pH meter measures the voltage between
two electrodes placed in the solution. The
important part of the system is an electrode whose potential is pH
dependent. The most commonly used pH dependent unit is glass electrode. The
action of this electrode is based upon the fact that the certain types of
borosilicate glass are permeable to H+ ions but not other cat ions
or anions. Therefore, if thin glass layer of such glass is interposed between
two solutions of different H+ ion concentration H+ ions
will moves across the glass from the solution of high to that of low H+
concentration. Because passage of H+ ions through the glass adds a
positive ion to the solution of low H+ concentration and leaves
behind (high H+ concentration) a negative ion an electric potential
develops across the glass.
|
If inner H+ moves to outer, a negative
ion at inner wall and a positive ion at outer wall developed.
If outer H+ moves to inner, a negative
ion at outer wall and a negative ion at outer wall developed
|
The magnitude of this potential is
given by the equation
Potential=E= {2.303 RT/F}{Log [H+]1/
[H+]2}
R=Gas constant
T=Absolute temperature
F=Faraday constant
[H+]1 & [H+]2
= The H+ concentration of inside and outside of the glass
respectively.
Clearly if H+ concentration
of one of the solution is fixed, the potential will be proportional to the pH
of the other solution. So the second part of the pH meter is a reference
electrode whose concentration is fixed. (most commonly contains Hg-HgCl2 paste
in saturated KCl. If high temperature is required Ag-Agcl2 is required instead
of Hg-HgCl2). This is called a calomel electrode. KCl serves to make contact
between Hg-HgCl2 or Ag-Agcl2 unit and the solution being measured. The calomel
electrode tube is made of glass that is impermeable to H+ ions (so
that potential is pH independent
So the pH measured by such system is
primarily the difference between the potentials of that of the glass and
reference electrodes. And then the potential is converted to pH.
1 pH=59.12 mv
However there are
three other potentials present in the circuit.
1.The liquid junction potential of the
reference electrode resulting from the fact that K+ & Cl- do not diffuse at
the same rate so that a charge is generated at the interface between the KCl
solution in the reference cell and the sample.
2. Asymmetry potential which develop
across glass even when the pH on both sides is the same.
3. Potential of Ag-Agcl in glass
electrode unit which is itself an electrode because of its contact with the CL-
of the HCl.
These three potentials and that
the reference electrodes itself are relatively independent of pH and of ionic
strength in the range normally encountered. Hence voltage V, measured with the
total system may be expressed as the difference between the fixed potentials
and that of the glass electrode. Therefore the voltage generated is linearly
related to the ph of the solution.
pH electrode
standardization – Calibration:
Before measuring the pH you have to
calibrate (standardize) electrode. To calibrate the electrode you need at least
two solutions of known pH. Most commonly used commercially available
calibration buffers have pH of 4.01, 7.00 and 10.00.
Details of calibration procedure depend
on the pH meter model.
First step is usually related to temperature
correction. Some models will measure temperature by itself, others need
external temperature probe, or you will have to enter temperature measured by
others means using dials or buttons. Buffer pH changes with temperature.
Check the pH of buffer 4.01, 7.00 and
10.00 at 25. If they are not in 0.02 ranges adjust with up and down arrows. (If
desirable check the buffers of pH 1.00 and 12.45 at 20°)
Then observe the slope value which displays automatically whether is in specified limit
of 80-120 or not.
pH electrode test procedure or Slope
calculation:
Mathematical
difference between two buffer mill volts (Electrode span)
Slope = --------------------------------------------------------------------
X100
Theoretical span (Theoretical
span)
Electrode
span= 7 buffer millivolts reading – 4 buffer millivolts reading (at 25°)
Theoretical span= 176.9
Cleaning of pH
electrode:
·
General
·
Soak in 0.1M HCl for
half an hour.
·
Drain and refill the
reference solution.
·
Soak the electrode in
filling solution for one hour.
·
Inorganic
Contaminants
·
Soak in 0.1M tetra
sodium EDTA solution for 15 min.
·
Drain and refill the
reference solution.
·
Soak the electrode in
filling solution for one hour.
·
Protein Contaminants
·
Soak in 1%
pepsin/0.1M HCl for 15 min.
·
Drain and refill the
reference solution.
