Category Archives: Drug Substance

Sample Quantity for Stability Studies

In the stability studies the sample must be stored in the stability chambers either in accelerated or in long term. So the sample quantity must be selected prior to stability studies.

The quantity of the sample depends on the tests to be performed during the stability studies. For example consider the following specification and calculate the sample quantity for each time point study.
Test
Specification
Description
White to off-white powder.
Identification by Infrared absorption
The IR absorption spectrum of the sample shall be concordant with that of Ranolazine standard spectrum.
Loss on drying [% w/w][Dry it in vacuum at 60±2°C for 3 hours]
Not more than 0.50
Related substances by HPLC [%]
Monoamide impurity
Not more than 0.15
Dimer impurity
Not more than 0.15
Major unknown individual impurity
Not more than 0.10
Total impurities
Not more than 1.0
Assay by HPLC [% w/w][on dried basis]
Not less than 98.0 and not more than 102.0
Calculation of sample quantity:
Test
Specification
Description
1g ( reusable)
Identification by Infrared absorption
300 mg
Loss on drying [% w/w][Dry it in vacuum at 60±2°C for 3 hours]
1g
Related substances by HPLC [%]
Monoamide impurity
1g
Dimer impurity
Major unknown individual impurity
Total impurities
Assay by HPLC [% w/w][on dried basis]
1g
Total quantity 3.3 g( rounded to 4 grams)
* For Description of drug substance we used one gram, as this can be re-used, we are not calculated in the total quantity.
For accelerated studies 
Time points: 0,1,2,3 and 6 months
Samples to be stored( 1M, 2M,3M and 6Months), so 4 points
For each point : 4 grams
for total accelerated studies: 4(time points) x 4 grams=16 g 
For Long Term studies 
Time points: 0,3,6,9,12,18,24,36,48 and 60 months
Samples to be stored (3,6,9,12,18,24,36,48 and 60 months ), so 9 points
For each point : 4 grams
for total Long term studies: 9(time points) x 4 grams=36 g 
Total sample:
Accelerated: 16 g + 5 g extra = 21 grams
Long term   : 36 g + 10g extra= 46 grams
Total sample = 67 grams (rounded to 70 grams)
This is an example, the specifications for each substance may vary. So based on the specification, sample quantity is going to change.
So treat each drug substance separately and calculate the sample quantity accordingly.
In some cases microbiological attributes required to be analysed in such cases aroung 10-15 grams of sample should be stored for each study
Store the sample in container closures same as marketed container.

Article Contributed by
Mr. Srikanth Nagabiru
M.Sc., DAC

Storage Conditions

The storage conditions of a drug substance play a vital role in the stability studies. These conditions are temperature and moisture and may vary from drug substance to drug substance and can be obtained by literature search or Merck index or pharmacopoea or any other relevant source.
The length of the stability study may vary from drug to drug. Generally this may vary from three years to five years.
The stability conditions such as temperature and humidity can be categorized into three. There are (a) long term (b) accelerated and (c) intermediate conditions.
Data from accelerated studies are used to evaluate the effect of short term excursions outside the label storage conditions( during shipping or handling) where as long term data is useful in assigning the retest period of the drug substance.
Study

General Case

Long term : 25°C ± 2°C/60% RH ± 5% RH or

Accelerated : 40°C ± 2°C/75% RH ± 5% RH

Intermediate : 30°C ± 2°C/65% RH ± 5% RH*

Refrigeration

Long-Term : 5°C ± 3°C

Accelerated: 25°C ± 2°C/60% RH ± 5% RH

Intermediate  : NA

Freezer Storage

Long term : – 20°C ± 5°C

Intermediate and Accelerated Conditions are not applicable for freezer storage
*It is up to the applicant to decide whether long term stability studies are performed at 25 ±2°C/60% RH ± 5% RH or 30°C ± 2°C/65% RH ± 5% RH.
**If 30°C ± 2°C/65% RH ± 5% RH is the long-term condition, there is no intermediate conditions
General case:
The conditions such as temperature and Humidity are give in the above table.
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. Testing at the intermediate storage condition should include all tests, unless otherwise justified.
“Significant change” for a drug substance is defined as failure to meet its specification.

Drug substances intended for storage in a refrigerator
The conditions such as temperature and Humidity are described in the above table .

Drug substances intended for storage in a freezer
The conditions such as temperature and Humidity are described in the above table . As described in the above table-I there is no accelerated storage conditions for drug substance. So testing on a single batch at an elevated temperature for an appropriate time should be conducted to address the effect of short term excursions outside label storage conditions(during shipping or handling)
Drug substances intended for storage below -20°C
Drug substances intended for storage below -20°C should be treated on a case-by-case basis.

