preclinical development handbook || drug impurities and degradants and their safety qualification

1015

30 DRUG IMPURITIES AND DEGRADANTS AND THEIR SAFETY QUALIFICATION

Robin C. Guy Robin Guy Consulting, LLC, Lake Forest, Illinois

Preclinical Development Handbook: Toxicology, edited by Shayne Cox GadCopyright © 2008 John Wiley & Sons, Inc.

Contents

30.1 Introduction 30.2 Types and Sources of Impurities

30.2.1 Manufacturing Impurities 30.2.2 Degradants 30.2.3 Leachables and Extractables

30.3 Regulations 30.3.1 International Conference on Harmonisation (ICH) Guidance 30.3.2 U.S. FDA (CDER)

30.4 Technigues 30.4.1 General 30.4.2 Ultraviolet and Visible (UV/Vis) Spectroscopy 30.4.3 Atomic Absorption Spectroscopy 30.4.4 Thin Layer Chromatography (TLC) 30.4.5 High Pressure Liquid Chromatography (HPLC) 30.4.6 Mass Spectrometry (MS) 30.4.7 Gas Chromatography (GC) and Combination of GC and MS 30.4.8 MS/MS 30.4.9 HPLC/Mass Spectrometry (LC/MS or LC/MS/MS) 30.4.10 Common Problems

30.5 Toxicity Assessment 30.5.1 Literature - Based Approaches 30.5.2 Common Testing Requirements for New Impurities 30.5.3 Study Design Based on Exposure, Route, Dosage, and Other Factors 30.5.4 Documentation Content and Format


30.6 Bridging Studies 30.7 Discussion Regarding Testing the Drug with the Impurity Versus Actual Impurity Acknowledgments References

30.1 INTRODUCTION

In the United States, since very early times, traveling salespeople touted their latest miracle cures with enough publicity and fanfare to attract many to their circus - like shows. These cures were ointments, elixirs, and creams that would cure everything including obesity, diabetes, tuberculosis, and cancer [1] . Unfortunately, almost all of these products were either toxic or not effi cacious. Even some vaccines were not safe, as evidenced in the deaths of thirteen children from St. Louis, Missouri after receiving an inoculation from a diphtheria antitoxin that was contaminated with tetanus [2] . This incident prompted Congress to enact the Biologics Control Act of 1902, a law that required inspections of the manufacturers and sellers of biological products, including testing of these products for purity and strength [3, 4] .

In 1906, due to the outcry from the public after publication of Upton Sinclair ’ s novel, The Jungle , the original Food and Drug Act was passed by Congress. The law prohibited adulteration and misbranding of food and drugs. Offending products could be seized and condemned and offending persons could be fi ned and jailed. Drugs had either to abide by standards of purity and quality set forth in the United States Pharmacopoeia (USP) and the National Formulary (composed of committees of physicians and pharmacists; these are now combined) or meet individual stan-dards chosen by their manufacturers and stated on their labels. Adulteration was defi ned as the removal of valuable constituents, the substitution of ingredients so as to reduce quality, the addition of deleterious ingredients, and the use of spoiled animal and vegetable products. Misbranding was making false or misleading label statements regarding a food or a drug as well as the failure to provide required information in labeling.

Sulfanilamide, a drug used to treat streptococcal infections, had been used safely for years in tablet and powder form. For marketing to children, a company prepared the drug in liquid form after dissolving it in diethylene glycol and adding a raspberry fl avor. Unfortunately, the 1906 law did not address testing products for toxicity. Diethylene glycol, a chemical normally used in antifreeze, is a deadly poison. Over 100 people died, most of whom were children. Another public outcry from this incident helped pass the Food, Drug, and Cosmetic Act in June 1938. This new law included cosmetics and medical devices and was instrumental in providing evidence of safety in pharmaceutical products prior to administration in humans. It also authorized factory inspections and gave the agency the ability to use injunctions to enforce the law.

