Starting materials, Byproducts, Intermediates, Reagents, ligands, and catalysts. Impurities which are not increased in stability studies and not formed during forced degradation studies are considered as process related impurities.

Process related impurities are formed during the synthesis, purification, and storage of the drug substance. Most of these impurities will be expected during the synthesis based on knowledge on synthesis process, type of chemicals/reagents used, also these should be confirmed with stability studies [15] and degradation studies. The impurity selection should be cross verified or challenged by stress studies or force degradation studies [16] .

In drug substances, impurities in final product specifications may be proposed based on the impurities detected in commercial process for setting better process control. Impurities which are above the qualification limits should be considered in specification with reporting and qualification thresholds based on its safe levels.

Where as in drug product demonstrating specificity (usually separation in chromatographic methods) for process related impurities with degradation impurities is requirement for a stability indicating method but there is no requirement to report process related impurities (as they are controlled in applicable raw materials) unless it is mentioned in respective pharmacopeial monograph.

Forced degradation studies should be performed to identify potential degradation products, characterization of degradation product, stability of active molecule, to understand the degradation pathways of drug, rate of degradation, to develop a stability indicating analytical method and to define shelf life.

Impurities which are increased during stability studies (Q1A) and/or formed during forced degradation studies are classified as degradation impurities/products.

The ICH Q1A guideline defines that the stress testing is designed to help “determine the intrinsic stability of the molecule by establishing degradation pathways in order to identify the likely degradation products and to validate the stability-indicating power of the analytical procedures used” [17] .

The selection of conditions for degradation studies and levels of target degradation is practiced as per common industry practices and available peer reviewed publications. ICH guidance Q1B “Photostability testing of new drug substances and products” serves as a guidance document on photo stability [18] .

WHO guidance document “Guideline on Submission of Documentation for Prequalification of Multi-source (Generic) Finished Pharmaceutical Products (FPPs) Used in the Treatment of HIV/AIDS, Malaria and Tuberculosis―GuideGenericSubmitDocFPPs_08_2005_WoAnnexes” [19] defines stress testing conditions in below Table 2.

The industry practices conditions include Hydrolysis at various pH (Oxidative, acid and base), photo degradation, and thermal degradation and humidity studies [20] [21] . Since the conditions varies for individual molecule, based on type of molecule and its physicochemical properties to attain degradation to a small extent, typically 10% - 30% loss of active by assay [19] . The degradation should not be performed by exposing to harsher conditions to form secondary degradation products (Secondary degradation products are degradation products of degradation products and in most cases, are not observed during stability studies). Most of the common degradation pathways include chemical reactions such as, hydrolysis/dehydration, oxidation, isomerization/epimerization, rearrangements, decarboxylation, dimerization/polymerization, photolysis and transformation products involving reaction with excipients/salt forms. Some of the most common reactive impurities include aldehydes/reducing sugars, peroxides, nitrates, nitrites, metals and solvents [22] . In some cases, compounds with Halogens (Chlorine, Fluorine, Bromine) are considered as Structural alerts, carcinogenic substances which require genotoxicity evaluation to fix the specification.

Enantiomers, non-superimposable mirror image stereoisomers with identical physico-chemical properties except that they rotate the plane of polarized light in opposite directions and by equal amounts [27] .

If one of the enantiomer is considered as active ingredient, then the other may/shall be considered as impurity/undesired.

In earlier days, there was not much focus on individual enantiomers and were not well studied or characterized due to limitation of analytical techniques. Current advanced techniques allow to define, study completely, separate and quantify stereoisomeric impurities [28] .

If applicable, stereoisomeric purity of starting material should be studied with a well resolved and validated stereoselective test procedure and possible formation of stereogenic center in individual synthetic process details and controls should be established [29] .

For a drug substance stability indicating method, it is required to perform forced degradation studies for chiral impurity methods and confirm its formation. If the chiral impurity increases in stability studies, then it should be monitored in drug product.

Based on decades of studies, knowledge of toxicological effects and to improve safety and efficacy of drugs, current requirements changed and replaced nonspecific heavy metal tests with specific quantifying techniques [43] and specifications [44] were included. The classical color comparison test in Pharmacopeia is deficient in detecting few metallic impurities. The standard solutions of all the metals are also of different solubilities due to the variable levels of reactivities of metals with sulfide ion, which also creates a problem in visual comparison with lead standard as shown in Figure 5 [45] .

Process catalysts and environmental contaminants may be present in drug substances, excipients, or drug products. These impurities may occur naturally, be added intentionally, or be introduced inadvertently. As elemental impurities do not provide any therapeutic benefit to the patient, besides continuous exposure might be toxic (as the toxicity is related to its extend of exposure), so their toxic levels in the drug product should be controlled within acceptable limits [46] .