·
Soak the electrode in
filling solution for one hour.
·
Grease and Oil
·
Rinse with detergent
or ethanol solution.
·
Drain and refill the
reference solution.
· Soak the electrode in
filling solution for one hour. Electrode response may be enhanced by
substituting a mixture of 1:1 pH 4 buffer and filling solution for the soaking solution.
Cleaning of clogged
junction
·
Pollution by sulfides
· Use a solution of 8%
thiocarbamide in 1mol/L HCl.
· Keep the electrode in
the above solution till junction color turns pale.
·
Pollution by Silver
chloride
·
Use concentrated
ammonia solution.
·
Keep the electrode
above solution for about 12 hours.
·
Rinse and put into pH
4 buffer for at least 1 hour.
Other contaminants have to be removed by cleaning with
distilled water, alcohol or mixture of acids .If nothing else help you may
consider use of ultrasonic cleaners as last resort.
Regenerating pH electrode:
Following procedure is the last resort. They may work, they
may won’t .You may try them before throwing electrode away.
First of all clean
the electrode as described in electrode cleaning section then
·
Soak the electrode
for 4-8 hours in 1M HCl solution.
·
Rinse it and move to
pH 7 buffer for an hour.
If electrode is still
not working
·
Fill the electrode
with filling solution.
·
Move to the fume hood
·
Place the electrode
in the 10% nitric acid solution on a hotplate. Heat to boiling and keep it in
the solution for 10 min.
·
Place 50ml of filling
solution in a second clean beaker. Heat although boiling is not necessary.
·
While the electrode
is still hot, transfer it to the beaker of heated filling solution, set aside
to cool.
·
Some manufacturers
suggest the electrode may be reactivated by treating with diluted solution of
hydrofluoric acid followed by subsequent conditioning in filling solution.
(Before going to HF, remember it is highly dangerous and dissolves glass).
pH electrode maintenance:
·
Handle electrode with
care-it is fragile (easily broken or damaged).
· Keep electrode always
immersed. Use the solution recommended by manufacturer or neutral solution of
KCl (3M-4M).
· Remember to always
keep internal level of filling solution above the level of measured solution.
· Don’t put electrode
in a solution that can dissolve glass-HF (or acidified fluoride solution),
concentrated alkalies.
· Don’t put electrode
into dehydrating solution such as ethanol, sulphuric acid, etc.
· Don’t rub or wipe
electrode bulb, to reduce chance of error due to polarization.
·
Fill the electrode
with correct filling solution to not let it dry internally.
· If you are using
electrode in a solution containing substances able to clog the junction or
stick to the glass bubble clean the electrode as soon as possible after use.
· If electrode will not
be used for long time, you may store it dry to prevent aging (aging take place
only when electrode is wet).
· If dried
incidentally, or after storing-soak for at least 24 hrs before using.
pH electrode storing
· A wet stored
electrode allows an immediate use and a short response time, which is not true
for dry storage ones. Unfortunately, the wet stored electrode is aging faster,
because the process of aging (changing the structure in the membrane) proceeds
also in the case of non-use. Keeping the electrodes wet should preferably be
made in KCl solution. Most electrodes have preventive cap that can be filled
with storage solution before placing.
· To store electrode
dry you must first remove internal solution, rinse the electrode in DI/RO
water, and let it dry. (in fact electrodes that can be stored dry are getting
more and more rare)
· Always check
electrode owner manual for details, as these may depend on the electrode make.
USP Criteria for pH measurement:
·
Measure at Temperature
25 +2o.
·
Reproducibility
0.02.
·
To standardize the
p H meter, select two buffers whose difference does not exceed 4 units and such
that the expected pH of the test solution should falls in them.
·
Check the first
buffer and set the value to std and check for second buffer, if more than +0.07
units examine the electrode.
·
If electrode is
good, adjust slope or electrolyte changing etc.
·
Repeat the buffers
checking until the pH values within+0.02
STABILITY STUDY
What
is Stability?
Stability is a study to
provide evidence on how the quality of product varies with time under the
influence of a variety of environmental factors such as temperature, humidity
and light.
i.e. evaluation of product
under recommended storage conditions (with appropriate tolerances) that test
its thermal stability and, if applicable, its sensitivity to moisture.