Impurities in drug products have triggered numerous laws and amendments. In 1941 almost 300 people were killed or injured by ingestion of sulfathiazole tablets that were contaminated with phenobarbital. This caused the FDA to revise manu-

facturing and quality control requirements, leading to what would later be called Good Manufacturing Practices (GMPs) [5] . In 1945, batch certifi cation by the FDA became a requirement for penicillin. The Penicillin Amendment requires FDA testing and certifi cation of safety and effectiveness of all penicillin products; later amendments extended this requirement to all antibiotics. In 1983 FDA testing was found to be no longer necessary and was abolished [6] .

Through the years, the U.S. Food and Drug Administration (FDA) and other international regulatory agencies, working alone or with industry representatives, have set regulations and guidelines for the handling of impurities in pharmaceuti-cals. As per the GMPs, it is essential that the purity of the pharmaceuticals is pre-served from manufacturing through delivery to the consumer.

Most drugs are not administered as neat chemicals but are mixed with inactive ingredients to form the specifi c drug product. Many have impurities, due to manu-facturing, leachables, or degradants. Impurities are chemicals contained in raw mate-rials or formed during manufacture, storage, or use. Their properties may be different from the desired product with respect to activity, effi cacy, and safety. These impuri-ties may be either intentional or unintentional. Intentional impurities may be due to the raw material itself or excipients. Unintentional impurities may occur due to a few determinants, including interactions between the other materials in the drug product, leaching from containers, or degradation due to environmental conditions. Regulations and guidances exist for the safety assessment of impurities. Many focus on different levels of testing, based on the amount of impurity detected. There are many different methods and equipment available for the analysis of impurities. Safety or toxicology studies to test the impurity may be needed, and there are guid-ances for the selection and design of these studies.

30.2 TYPES AND SOURCES OF IMPURITIES

Impurities may come from a variety of sources. Manufacturing impurities, leachable impurities, and degradants are the most common.

30.2.1 Manufacturing Impurities

Manufacturing impurities can come from raw materials, active ingredients, excipi-ents, process impurities and packaging impurities.

Raw Material Impurities Contamination from raw material sources is important to control. The highest quality starting material is essential to obtain a high quality product. Raw materials with impurities may be cleaned up, and it is important to do so before they have a chance to interact with other materials in the formulation that can lead to additional impurities. Cleaning up a raw material such as a chemical is usually easier than cleaning up natural materials. The latter may contain complex sample matrices that include similar chemical compounds. In addition, the pharma-cologically active compounds are usually present in small amounts and therefore may be diffi cult to separate out from the impurities. Thus, highly sophisticated ana-lytical methodologies must be developed to ensure that the natural drug is uniform and pure.

TYPES AND SOURCES OF IMPURITIES 1017


Compounds Related to Parent Pharmaceuticals may contain impurities that are similar in structure to the parent material. These impurities may be more diffi cult to detect and isolate depending on how similar the structure is as compared to the parent material. A chromatogram may indicate a “ shoulder ” on the main peak or an eluting peak indicating such an impurity. An example is given in Fig. 30.1 .

Spurious Compounds Although each known degradation pathway is considered, and every attempt is made to decrease or obliterate the impurities, pharmaceutical chemistry can still be fi ckle. The additional unpredicted peak found on the chro-matogram or the inexplicable tan fl ecks in a powder material could be an extract-able – leachable, a residual solvent, or an unforeseen degradant. These must be investigated and, depending on the amount of the impurity, must be identifi ed. The active pharmaceutical ingredient (API) from a natural material is an example of how complicated raw materials and impurities can become.

Active Ingredients Impurities arise from the manipulation of an active ingredient in a pharmaceutical. These impurities may be formed due to process or storage conditions.

Excipients An excipient is any inactive ingredient that is intentionally added to therapeutic or diagnostic product, but is not intended to exert a therapeutic effect at the intended dosage. Excipients may act to improve product delivery (e.g., enhance absorption or control release of the drug substance) or allow the product to be for-mulated (e.g., tablet). Examples of excipients include fi llers, extenders, diluents, wetting agents, solvents, emulsifi ers, preservatives, fl avors, absorption enhancers, sustained - release matrices, and coloring agents [7] . Within the context of this guid-ance, the term excipient applies to macromolecular substances, such as albumin, or substances such as amino acids and sugars that are used in drug and biological products. It does not, however, apply to process - or product - related impurities (e.g., degradation products, leachates, residual solvents) or extraneous contaminants. Excipients may interact with the active ingredient or container to form impurities or they may be formed due to process or storage conditions.

