Analytical Method Development

Purpose

The purpose of this SOP is to develop an analytical method to test defined characteristics, of the finished pharmaceutical product, derived from product specific literature review report, technical feasibility report and lab manual to achieve quality target profile of the product.

Scope

Analytical method development will derive from the product specific literature review report, technical feasibility report and lab manual and applicable to develop the methods of finished pharmaceutical products of all dosage forms.

Procedure

Analytical method describes how chemical components in FPP are identified, separated and quantified. The purpose of AMD for a product is to verify or check various appropriate attributes of the product against a suitable acceptance criterion in order to establish its quality. 

Analytical method to be developed for certain set of FPP parameters shall be derived from product specific literature review report. 

The choice of analytical instrumentation and methodology is based on intended purpose.

Following factors are important in analytical method development.

Dosage Form

If product is compendia, the analytical method, described in the compendia monograph shall preferably be accepted without change, unless any definite benefit in using an alternate method is established. If the compendia method is accepted without change or any significant change within the acceptance criteria as per compendia, analytical method verification shall be done as per Verification SOP. If an alternate method is to be used, complete analytical method validation shall be performed as per Validation SOP.

If the product is non compendia, general monograph of dosage form and ICH Q6A will be used to select the FPP testing parameters and the acceptance criteria. All the test for the specific dosage form like appearance, DT, pH, viscosity, weight variation, optical rotation, surface tension, dissolution profile results of Reference Drug shall be used to set the time point and the acceptance criteria. AQbD approach shall be used.

Analytical Quality by Design (AQbD)

AQbD approach shall be used in the method development process to ensure method robustness during development stage. The following steps are involved in AQbD.

Analytical target profile (ATP)

ATP [ICH Q8 R (2)] defines the method requirements i.e. acceptance criteria and the level of measurement i.e. performance level characteristics such as precision, accuracy, range, sensitivity, robustness and the associated performance criterion.

ATP for analytical procedures comprises of 

a. Selection of target analyte (API and impurities), 

b. Selection of analytical technique (UV, GC, HPLC, Ion Chromatography, chiral HPLC, etc.), 

c. Choice of method requirements (assay, impurity profile or residual solvents). 

A method cannot be accurate and precise without acceptable specificity, linearity & range, robustness, sufficient peak resolution for accurate integration, and repeatability of injections, etc. To achieve an accurate and precise method the above important characteristics (specificity, precision, accuracy, and robustness) must be evaluated during method development as they provide an extensive data for setting method controls.

CQA (Critical Quality Attributes) 

CQA for analytical methods comprises of method attributes and method parameters that need to be controlled to ensure product quality. It can differ from one analytical technique to another, for example; 

a. CQA for HPLC (UV or RID) are buffers used in mobile phase, pH of mobile phase, diluent, column selection, organic modifier and elution method. 

b. CQA for GC method is oven temperature and its program, injection temperature, flow rate of gas, sample diluent and concentration. 

Physical and chemical properties of the drug substance and impurities can also describe CQA for analytical method development such as polarity, charged functional groups, solubility, pH value, boiling point and solution stability.

Determination of CQAs is based on the impact of quality attribute on the safety, efficacy and quality of the product.

Risk Assessment 

This step is vital in order to reach a confidence level that the method is reliable. Risk assessment is a systematic process for the assessment, control, communication and review of risks to the quality across the product lifecycle”. 

Once the technique is identified, risk assessment of the factors that may lead to possible variability in the method, like analyst methods, instrument configuration, measurement and method parameters, sample characteristics, sample preparation, and environmental conditions shall be considered. Risk assessment helps to increase the quality of a method which eventually links to the protection of the patient.

According to ICH Q9 (Quality Risk Management; QRM), risk assessment can be carried out in three steps viz., risk identification, risk analysis and risk evaluation. 

Before setting up method control strategy, method verification by accuracy, precision and robustness help in confirming the conformances to the pre-defined method performance requirements (ATP). 

Design of Experiments (DoE) in Analytical procedure

After determination of critical analytical method variables with initial risk assessment, design of experiments (DoE) shall be performed to confirm the variables based on the statistical data. DoE is an important tool of AQbD where the raw material attributes (e.g. solubility, pH) and process parameters (e.g. injection volume, run time, detection wavelength) are considered as input variables and output variables shall be the critical attributes (e.g. tailing factor, resolution, theoretical plates, area under curve etc.). 