To establish a re-test
period for the drug substance or a shelf life for the drug product at
recommended storage conditions.
In
simple words Stability can be also defined as
The capacity of a drug to remain within
specifications established to assure its identity, strength, quality and
purity”
Stability study Lifecycle:
Stability studies are incorporated at all
stages of drug product life cycle from early stages of product development to
last stage follow-up studies. In particular the life cycle can be divided in to
6 different stages as follows.
Stage-1: Early stage stress and accelerated
testing with drug substance.
Stage-2: Accelerated & Long term testing
of drug substance
Stage-3: Stress testing on scale up batches of
drug products
Stage-4: Accelerated & Long term testing
for registration purpose
Stage-5: On-going stability testing.
Stage-6: Follow-up stabilities.
Stress Testing:
Ø Stress testing is to be
carried out on a single batch
Ø The testing should include
the effect of temperature (50/60 oC, i.e. 10 oC
increment) above that of an accelerated testing, humidity (e.g.: 75% RH or
greater) where appropriate oxidation & photolysis on the product has to be
performed.
Ø Stress testing can help
identify the likely degradation products which can help to establish:
i. The degradation pathways.
ii. The intrinsic stability of the molecule.
iii. Developing & validating the suitable analytical procedures.
Ø Photo stability Testing (ICH
Q1B) should be an integral part of stress testing.
Selection
of batches:
Ø Data from at least 3 primary
batches required.
Ø Primary batches could be
from pilot / plant scale.
Ø Plant / pilot batches should
be Apple to Apple (process, equipment, route should be similar).
Ø The overall quality of the
batches of drug substance placed on formal stability studies should be
representative of the quality of the material to be made on a production scale.
Container
Closure system:
The stability studies should be conducted on
the drug substance packaged in a container closure system that is the same as
or simulates the packaging proposed for storage and distribution.
Specification:
Ø Specification, which is a list of tests, reference to
analytical procedures, and proposed acceptance criteria, is addressed in ICH
Q6A and Q6B.
Ø specification for degradation products in a drug
substance is discussed in Q3A
Ø Stability studies should
include testing of those attributes of the drug substance that are susceptible
to change during storage and are likely to influence quality, safety, and/or
efficacy. The testing should cover, as appropriate, the physical, chemical,
biological, and microbiological attributes.
Ø Validated
stability-indicating analytical procedures should be applied. Whether and to
what extent replication should be performed will depend on the results from validation
studies.
Ø The testing should cover as
appropriate : chemical, physical, biological & microbiological parameters
Testing
Frequency:
Long Term
: First year
: Every 3 months.
Second year : Every
6 months.
Thereafter : Annually.
Intermediate
: 0, 6, 9, 12 months. (Min 4
time point)
Accelerated
: 0, 3, 6 months. (Min 3 time point)
Ø If results from accelerated studies are likely to
approach significant change criteria, increased testing should be conducted
either by adding samples at the final time point or by including a fourth time
point in the study design.
Ø When testing at the intermediate storage condition is
called for as a result of significant change at the accelerated storage
condition, a minimum of four
time points (0, 6, 9, and 12) study is recommended.
Storage Condition:
A very important point in conducting
stability studies are storage conditions.