Elemental impurities are classified into 4 categories [1, 2A, 2B and 3] based on the route of administration, limits and requirements for control varies. Risk assessment should be done for elements: Cd, Pb, As, Hg, Co, V, Ni, Tl, Au, Pf, Ir, Os, Ph, Ru, Se, Ag, Pt, Li, Sb, Ba, Mo, Cu, Sn, Cr as per ICH Q3 D [47] and USP <232> based on potential source, Evaluation of toxicity data with respect to route of administration, Establishment of a Permitted Daily Exposure (PDE) and Justification for higher levels than established PDE.

In drug product risk evaluation, potential sources of elemental impurities should be considered are elements intentionally added such as process catalysts, elements potentially present in the materials used to prepare the drug product, and elements potentially introduced from manufacturing equipment or container closure systems [46] [47] .

Based on risk assessment, it should be concluded to include testing of elemental impurities in routine testing for components of drug product or for drug product i.e. if elemental impurities are controlled in raw material (active and inactive) as part of routine testing, in drug product routine testing may not be required. ICH Q3 D defines different options (Option 1, Option 2a, Option 2b and Option 3) for risk assessment and control strategy (If batch analysis results are consistently less than 30% of the PDE then additional controls are not required).

As per FDA guidance, Elemental impurity risk assessment for ANDA’s filed and approved before Jan 1^st 2018 should be updated in annual updates, if any process changes are needed to be made to meet the Elemental impurities requirements they should follow appropriate regulatory filing strategy and for ANDA’s to be filed, it is a requirement to be considered for filing [46] . An overview of elemental impurities as per ICH Q3D to consider for risk assessment, presented below Figure 6.

ICH Q3 D classifies metallic impurities into Class 1, 2A, 2B and 3 with decreasing level of toxicity.

The specifications and class of impurities to be controlled varies on route of administration.

In general, typical analytical techniques followed for elemental impurities analysis

are ICP, ICP-MS. These can be analyzed with the method listed in USP <233>, if alternate methods are used those are to be validated as per ICH Q2, USP <1225>. There are few alternate analytical methods available and reported in research articles [45] .

・ It is an emerging topic.

・ Quality risk assessment complete documentation not provided.

・ Complete assessment report and related documents for ANDA’s filed before Jan 1^st 2018.

・ Excipient batch analysis data.

・ Consistency between batches which contains excipients like Sodium bicarbonate, Calcium carbonate.

・ More batch data is required when natural source excipients like sodium carbonate and sodium bicarbonate are used.

Usually GC and GC-HS methods are employed and in some cases HPLC, IC and other techniques are employed. If residual solvent method is specified in a monograph, the same can be followed or method specified in USP <467> can be used. USP <467> does not specify a method for class-3 solvents but FDA cites to control and report class 3 solvents also with a validated method. Usually residual solvents analysis is done by gas chromatography technique, but some of the class-3 solvents like Acetic acid, triethylamine, formic acid etc might not be accurately quantified with Head space sampler Gas chromatography, even in auto liquid sampler acetic acid may elute but the improper peak shape may pose problems in meeting the analytical method validation criterion (Figure 7) [Chromatogram eluted by GC -Auto Liquid sampler method on FID detector −250˚C using DB WAX column −30 m × 0.53 mm × 1.0 µm, Inlet temperature 240˚C, Carrier gas-Nitrogen 3.0 mL/min, Split ratio 1:5 and Makeup gas: 25 mL/min Nitrogen]. These solvents may be quantified using other techniques like High performance liquid chromatography (Figure 8) [Chromatogram eluted by HPLC, Reverse phase Grace Altima C18, 150 × 4.6 mm, 3 µm at 210 nm, 0.8 mL/min gradient flow; Mobile phase-A: 0.01 M sodium phosphate monohydrate

pH 2.0, Mobile phase-B Acetonitrile: Water −80:20 v/v], Ion exchange chromatography. Some of the drug substances manufacturing process use non-listed solvents, for those solvents there may be published research articles available or USP <467> methods can be modified. Scientists achieved separation and Quantification of Cyclopropylamine, Diethylamine and Triethylamine in Active Pharmaceutical Ingredients [50] .

As per ICH Q3C classification.

As the residual solvents limits were set as per the guidelines and quantified in individual excipient and drug substance, testing for solvents in drug product is not required. Compliance may be demonstrated through comprehensive risk assessment based on quantified results of residual solvents present in individual drug substance and excipients [51] .

If residual solvents are used in the manufacturing process of drug product, they should be quantified with validated analytical methods with ICH Q3C limits.

If a non-listed solvent is used in the process, then justification for limits proposed, control strategy and toxicity evaluation should be presented/executed.

・ Specification not included for Residual solvents used in drug product formulation.

・ Improper justification for residual solvents with limits not as per ICH Q3C, USP <467> and their effect on human drug quality.