FIGURE 30.1 Chromatogram showing eluting peak.

Chromatogram of a sample with an impurity (A) vs. sample with the impurity removed (B)

shoulder

0 1 2 minutes 0 1 2 minutes

(a) (b)

Process Impurities Process impurities, primarily residual solvents, are organic volatile impurities that remain in pharmaceuticals, even after processing, as well as oxygen left in lyophilized vials. Both the United States Pharmacopoeia (USP), the offi cially recognized drug standard - setting compendium [8] , and the International Conference on Harmonisation of Technical Requirements for Registration of Phar-maceuticals for Human Use (ICH), Q3C [9] , address commonly used chemical sol-vents, the latter of which has been adopted by the European and Japanese pharmacopoeias. Other process impurities may be due to by - products of synthetic reactions, chiral or stereoisomeric impurities, contamination, and residual process intermediates or reagents.

Biologics and biotechnology products have similar process impurity issues. The process needs to ensure that there are no residual cellular components in the bio-logic product. Biotechnology processing needs to avoid causing structural deformi-ties to the protein. In all cases, the process must be scrutinized closely. Checking for impurities at various steps throughout the manufacturing phase may help to pin-point where the impurities are produced.

Packaging Impurities Packaging impurities can also be an issue in pharmaceuti-cals. These impurities can occur when chemicals from the packaging of the pharma-ceutical are found in the product itself. These are discussed further in Section 30.2.3 .

30.2.2 Degradants

Degradants are formed from the breakdown of the drug substance or product, pos-sibly due to storage conditions, interaction of ingredients from the container, label or closing systems, or treatment of the material.

The ICH has published guidelines for the identifi cation and qualifi cation of degradant - related impurities. These guidelines take into account new and existing drugs and formulations. A forced degradation test is essential to assist in the deter-mination of identifying any degradants and their degradation pathways. Forced degradation studies can be useful in the determination of the chemical and physical stability of crystal forms, the stereochemical stability of the drug substance alone and in the drug product, and for differentiating drug substance – related degradation products in formulations. Forced degradation tests are important to determine if extreme conditions will cause any impurities in the drug product. Forced degrada-tion conditions can include storage under high temperatures, high humidity, extreme pH, freeze – thaw cycles, oxygen exposure, or UV light for specifi c time intervals. At certain timepoints, samples of the drug product are taken and analyzed for purity. One important consideration is that forced degradation experiments may only produce or release impurities into the drug product that would not generally be present during normal storage and handling conditions. A limitation of forced deg-radation testing is that a compound may not necessarily degrade under a given stress condition.

There are no specifi cs in the ICH guidelines for pH levels, temperature, conditions, or light intensity for forced degradation studies. However, for photodegradation, ICH Q1B [10] suggests that the light source should produce combined visible and ultraviolet (UV, 320 – 400 nm) outputs, and that exposure levels should be justifi ed.

TYPES AND SOURCES OF IMPURITIES 1019


Degradation products if detected in signifi cant amounts should be identifi ed, tested for, and monitored against appropriately established acceptance criteria, according to ICH guidances [11, 12] . Investigation and identifi cation of some specifi c degradation products may not be necessary if it has been proven that they are not produced under accelerated or long - term storage conditions [10, 13] . In addition, forced degradation may not be necessary when the routes of degradation and the suitability of the analytical procedures can be determined through use of data from stress testing of drug substance, reference materials for process impurities and degradants, data from accelerated and long - term studies on the drug substance, and data from accelerated and long - term studies on the drug product [14] . Additional corroborative information on the specifi city of the analytical methods and on the degradation pathways of the drug substance may be available from literature sources.

Degradants will obviously vary for different drug products. Small chemicals may produce degradants that are similar to the parent compound. Biotechnological and biological products may encounter protein variants including truncated fragments, deamidated, oxidized, isomerized, aggregated forms, and mismatched disulfi de links [15] . In these studies, there may also be diffi culties in working with unstable degra-dation products.