World has been divided in to 4
stability zones based on real climate conditions in the respective regions
ICH Stability zones
Zone
|
Type of climate
|
Kinetic Average temperature
|
Average relative Humidity
|
Zone I
|
Temperate Zone
|
21°C
|
45 %RH
|
Zone II
|
Mediterranean/ Sub
tropical zone
|
25°C
|
60 %RH
|
Zone III
|
Hot dry zone
|
30°C
|
35 %RH
|
Zone IV
|
Hot / Humid
(Tropical zone)
|
30 °C
|
65 %RH
|
Zone IVb
|
Hot / Very Humid
|
30 °C
|
75 %RH
|
Long term testing Conditions:
Climate Zone
|
Temperature
|
Humidity
|
Minimum Duration
|
Zone I
|
21°C±2°C
|
45% RH ± 5% RH
|
12 Months
|
Zone II
|
25°C±2°C
|
60% RH ± 5% RH
|
12 Months
|
Zone III
|
30°C±2°C
|
35% RH ± 5% RH
|
12 Months
|
Zone IV
|
30°C±2°C
|
65% RH ± 5% RH
|
12 Months
|
Zone IVb
|
30°C±2°C
|
75% RH ± 5% RH
|
12 Months
|
Refrigerated
|
5°C±3°C
|
No Humidity
|
12 Months
|
Freezer
|
-20°C±5°C
|
No Humidity
|
12 Months
|
Accelerated testing conditions:
Climate Zone
|
Temperature
|
Humidity
|
Minimum Duration
|
Zone I to 1Vb
|
40°C±2°C
|
75% RH ± 5% RH
|
6 Months
|
Refrigerated
|
25°C±2°C
|
60% RH ± 5% RH
|
6 Months
|
Freezer
|
5°C±3°C
|
No Humidity
|
6 Months
|
Intermediate testing conditions:
Climate Zone
|
Temperature
|
Humidity
|
Minimum Duration
|
All zones
|
30°C±2°C
|
65% RH ± 5% RH
|
6 Months
|
Storage condition
|
Long term
|
Intermediate
|
Accelerated
|
Ambient
|
25°C ± 2°C/
60% RH ± 5% RH
(or)
30°C ± 2°C/
65% RH ± 5% RH
|
30°C ± 2°C/ 65% RH ± 5% RH
|
40°C ± 2°C/
75% RH ± 5% RH
|
Below 30°C
|
30°C ± 2°C/
65% RH ± 5% RH
|
NA
|
40°C ± 2°C/
75% RH ± 5% RH
|
Cool (15°C-25°C)
|
25°C ± 2°C/
60% RH ± 5% RH
|
30°C ± 2°C/
65% RH ± 5% RH
|
40°C ± 2°C/ 75% RH ± 5% RH
|
Refrigerated
|
5°C ± 3°C
|
NA
|
25°C ± 2°C/ 60% RH ± 5% RH
|
Freezer
|
- 20°C ± 5°C
|
NA
|
NA
|
Ø If 30°C ± 2°C/65% RH ± 5% RH is the long-term
condition, there is no intermediate condition.
Ø If long-term studies are
conducted at 25°C ± 2°C/60% RH ± 5% RH and
“significant change” occurs at any time during 6 months’ testing at the
accelerated storage condition, additional testing at the intermediate storage
condition should be conducted and evaluated against significant change criteria
Ø If “significant change”
occurs between 3 & 6 months of accelerated study, data on long term study
should be submitted.
Ø If “significant change”
occurs within 3 months of accelerated study, it is unnecessary to continue
further testing.
Ø In the absence of an
accelerated storage condition for drug substances intended to be stored in a
freezer, testing on a single batch at an elevated temperature (e.g., 5°C ± 3°C
or 25°C ± 2°C) for an appropriate time period should be conducted to address
the effect of short term excursions outside the proposed label storage
condition, e.g., during shipping or handling.
Ø Drug substances intended for
storage below -20°C should be treated on a case-by-case basis.
Stability Commitment:
Ø When available long term
stability data on primary batches do not cover the proposed re-test period
granted at the time of approval, a commitment should be made to continue the
stability studies post approval in order to firmly establish the re-test
period.
Ø If the submission includes
data from stability studies on at least three production batches, a commitment
should be made to continue these studies through the proposed re-test period.
Ø If the submission includes
data from stability studies on fewer than three production batches, a
commitment should be made to continue these studies through the proposed
re-test period and to place additional production batches, to a total of at
least three, on long term stability studies through the proposed re-test
period.
Ø If the submission does not
include stability data on production batches, a commitment should be made to
place the first three production batches on long term stability studies through
the proposed re-test period.
The stability protocol used
for long term studies for the stability commitment should be the same as that
for the primary batches, unless otherwise scientifically justified.
Statement
& Labelling:
Ø A storage statement should
be based on the stability evaluation.
Wherever applicable, specific instructions should be provided. For eg:
drug substances that cannot tolerate freezing.
Ø Avoid use of “ambient condition” or “Room
temperature”.
Ø Need direct link between the label storage
statement & the demonstrated stability.
Ø A retest period for drug substance should be
derived from stability information, and should be displayed on the container
label as appropriate.