・ All residual solvents not included in specification.

・ Drug substance residual solvents specification in line with ICH Q3C, USP <467> and DMF.

・ Excipient residual solvents not considered for assessment/ specification.

・ Excipient residual solvents methods not validated/ verified.

・ Tighten the solvents limits based on trend data.

・ Establish LOQ, LOD limits for all residual solvents and LOQ limits should be at least 50% of specification.

・ Unidentified peaks in samples which are to be analyzed with validated method.

Common analytical technique used is polymorphism by p X-ray diffraction. Method adopted should be specific to desired crystalline form of the compound based on drug substance manufacturing process. Method should be validated as per USP <1225> [23] .

In some cases XRD pattern of different polymorphic forms might be similar, then other techniques like Thermal analysis (DSC) or Raman spectroscopy should be used.

Omeprazole form A and form B can be identified by different techniques (e.g.: XRPD, FTIR and Raman spectrometer) among them Raman spectrometer technique is more specific as it can also identify amorphous forms along with crystalline forms. Polymorphism evaluation in Omeprazole is reported by Raman spectroscopy that Omeprazole™ form A is easily characterized by the absence of a band at 1364 cm⁻¹, which is observed in Omeprazole form B, and by the ratio of the relative intensities of 840.87 and 821.95 cm⁻¹ bands.

The ratio (intensity of 840.87 cm⁻¹ band/intensity of 821.95 cm⁻¹ band) is <1 for Omeprazole Form A, while the ratio is >1 for Omeprazole form B (Figure 9) [58] .

Fexofenadine hydrochloride form identification can be done by DSC (Melting point: 142˚C is Form-VIII, Melting point: 155.35˚C is Form X, Melting point: 138.61˚C is Form IX) [59] .

Very few monographs in USP have the specification for identification by polymorphism (e.g.: Colloidal Activated Attapulgite, Indomethacin).

・ Include test for polymorphism in specification.

・ Polymorphic stability of the molecule in drug substance and drug product to be demonstrated.

・ Demonstrate polymorphic stability throughout the drug substance and drug product stability.

・ Evaluate polymorphic stability effect on dissolution / drug product blending and/or unit operations.

・ Demonstrate possibility of existence of other forms in drug substance.

・ Validation of analytical technique used for polymorphic study.

LCMS, GC MS are normally used for genotoxic impurity detection and quantification to achieve desired limit of quantification. HPLC, UPLC and GC methods may be used in some cases.

Threshold of Toxicological Concern (TTC) concept was developed to define an acceptable intake for any unstudied chemical that poses a negligible risk of carcinogenicity or other toxic effects. The methods upon which the TTC is based are generally considered to be very conservative since they involve a simple linear extrapolation from the dose giving a 50% tumor incidence (TD50) to 1 in 10⁶ incidence cases, using TD50 data for the most sensitive species and most sensitive site of tumor induction [60] .

The concentration limits in ppm of genotoxic impurity in drug substance derived from the threshold of toxicological concern (TTC) can be calculated based on the expected daily dose to the patient using equation [60] [65] .

Concentration limit (ppm) = TTC [μg/day]/dose (g/day)

An overview of the genotoxic impurities guidance is presented in Figure 10. ICH guidance M7 classifies possible genotoxic impurities into 5 classes, which is presented in Table 3 below.

Standard risk assessments of known carcinogens assume that cancer risk increases as a function of cumulative dose. Thus, cancer risk of a continuous low dose over a lifetime would be equivalent to the cancer risk associated with an identical cumulative exposure averaged over a shorter duration.

ICH M7 defines limits for single impurity and for total of two or more impurities as per duration of treatment as per below Figure 11 limits [60] .

*Or other relevant positive mutagenicity data indicative of DNA-reactivity related induction of gene mutations (e.g., positive findings in in vivo gene mutation studies).

ICH M7 defines different control strategies with Options 1 - 5. Option 3 allows manufacturers to avoid release testing if the impurities are less than 30% of control in few batches.

Pharmacopeia’s are embracing this concept slowly however regulatory authorities are watchful and issuing deficiency to control the impurities. Bromochloropropionphenone impurity in Bupropion Hydrochloride, USP specifies this impurity at NMT 0.1% but as per literature impurity should be controlled at 4 ppm, FDA has cited this as a deficiency for the filers.

As per ICH M7 and S9 or respective impurity toxicological limits.

・ Possible limits not demonstrated: Genotoxicology study of a compound not demonstrated.

・ USP limits not adequate as compound has structural alerts.

・ Study or cut off for all possible structural alerts not demonstrated.

・ Limits should be tightened as per toxicological limits.

・ One-time study not adequate for genotoxic impurity and to be included in specification.

・ Spike & purge studies data to be presented for control option selected.