As previously mentioned, many factors play a role in the formation of degradant products. Raw material handling is important to control early in the production phase. There should be no alteration of the material in any manner by chemicals or processing during manufacture. Changes in products can even lead to mutagenic degradants or degradants with increased immunogenicity, especially in biologics. The packaging used in various stages of the process may produce degradants. In addition, the choice of sterilization techniques may also play a role in producing degradation products, as different techniques may produce different degradants. Instability can lead to degradation over time. These factors would need to be taken into consideration prior to fi nalizing manufacturing, handling, and storage procedures.

30.2.3 Leachables and Extractables

Leachables and extractables are chemicals that may migrate from packaging or labeling, producing contamination in the drug product. These chemicals may be released throughout different stages of manufacturing, during storage or shipment of the drug components, during contact with processing materials in the plant, and even at the fi nal stage while the drug product is in its primary storage container or in contact with labeling materials.

Extractables are compounds that can be extracted from the container, from the coatings of the container closure system, or from the label during forced degradation conditions including contact with an appropriate solvent or high temperature [16] . Forced degradation experiments may only release impurities into the pharmaceuti-cal that would not generally be present during normal storage and handling conditions.

Leachables are compounds that migrate into the formulation from the label, container components, or coatings of the container and closure system as a result of direct contact with the formulation [17] .These are impurities that have formed

REGULATIONS 1021

during normal storage and handling conditions. Certain types of storage or treat-ment conditions may also produce leachables more readily, including sterilization procedures.

Sources of leachable and extractable contamination from packaging may include accelerators, adhesives, antioxidants, coatings, components of the container or closure system, excipients, plastic or rubber components, plasticizers, processing aids, and vulcanizing agents. Plasticizers are used to make plastics more fl exible. Plasticiz-ers such as bis(2 - ethylhexyl)phthalate (BEHP) may be present throughout package manufacturing. Acrylonitrile is a leachable that may be present in manufactured products and is found in a number of pharmaceuticals [16] . Another potential source of leachable or extractable contamination is nitrosamines, which are found in prod-ucts made of rubber. Rubber and rubber products are used in packaging, in closure components, and in devices, primarily in O - rings. Inhalers are a medical device that is prepared with various plastic, rubber, and stainless steel components. The FDA has a draft guidance for inhalers, which basically states that the levels of degradation products and impurities should be determined by means of stability indicating methods, and that any impurities or degradation products appearing at levels 0.10% or greater should be specifi ed [18] . Many drug products are distributed or adminis-tered in packages made of plastic and rubber components; therefore, phthalates and nitrosamines could come into contact with the drug product.

30.3 REGULATIONS

Both the U.S. Food and Drug Administration (FDA) and the ICH offer a plethora of information on dealing with impurities in pharmaceuticals. While the FDA is the regulatory authority in the United States, the ICH is an organization that brings together regulatory authorities and pharmaceutical associations of Europe, Japan, and the United States, in addition to experts from other non - ICH countries to discuss scientifi c and technical aspects of product registration. The purpose of the ICH is to make recommendations to achieve greater harmonization in technical guidelines and requirements for product registration in order to reduce or obviate the need to duplicate the testing carried out during the research and development of new pharmaceuticals.

It is essential that proper analyses be conducted for impurities. Knowledge gained from these analyses can be used to guide formulation development and improve manufacturing and packaging processes.

There are consequences when impurities are undetected or observed at higher levels than reported. For regulatory submissions for marketing applications, current FDA and ICH guidance recommends inclusion of the results, including chromato-grams of stressed samples, demonstration of the stability - indicating nature of the analytical procedures, and the degradation pathways of the drug substance in solu-tion, solid state, and drug product. The structures of signifi cant degradation products and the associated procedures for their isolation and/or characterization also are expected to be included in the application. For the latest updates on these guidances and for more details, check the FDA or ICH websites ( www.fda.gov , www.ich.org ). The following discussions are summaries of the guidances and include authentic excerpts but are not intended to be all inclusive.