Method Control Strategy

The method control is important while ensuring that the method is performing planned set of controls aimed at minimizing the variability in the process. The data generated during method development and method verification forms the basis of the control strategy and the factors identified to have risk shall be controlled. Method control strategy includes components like in-process controls, finishes product specifications and the associated methods and frequency of monitoring and control determined during development. 

Lifecycle Management  

For a particular analytical method AQbD ensure fitness of the method for its intended use. Method validation, verification and transfer is termed as ‘lifecycle management of analytical procedure’, which commence with establishment of ATP and continues till the methods are in use. Continual verification involves activities, which provide the assurance that the method is under control throughout its lifecycle.

API/ Excipients Characteristics 

Note: Prior to handling any API/Excipient and the chemical required for analysis of them, the Officer R&D shall train himself/herself on the MSDS of such chemical and shall follow the required safety procedure including the use of appropriate PPE, while handling them.

Officer R&D will collect the information about physico-chemical properties of APIs and excipients from literature review report which includes at least the following information:

Category

Chemical Name

IUPAC Name

Structure

CAS#

Molecular Formula

Molecular Weight

Appearance

Solubility

Identification

Ph

pKa value/s

Melting Point

Optical Rotation

Water determination

Sulfated Ashes

Assay

Impurities

Residual Solvents

Other

Container Closure System Information (CSS)

Testing parameter for container closure system shall be collected from literature review report and developed as per Container Closure System SOP. For the procurement of any material related to CCS qualification URS (User Requirement Specs) shall be raised. 

Literature Search for FPP Method Development

If the Finished pharmaceutical product is non compendia then,

Officer RND/Sr. Officer RND shall collect data about the method from official books, internet, journals, patents, API analysis method and previous experience of same class products.

All literature information related to specific API and FPP for the performance of physical, chemical and biological attributes should be searched before starting analysis.

Development of product specific parameters

Design of experiment will be implemented in the development of product specific testing parameters. All testing variables will be identified during method development. 

Some variables are described below:

Sr. Parameter Variables

1 Appearance Color

2 Identification Chemical Analysis, UV/IR Spectrum, Retention Time on HPLC Chromatogram, Retention Factor (RF) on TLC

3 pH Temperature and pH Value

4 Viscosity Time & Temperature, Used Spindle, 

5 Specific Gravity/Density/ Weight per Volume Temperature 

6 Surface Tension N.A

7 Extractable Volume N.A

8 Disintegration Medium

9 Dissolution Medium, Volume, Time, Speed, Class of Apparatus

10 Assay by UV Concentration, Wavelength

11 Assay by Titration Titration Type, Used Titrant, Volume of Titrant, Reagent/Indicator Type

12 Assay by HPLC Diluent, Mobile Phase, System Type (Gradient or Isocratic), Column Type and Temperature, Detector, Flow Rate, Retention Time

Appearance

A qualitative description of the drug product should be provided based on the comparison with RLD available in literature review report. The method used for observing the appearance shall depend upon the type of the dosage form e.g. tablet, capsule, suspensions, clear liquid, etc.

Identification

       The identification test should establish the identity of the drug or drugs present in the dosage forms. Identity test should be specific to the drug substance(s) [e.g. infrared (IR) spectroscopy]. If the test is non-specific, at least two orthogonal nonspecific tests should be used including Chromatographic procedure, UV/Visible spectroscopy, or IR.   

pH

pH is measured for liquid solutions, suspensions and sometimes for semi-solid preparations too with the help of suitably calibrated pH meter. The pH tolerance for ophthalmic dosage forms ranges from 3.0 to 8.6, depending upon buffering capacity of the formulation. 

The acceptance criteria for pH value of the formulation should be the one where the drug product is most stable.  

Extractable Volume

Extractable volume test is developed according to Q4B Annex 2(R1), USP<755>.

The test for extractable volume ensures that the amount of material filled into the product conforms to the labeled amount. The fill determination is performed by mass from which the volume is calculated by use of the density of the preparation.

The acceptance criteria is met if the net weight of the content of each of the 10 containers is not less than the labeled amount.

Disintegration Time (DT)

Disintegration USP<701> and BP Appendix XII A1 provide sound knowledge on how to determine disintegration time of a tablet. It is an important quality attribute to check the efficacy of excipients.