Article Contributed by
Mr. Srikanth Nagabiru
M.Sc., DAC

Time Window for stability studies

Stability samples analysis should conducted for long term, accelerated and also for intermediate conditions. The stability samples should pull out from the respective chambers on the date of analysis mentioned in the stability protocol.

The analysis of the sample should be completed on the day that is mentioned in the stability protocol. This is general rule but it may not be possible to conduct studies on the same day due to some reasons. The reasons may be busy schedule of the chromatographic systems and man power availability and so on.

Then there is a need to keep some acceptance criteria conduct stability sample analysis. Then within how many days these studies should be conducted? what is the acceptance criteria? Is the acceptance criteria is same for all the conditions such as long term, accelerated and intermediate condition? what is the industry general practice? What is acceptance criteria for regulatory authorities?
Before answering to all the questions, regulatory authorities does not have a mention on the time window for stability samples analysis.
Then how to handle this issue……the solution to this problem is as follows…

Time window for analysis should be controlled at lab level by writing and following the acceptance criteria in the Standard operating procedure(SOP).

Samples analysis time window
The sample should be analyzed with in
7 days of due date for long term samples.
2 days of due date for accelerated samples.

Article Contributed by
Mr.Srikanth Nagabiru
M.Sc., DAC

Testing Frequency

For long term studies, frequency of testing and duration should be sufficient to establish the stability profile of the drug substance. The duration may vary from 3 years to five years depending on the stability of the drug substances. For drug substances with a proposed re-test period of at least 12 months, the frequency of testing at the long term storage condition should normally be every 3 months over the first year, every 6 months over the second year, and annually thereafter through the proposed re-test period.

In practice, testing of the samples for long term shall be done at initial, 3M,6M,9M and 12th month of the first year, 18 and 24th month of second year and 36, 48 and 60th month from third year onwards.

At the accelerated storage condition, a minimum of three time points, including the initial and final time points (e.g., 0, 3, and 6 months), from a 6-month study is recommended. Where an expectation (based on development experience) exists that 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.

In practice testing for accelerated stability shall be conducted at 0,1,2,3, and 6th months.

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, including the initial and final time points (e.g., 0, 6, 9, 12 months), from a 12-month study is recommended.

In practice testing at intermediate conditions shall be done at 0,3,6,9 and 12 th months.

Type of study Testing Frequency(in Months)
Accelerated 0,1,2,3 and 6 M
Long term 0,3,6,9,12,18,24,36,48 and 60 M
Intermediate 0,3,6,9, and 12 M

* Based on Duration of stability studies

Article Reviewed by
Mr.Srikanth Nagabiru
M.Sc., DAC

Stability Indicating Quality Parameters(Specifications)

Specification is a list of tests, reference to analytical procedures, and proposed acceptance criteria.

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 following

Physical
Appearance, melting point, clarity and color of solution, water, crystal modification(polymorphism), particle size and chirality
Chemical
increase in impurities and decrease in assay
Microbilogical
Total bacterial count, fungal count and absent for pathogenic microbes listed in the ICH

Note: The physical parameters such as polymorphism,chirality and particle size are specific to some drug substances. The quality unit must decide which parameter to be tested during stability studies based on the drug substance.

Analytical Procedures:
The analytical procedures used for stability studies are either from Pharmacopoea or In-house tests. Analytical methods should be stability indicating. Limit tests do not need validation, but other tests such as Chemical and microbiological procedures need validation.

Example:
Description: White to off-white powder.
Identification by Infrared absorption:
The IR absorption spectrum of the sample shall be concordant with that of Ranolazine standard spectrum.
Loss on drying [% w/w][Dry it in vacuum at 60±2°C for 3 hours]: Not more than 0.50
Related substances by HPLC [%]
Monoamide impurity: Not more than 0.15
Dimer impurity: Not more than 0.15
Major unknown individual impurity: Not more than 0.10
Total impurities : Not more than 1.0
Assay by HPLC [% w/w][on dried basis]: Not less than 98.0 and not more than 102.0

Physical and chemical attributes testing is performed regularly where as Microbiological attributes may be performed based on the customer requirement.

Article Contributed By
Mr.Srikanth Nagabiru
M.Sc., DAC

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.