30.3.1 International Conference on Harmonisation ( ICH ) Guidance

Q 3 A The ICH Guidance for Industry, Q3A Impurities in New Drug Substances , Revision 1, was published February 2003 [11] . It is intended to “ provide guidance for registration applications on the content and qualifi cation of impurities in new drug substances produced by chemical syntheses and not previously registered in a region or member state. ” A new drug substance is not the fi nal marketed product, but the active ingredient used in the marketed product. An active ingredient is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the human body. The active ingredient does not include intermediates used in the synthesis of such ingredient. The term includes those components that may undergo chemical change in the manufacture of the drug product and be present in the drug product in a modifi ed form intended to furnish the specifi ed activity or effect (21 CFR 210.3(b)(7) and 21 CFR 314.3(b)). Impurities in new drug substances are addressed from both a chemistry and a safety perspective.

The guidance is not intended to apply to new drug substances used during the clinical research stage of development. Nor does it cover the following types of drug substances: biological/biotechnological, peptide, oligonucleotide, radiopharmaceuti-cal, fermentation products and associated semisynthetic products, herbal products, or crude products of animal or plant origin. The guidance does not cover extraneous contaminants that should not occur in new drug substances and are more appropri-ately addressed as good manufacturing practice (GMP) issues, polymorphic forms, and enantiomeric impurities.

The guidance divides impurities into three categories: organic impurities (process – and drug - related), inorganic impurities, and residual solvents. These are defi ned in the guidance, along with their respective rationale for the reporting and control of impurities. The guidance further describes the circumstances in which impurities need to be reported, identifi ed, and qualifi ed.

The rationale for the reporting and control, identifi cation, and qualifi cation of impurities is discussed in the guidance. Organic impurities need to be summarized based on the actual and potential impurities most likely to arise during the synthesis, purifi cation, and storage of a new drug substance. This discussion can be limited to those impurities that might reasonably be expected based on knowledge of the chemical reactions and conditions involved.

In addition, laboratory studies conducted to detect impurities in the new drug substance need to be summarized for the regulatory application. According to the guidance, “ this summary should include test results of batches manufactured during the development process and batches from the proposed commercial process, as well as the results of stress testing used to identify potential impurities arising during storage. The impurity profi le of the drug substance batches intended for marketing should be compared with those used in development, and any differences discussed ” [13] .

Studies conducted to characterize the structure of impurities present in a new drug substance at a level greater than the identifi cation threshold (Table 30.1 ) should be described and any impurity from any batch or degradation product from stability studies should be identifi ed. If identifi cation of an impurity or degradant is not feasible, a summary of the laboratory studies demonstrating the unsuccessful effort should be included in the application. If an impurity is pharmacologically or

REGULATIONS 1023

toxicologically active, identifi cation of the compound should be conducted even if the impurity level is below the identifi cation threshold.

The guidance also states that “ qualifi cation is the process of acquiring and evalu-ating data that establishes the biological safety of an individual impurity or a given impurity profi le at the level(s) specifi ed. The applicant should provide a rationale for establishing impurity acceptance criteria that includes safety considerations. The level of any impurity that is present in a new drug substance that has been ade-quately tested in safety and/or clinical studies would be considered qualifi ed. Impu-rities that are also signifi cant metabolites present in animal and/or human studies are generally considered qualifi ed. A level of a qualifi ed impurity higher than that present in a new drug substance can also be justifi ed based on an analysis of the actual amount of impurity administered in previous relevant safety studies. If data are unavailable to qualify the proposed acceptance criterion of an impurity, safety studies to obtain such data can be appropriate when the usual qualifi cation thresh-olds are exceeded. ”

The “ Decision Tree for Identifi cation and Qualifi cation, ” as revised for Q3B(R) (Fig. 30.2 ) describes considerations for the qualifi cation of impurities when thresh-olds are exceeded. If the level of impurity cannot be decreased to below the thresh-old, or if adequate data is not available in the scientifi c literature to justify safety, then additional safety testing should be considered. The studies considered appro-priate to qualify an impurity will depend on a number of factors, including the patient population, daily dose, and route and duration of administration. Toxicology studies are discussed briefl y later in this chapter and in more detail in other chapters in this volume. Such studies can be conducted on the new drug substance containing the impurities to be controlled, although studies using isolated impurities can some-times be appropriate.