For this purpose Disintegration apparatus is used. The DT beaker must be filled the fluid that at the highest point of the upward stroke the wire mesh remains at least 15 mm below the surface of the fluid and descends to not less than 25 mm from the bottom of the vessel on the downward stroke. At no time should the top of the basket-rack assembly become submerged. The temperature of the fluid must be maintained at 37°C ±2°C prior to the DT test performance.

Dissolution

USP chapter <711> & <1092> define the dissolution procedure and will be followed during development of non-pharmacopoeia products, under the SOP for Dissolution Test.

Dissolution Test apparatus is used to perform the dissolution test required for various dosage forms for product release testing and its in vivo performance. 

Sterility Testing

The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any micro¬organisms which are to be revealed in the test.

 Development of assay testing procedure by UV

   Quantification of drug substance using spectrophotometer may be carried out by preparing solution in transparent solvent and measuring it’s absorbance at suitable wavelength. The wavelength normally selected is wavelength of maximum absorption (λmax), where small error in setting the wavelength scale has little effect on measured absorbance. Wavelength selection can be based on APIs wavelength that will be given in monograph or manufacturer’s method. The concentration should be adjusted to give an absorbance of approximately, 0.9, around which the accuracy and precision of the measurements are optimal.

The sample and standard of same concentration shall be prepared. Filtration of sample solution shall be carried out to eliminate the possible interferences.  

USP general chapter <857> can be used to develop assay procedure for finished pharmaceutical products

Development of assay testing procedure by titration

To develop assay by titration, nature of API, excipient and there compatibility is very important. The API testing method of titration shall be followed to develop the drug assay content (if required).

A blank titration/placebo titration is performed along with sample to find the response factor defined as the relation between consumed volume and ion quantity. 

The characteristics to be used to select the type of titration, the equivalence factor of the titrant and the titrant shall be used in calculations.

Titrations are usually not preferred for drug product assay because of non-specific nature of the technique particularly with regard to stability indicating specificity.

USP general chapter <541> and BP Appendix VIII explain the steps required for development and selection of titration method.

Development of assay and impurities testing procedure by HPLC

Development of assay and impurities is based on method that is available in API monograph or manufacturer’s method.

As far as possible, efforts shall be made to arrive at chromatographic conditions that are common to the Assay, impurities and dissolution methods. Difference in the injection volume may be considered as minor difference. However, if the specificity, detection limits and accuracy attributes of the tests require setting up totally different chromatographic parameters, the requirements of the tests shall have preference.

Following are the important and crucial steps that need to be followed during development of assay, impurity and preservatives.

Chromatographic conditions & system suitability

System suitability tests are an integral part of analytical method to verify its suitability for intended use. USP chapter <621> defines chromatographic conditions and system suitability parameters with acceptable limits. 

The system suitability parameters, like tailing factor, theoretical plates, and resolution are automatically computed by the chromatographic software while processing the data.

 Impurities

Identify and test the impurities during method development.

Following types of impurities can be found

Organic Impurities

They can be identified or unidentified, volatile or non-volatile, and include:

a. Process impurities

b. Impurities/Related compounds

c. Intermediates

d. Degradation products

 Residual Solvents

Organic volatile chemicals used in the making of drugs substances or excipients or in the preparation of drug products

Inorganic Impurities

They are normally known and identified and include:

a. Reagents, ligands and catalysts

b. Heavy metals and other residual metals

c. Inorganic salts

d. Other materials e.g. filter aids, charcoal

   Check parameters before starting analysis

Safety

The procedural and engineering control should be in place to make the execution of the method safe. The Analyst should be trained to follow the instructions that make the analysis safe. The PPE, as applicable should be sufficiently available.

Apparatus/Equipment

Calibrated and qualified equipment, instruments and other devices should be used for analytical method operations and sample preparation.

Training and qualification

Analyst should be trained and qualified to operate the apparatus and to carry out the test.

Operating Parameters

Qualified experimental configuration and ranges (including allowed adjustments supported by compendial sources or development and/or validation studies) critical to the analysis (like in case of HPLC: flow rate, column temperature, runtime, detection wavelength, gradient, head space sampler for GC etc.) should be applied. I.e. the instrument/equipment used for the analysis should be qualified in such a range that the parameter values to be set fall within it, e.g. if the method specifies weighing of 20 mg of a substance, a balance qualified in the range of 50 mg to 200 g cannot be used for the analysis.