For packaging of drug substances there are usually two types of materials used. 1. Primary packaging material and secondary packaging material.
Primary packaging material is the one which is direct contact with drug substance. It provides protection from air and moisture.
Eg: Transparent LDPE bags.
But LDPE bags are not good at moisture protection so use heat sealable laminate bag with low rate of water transmission.
In practice primary packing material should be of food grade.
Secondary packing materials are the one which provide protection from light and mechanical stress. Generally black LDPE bags and fiber drums or HDPE drums are used for this purpose.
For some drug substances, we may see usage of fillers or desiccant (silica) packs in HDPE drums.
The choice of packing material and mode of packing varied with the drug substances under study. In some drug substances there is need for vacuum packing or nitrogen packing or Helium packing.
Examples for container closure systems of different drug substances:
Example 1:  Rosuvastatin Calcium is packed in a clear low-density polyethylene bag under Nitrogen atmosphere and this bag is placed inside a Black color low-density polyethylene bag. Each bag is individually tied with a thread / plastic strip. This double polythene bag is placed inside a Triple Laminated Bag, and then this Triple laminated bag is sealed and kept inside a Blue Colour HDPE Container to simulate the commercial pack.
The above packing conditions may be used for Rosuvastatin Calcium, Memantine Hydrochloride, Ezetimibe etc..
Example 2: Eplerenone is packed in clear low-density polyethylene bag and tied with a thread / seal. This bag is placed inside a black colour low-density polyethylene bag and tied with a thread / seal. This double polythene bag is placed. inside a triple laminated bag and sealed. This sealed triple laminated bag is placed inside a HDPE container with lid and this outer container is sealed to simulate the commercial pack.

The above packing conditions may be used for Pregabalin, Itraconazole, Omeprazole, Ranolazine etc…

Article contributed by
Mr. Srikanth Nagabiru
M.Sc., DAC

Batch Selection for drug substances

Stability studies shall be conducted on at least three batches of the drug substance. The batches may be either produced in pilot scale or commercial scale. The pilot batch should be one eight of the production batch. But it should be of same synthetic route and overall quality of the batches should be the same for rest of the production batches.

Three commercial batches may be used for the stability studies. Commercial batch means batches which are commercially distributed to the customer. It may be either direct production batch(as is) or blended batch(more than one production batch).

General batches
In practice, three process validations batches shall be taken for stability studies. If the blending batch size is not same as production batch, then manufacturer may go for other batches.

For example
Each production batch size 30Kg.
Blending batch size 50Kg
Three production batches(1,2,3) 3*30=90 kg
For 3 blending batches require 150kg
In such cases next consecutive production batches to process validation, will go to blending.
Next two production batches(4,5) 2*30=60kg
Total production batches(5) 5*30=150kg

In addition to the three batches as above, one commercial batch shall be added to the long term stability on every year [addon batch], even after stability studies completion. This is generally first batch of the current year.

Micronized batches
If there is any physical modification to the drug substances such as particle size changes (micronization), these changes should be taken into account and stability studies should be conducted separately. Three successive batches should be selected and placed under stability.This should be treated as new product and stability studies should be conducted similar to the new drug. For this also manufacturer must add new batch to the stability studies[add on batch]

For example, Omeprazole is having particle size modification such as Micronization, so manufacturer should keep the separate stability testing for micronized material, in addition to the original material stability studies.

Reprocessing batches
During the manufacturing process the drug substance may fail to comply with specifications, in such cases the material may go for reprocessing or reworking.

Reprocessing means repeating the manufacturing process steps(repeating a crystallization step or other appropriate chemical or physical manipulation steps (e.g., distillation, filtration, chromatography, milling)) required to get the material within the specifications.

As we are performing the reprocessing(additional processing) to the drug substance, these batch/batches must go for stability studies. For these batches only long terms stability studies are conducted.

Reworking Batches
Reworking is Subjecting an intermediate or API that does not conform to standards or specifications to one or more processing steps that are different from the established manufacturing process to obtain acceptable quality intermediate or API (e.g., recrystallizing with a different solvent).

For reworking batches, stability studies should be conducted. Both accelerated as well as long term studies should conducted.

Batches manufactured with Recovery Solvents
Manufacturing process may some times use recovery solvents. .The solvents may be recovered from mother liquor or filtrates of reactants, intermediates, or the API.

If the batch is manufactured with recovery solvents, then also one batch of the samples should be verified to evaluate the effect of that change on the stability of the drug substance.

Change in the Route of synthesis
If there is any change in the route of synthesis of drug substances, there is a need for separate stability studies in-order to evaluate the effect of that change on the stability of the drug substance. This can be treated as new drug substances and do the stability studies.