Testing conditions where
stability has been shown
|
Required labeling
statement
|
Additional labeling
statement, where relevant
|
25 ± 20C / 60 ± 5% RH (long term)
40 ± 20C / 75 ± 5% RH (accelerated)
or
30 ± 20C / 65 ± 5% RH (long term)
40 ± 20C / 75 ± 5% RH (accelerated)
|
None
|
Do not refrigerate or
freeze.
|
25 ± 20C / 60 ± 5% RH (long term)
30 ± 20C / 65 ± 5% RH (intermediate)
or
30 ± 20C / 65 ± 5% RH (long term)
|
Do not store above 30 0C
or
Store below 30 0C.
|
Do not refrigerate or
freeze
|
25 ± 20C / 60 ± 5% RH (long term)
|
Do not store above 25 0C
or Store below 25 0C.
|
Do not refrigerate or
freeze.
|
5 ± 30C (long
term)
|
Store in a refrigerator or
Store & transport refrigerated
|
Do not freeze
|
Below zero
|
Store in a freezer or
Store & transport frozen
|
None
|
S.No
|
Storage problem
|
Additional labeling statements* depending on the
packaging
|
1.
|
Sensitivity to moisture.
|
Keep the container tightly
closed
|
2.
|
Sensitivity to moisture.
|
Store in the original
package.
|
3.
|
Sensitivity to light
|
Store in the original
package.
|
4.
|
Sensitivity to light
|
Keep the container in the
outer carton.
|
EVOLUTION OF STABILITY DATA TO ESTABLISH RETEST:
·
Retest date shall be established
by extrapolation of real time data
·
Extrapolation is the practice of using a known data set to infer
information about future data. Extrapolation to extend the retest period or
shelf life beyond the period covered by long-term data can be proposed in the
application, particularly if no significant change is observed at the
accelerated condition.
EVOLUTION OF STABILITY DATA TO ESTABLISH RETEST FOR
DRUG SUBSTANCE INTENDED FOR ROOM TEMPERATURE STORAGE
Accelerated condition
|
Long term /ACC Data
|
Data willing to
statistical analysis
|
Retest period / Shelf life
(Y)
(X= available long term
data)
|
No significant change
|
Little or no variability
over time
|
-NA-
|
Y = 2x
(Should NMT X+12)
|
Showing variability over
time
|
Yes
|
Y = 2x
(Should NMT X+12)
|
|
No
|
Y = 1.5x
(Should NMT X+6)
|
||
Accelerated condition
|
Intermediate Data
|
Data willing to
statistical analysis
|
Retest period / Shelf life
|
Significant change
|
Significant change
|
-NA-
|
Y = x
|
No Significant change
|
Yes
|
Y = 1.5x
(Should NMT X+6)
|
|
No
|
Y = x + 3
|
If no significant change at accelerated stability data
for 6 months.
Long-term and accelerated data showing little or no
change over time and little or no variability
X
|
Y
|
|
Accelerated
(6months)
|
Long Term
(9 months OK)
|
y = 2x (Should not more than X+12)
re-test date is 18 Months
|
Accelerated
(6months)
|
Long Term
(12 months OK)
|
y = 2x (Should not more than X+12)
re-test date is 24 Months
|
Accelerated
(6months)
|
Long Term
(18 months OK)
|
y = 2x (Should not more than X+12)
re-test date is 30 Months
|
Accelerated
(6months
|
Long Term
(24 months OK)
|
y = 2x (Should not more than X+12)
re-test date is 36 Months
|
Accelerated
(6months)
|
Long Term
(36 months OK)
|
y = x
re-test date is 36 Months
(No extrapolation beyond 36 months)
|
If significant change at accelerated stability data
for 6 months
X
|
Y
|
|
Accelerated
(6months)
|
Intermediate
9 months OK
|
y = 1.5x (should not more than x+6)
re-test date
is 13.5 months
|
Accelerated
(6months)
|
Intermediate
12 months OK
|
y = 1.5x (should not more than x+6)
re-test date
is 18 months
|
Where significant change occurs at the intermediate
condition, the proposed retest period or shelf life should not exceed the
period covered by long-term data. In
addition, a retest period or shelf life shorter than the period covered by
long-term data could be called for.
|
||
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