Reagents& Chemicals

All reagents and chemicals including solvents and materials used in tests and assays, should be of appropriate quality.

In the preparation of reagent solutions or volumetric solutions, prescribed compendial procedures, if available, should be used.

The labels of reagent solutions or volumetric solutions used during analysis should include, but not limited to

• Name

• Date of preparation

• Expiry date or retest date

• Initials of the analyst

• Molarity or concentration (in case of volumetric solutions)

• Opening date (in case of containers)

• Storage conditions

The mobile phase prepared for the analysis should be labelled properly with Product name, Mobile phase mixture and ratio, date of  preparation and expiry date and enter in logbook every time.

The purified water should be used in analysis. 

Reference Standard and Materials

The primary standards or working standards will be used for the method development of assay, preservatives or impurities, wherever applicable.

The storage, usage conditions and handling instructions for primary/working standards should be strictly followed to avoid degradation and contaminations, which could result in additional impurities, lowering of potency and inaccurate analysis.

Retain the information supporting reference standards and materials which include qualification test reports and certificates of analysis (including relevant known impurity profile).

Confirmatory Testing

After the development of method, three replicate testing should be done by two different analysts. For the specific methods, such as HPLC methods, the placebo should also be analyzed. If no interference is observed from the placebo chromatogram at the retention times of the analytes (including the known impurities) and if repeat testing produces the results that meet the preset criteria for reproducibility (e.g. %RSD<2%), the method will be considered to be validatable.

Calculations

After analysis, perform the representative calculations for data analysis for tests based on label claim and specification. This includes a description of any mathematical transformations or formulas used in data analysis, along with a scientific justification for any correction factors used.

Analytical Method Development Protocol

Officer R&D (Analytical) shall prepare the product specific analytical method development protocol according to the protocol template.

Analytical method development protocol consists of following parameters:

a. Product information

b. Literature review 

c. Feasibility check

d. Analytical method development

e. Confirmatory testing

f. Summary and conclusions

Feasibility checklist  

The Technical feasibility report shall be prepared by RND department and the detail of availability of the instruments, chemicals and reagents and technique shall be checked from of the respective product. The detail shall be provided in the table below.  

Sr. No. Check Yes No Can be provided Target date Verification

1 Is Method of analysis available?

2 Is MSDS of API available?

3 Are the required safety controls available?

4 Are all the testing techniques available?

5 Are the Analysts trained for the type of technique?

6 Is Reference standard available?

7 Are impurities available?

8 Are all the equipment or column described in the method, available?

9 Are all the reagents and chemicals, used in the method, available?

10 Are all the consumables, used in the method, available?

11 Can the method be used to handle the expected load of samples in the QC within the required timeframe?

If answer to any one of the questions is ‘No’, then following should be done.

• Outsource the analysis or

• Carry out literature survey for finding an alternate method.

Analytical Method Development Report

All raw data (chromatograms, Testing Raw data sheets, print-outs, spectrums) of method development will be retained. Method development data will be attached with the method development report. 

Definitions and abbreviations

Sr. No. Terms/Words/

Statements Meaning/description

1. AMD Analytical method development

2. AMV Analytical method validation

3. DT Disintegration time

4. e.g. For example

5. etc Et cetera

6. FPP Finished Pharmaceutical product

7. GC Gas chromatography

8. HPLC High performance liquid chromatography

9. PD Product development

10. pKa Dissociation constant

11. PPE Personal Protective Equipment

12. QC Quality control

13. QA Quality assurance

14. RH Relative humidity

15. RT Retention time

16. SOP Standard operating procedure

17. UV/IR Ultraviolet/Infrared

18. USP United states pharmacopoeia

19. CAS Chemical Abstract Service

20. NA Not Applicable

References

Sr. No. Description

1. WHO good practices for pharmaceutical quality control laboratories Annex 1

2. ICH Guidelines; Q2R1, Q6b, Q2R1, Q8

3. ICH harmonized guideline impurities: guideline for residual solvents Q3 C (R6)

4. ICH Harmonized tripartite guideline impurities in new drug substances Q3 A (R2)