Article contributed by
Mr. Srikanth Nagabiru
M.Sc., DAC

Terminology of Drug substance Stability

Accelerated testing

Studies designed to increase the rate of chemical degradation or physical change of a drug substance or drug product by using exaggerated storage conditions as part of the formal stability studies. Data from these studies, in addition to long term stability studies, can be used to assess longer term chemical effects at non-accelerated conditions and to evaluate the effect of short term excursions outside the label storage conditions such as might occur during shipping. Results from accelerated testing studies are not always predictive of physical changes.

Bracketing

The design of a stability schedule such that only samples on the extremes of certain design factors, e.g., strength, package size, are tested at all time points as in a full design. The design assumes that the stability of any intermediate levels is represented by the stability of the extremes tested. Where a range of strengths is to be tested, bracketing is applicable if the strengths are identical or very closely related in composition (e.g., for a tablet range made with different compression weights of a similar basic granulation, or a capsule range made by filling different plug fill weights of the same basic composition into different size capsule shells). Bracketing can be applied to different container sizes or different fills in the same container closure system.

Climatic zones

The four zones in the world that are distinguished by their characteristic prevalent annual climatic conditions. This is based on the concept described by W. Grimm (Drugs Made in Germany, 28:196-202, 1985 and 29:39-47, 1986).

Commitment batches

Production batches of a drug substance or drug product for which the stability studies are initiated or completed post approval through a commitment made in the registration application.

Container closure system

The sum of packaging components that together contain and protect the dosage form. This includes primary packaging components and secondary packaging components, if the latter are intended to provide additional protection to the drug product. A packaging system is equivalent to a container closure system.

Drug substance

The unformulated drug substance that may subsequently be formulated with excipients to produce the dosage form.

Expiration date

The date placed on the container label of a drug product designating the time prior to which a batch of the product is expected to remain within the approved shelf life specification if stored under defined conditions, and after which it must not be used.

Formal stability studies

Long term and accelerated (and intermediate) studies undertaken on primary and/or commitment batches according to a prescribed stability protocol to establish or confirm the re-test period of a drug substance or the shelf life of a drug product.

Intermediate testing

Studies conducted at 30°C/65% RH and designed to moderately increase the rate of chemical degradation or physical changes for a drug substance or drug product intended to be stored long term at 25°C.

Long term testing

Stability studies under the recommended storage condition for the re-test period or shelf life proposed (or approved) for labeling.

Mass balance

The process of adding together the assay value and levels of degradation products to see how closely these add up to 100% of the initial value, with due consideration of the margin of analytical error.

Matrixing

The design of a stability schedule such that a selected subset of the total number of possible samples for all factor combinations is tested at a specified time point. At a subsequent time point, another subset of samples for all factor combinations is tested. The design assumes that the stability of each subset of samples tested represents the stability of all samples at a given time point. The differences in the samples for the same drug product should be identified as, for example, covering different batches, different strengths, different sizes of the same container closure system, and, possibly in some cases, different container closure systems.

Mean kinetic temperature

A single derived temperature that, if maintained over a defined period of time, affords the same thermal challenge to a drug substance or drug product as would be experienced over a range of both higher and lower temperatures for an equivalent defined period. The mean kinetic temperature is higher than the arithmetic mean temperature and takes into account the Arrhenius equation.

When establishing the mean kinetic temperature for a defined period, the formula of J. D. Haynes (J. Pharm. Sci., 60:927-929, 1971) can be used.

New molecular entity

An active pharmaceutical substance not previously contained in any drug product registered with the national or regional authority concerned.  A new salt, ester, or non-covalent-bond derivative of an approved drug substance is considered a new molecular entity for the purpose of stability testing under this guidance.

Pilot scale batch

A batch of a drug substance or drug product manufactured by a procedure fully representative of and simulating that to be applied to a full production scale batch. For solid oral dosage forms, a pilot scale is generally, at a minimum, one-tenth that of a full production scale or 100,000 tablets or capsules, whichever is the larger.

Primary batch

A batch of a drug substance or drug product used in a formal stability study, from which stability data are submitted in a registration application for the purpose of establishing a re-test period or shelf life, respectively.  A primary batch of a drug substance should be at least a pilot scale batch.  For a drug product, two of the three batches should be at least pilot scale batch, and the third batch can be smaller if it is representative with regard to the critical manufacturing steps.  However, a primary batch may be a production batch.

Production batch

A batch of a drug substance or drug product manufactured at production scale by using production equipment in a production facility as specified in the application.

Re-test date

The date after which samples of the drug substance should be examined to ensure that the material is still in compliance with the specification and thus suitable for use in the manufacture of a given drug product.

Re-test period

The period of time during which the drug substance is expected to remain within its specification and, therefore, can be used in the manufacture of a given drug product, provided that the drug substance has been stored under the defined conditions. After this period, a batch of drug substance destined for use in the manufacture of a drug product should be re-tested for compliance with the specification and then used immediately. A batch of drug substance can be re-tested multiple times and a different portion of the batch used after each re-test, as long as it continues to comply with the specification.  For most biotechnological/biological substances known to be labile, it is more appropriate to establish a shelf life than a re-test period. The same may be true for certain antibiotics.

Storage condition tolerances

The acceptable variations in temperature and relative humidity of storage facilities for formal stability studies. The equipment should be capable of controlling the storage condition within the ranges defined in this guideline. The actual temperature and humidity (when controlled) should be monitored during stability storage. Short term spikes due to opening of doors of the storage facility are accepted as unavoidable. The effect of excursions due to equipment failure should be addressed, and reported if judged to affect stability results. Excursions that exceed the defined tolerances for more than 24 hours should be described in the study report and their effect assessed.

Stress testing (drug substance)

Studies undertaken to elucidate the intrinsic stability of the drug substance. Such testing is part of the development strategy and is normally carried out under more severe conditions than those used for accelerated testing.

Supporting data

Data, other than those from formal stability studies, that support the analytical procedures, the proposed re-test period or shelf life, and the label storage statements.  Such data include (1) stability data on early synthetic route batches of drug substance, small scale batches of materials, investigational formulations not proposed for marketing, related formulations, and product presented in containers and closures other than those proposed for marketing; (2) information regarding test results on containers; and (3) other scientific rationales.

Introduction to stability studies on Drug substances

Stability is defined as the capacity of a drug substance to remain within the established specifications to maintain its identity, strength, quality and purity throughout the retest or expiration dating period

The purpose of stability testing is to provide evidence on how the quality of a drug substance varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a retest period for the drug substance and recommended storage conditions.

Stability is an essential factor of quality, safety and efficacy of a drug substance. A drug substance, which is not of sufficient stability, can result in changes in physical (like appearance, melting point, clarity and color of solution, water, crystal modification(polymorphism) or particle size) as well as chemical characteristics (increase in impurities and decrease in assay) and microbiological attributes (Total bacterial count, fungal count and for pathogenic microbes).

Three commercial batches to be selected for stability studies and are conducted in the container closures which simulate the market containers. There should be separate stability studies for drug substance physical modifications such as micronization.

The storage conditions for drug substances may be classified into accelerated and long term, where as intermediate conditions are required if the drug substances failed in accelerated stability studies.

Stability studies 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. Validated stability-indicating analytical procedures to be applied.

The testing frequency for stability studies determined by the type of stability, either long term or accelerated or intermediate. For accelerated testing there should be minimum of three time points for 6 months study, and for long term studies, every three months for first year, every six months for second year and every 12 months from third year onwards. In case of intermediate studies, 3,6,9 and 12 months for a 12 months study.

The stability data evaluation and extrapolation of stability data are performed by using ICH guidance (Q1E). In the data evaluation, we are going to verify the data variability during the stability studies and in extrapolation we need perform statistical analysis on stability data obtained.

The storage statements are drawn from the stability studies. For example if the drug substance is stored at 25°C in controlled condition, they we may go for storage statement like” Protected from light, at a temperature below 25 °C”, if the drug substances stored at refrigerator conditions, then we may go for statement like “Storage in a refrigerator 2 to 8 °C” and so on.

Stress testing is likely to be carried out on a single batch of the drug substance. Stress testing of the drug substance can help identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule and validate the stability indicating power of the analytical procedures used. The testing should generally include temperatures above than accelerated testing, humidity (>75%RH), hydrolysis (acid and base), oxidation and photolysis of the drug substance.  The testing should also evaluate the susceptibility of the drug substance to hydrolysis across a wide range of pH values when in solution or suspension.

From analysis stand point, stability studies are conducted on Photo Diode Array(PDA) detector for knowing the purity factor of the drug substance and impurities. In addition this is mass balance is another factor for consideration during stability studies.

These studies are very important during the regulatory audits. It is one of the parameter that every auditor is going to ask. So be cautious. A though knowledge about these studies are very important . Let begin to learn from industry experts.

Author is stability studies expert in reputed pharmaceutical industry

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