ICH Q3A states that “ safety assessment studies to qualify an impurity should compare the new drug substance containing a representative amount of the new impurity with previously qualifi ed material. Safety assessment studies using a sample of the isolated impurity can also be considered. ” The latter is especially important to consider for genetic toxicology studies and the importance of testing the isolated impurity is discussed in more detail at the end of this chapter.

Therefore, according to the guidance, if the maximum daily dose of the drug is less than 2 grams per day and the impurity intake is more than 0.15% or 1.0 mg/day, the Qualifi cation Threshold has been reached, meaning safety studies will need to be performed. Lower thresholds can be appropriate if the impurity is unusually toxic. In addition, the impurity will need to be reported and identifi ed. These studies include general and genetic toxicology studies, and possibly other specifi c toxicology

TABLE 30.1 Thresholds

Maximum Daily Dose a

Reporting Threshold b,c Identifi cation Threshold c Qualifi cation Threshold c

≤ 2 g/day 0.05% 0.10% or 1.0 mg/day intake (whichever is lower)

0.15% or 1.0 mg/day intake (whichever is lower)

> 2 g/day 0.03% 0.05% 0.05%

a The amount of drug substance administered per day. b Higher reporting thresholds should be scientifi cally justifi ed. c Lower thresholds can be appropriate if the impurity is unusually toxic.


FIGURE 30.2 The “ Decision Tree for Identifi cation and Qualifi cation, ” as revised for Q3B(R).

Is degradation product greater than

identificationthresholdc?

Yes

No

Yes

Structure identified?

No No action

No

Greaterthan qualification

thresholdc?

YesReduce

to not more than (≤) qualification

thresholdc?

No further action

Reduce to not more than (≤) identification

thresholdc?

Reduce to safe level

No

Anyknown human relevant risksd?

Yes NoNo action

Consider patient population and duration of use and consider conducting:

• Genotoxicity studies (point mutation, chromosomal aberration)a

• General toxicity studies (one species, usually 14 to 90 days)b

• Other specific toxicity endpoints, as appropriate

Yes

Yes

No

Qualified NoYes Reduce to

safe level

Anyclinically

relevant adverse effects?

Notesa If considered desirable, a minimum screen (e.g., genotoxic potential) should be conducted. A study to detect pointmutations and one to detect chromosomal aberrations, both in vitro, are considered an appropriate minimum screen.b If general toxicity studies are desirable, one or more studies should be designed to allow comparison of unqualifiedto qualified material. The study duration should be based on available relevant information and performed in thespecies most likely to maximize the potential to detect the toxicity of a degradation product. On a case-by-case basis,single-dose studies can be appropriate, especially for single-dose drugs. In general, a minimum duration of 14 daysand a maximum duration of 90 days would be considered appropriate.c Lower thresholds can be appropriate if the degradation product is unusually toxic.d For example, do known safety data for this degradation product or its structural class preclude human exposure atthe concentration present?

REGULATIONS 1025

endpoints, as appropriate. It also recommended to have a discussion of specifi c toxicity testing with the appropriate FDA division.

The genetic toxicology studies can include a minimum screen (a study to detect point mutations and one to detect chromosome aberrations, both in vitro ). The general toxicology studies should include one or more studies designed to allow comparison of unqualifi ed to qualifi ed material. The study duration should be based on available relevant information and performed in the species most likely to maxi-mize the potential to detect the toxicity of an impurity. On a case - by - case basis, single - dose studies can be appropriate, especially for single - dose drugs. In general, a minimum duration of 14 days and a maximum duration of 90 days would be con-sidered appropriate.

Inorganic impurities are normally detected and quantifi ed using pharmacopoeial or other appropriate procedures. The need for inclusion or exclusion of inorganic impurities in a new drug substance specifi cation should be discussed. Acceptance criteria should be based on pharmacopoeia standards or known safety data. The control of residues of the solvents used in the manufacturing process for a new drug substance should be discussed and presented according to ICH Q3C [9] .

A registration application should include documented evidence that the analyti-cal procedures are validated and suitable for the detection and quantifi cation of impurities [19, 20] . Organic impurity levels can be measured by a variety of tech-niques, including those that compare an analytical response for an impurity to that of an appropriate reference standard or to the response of the new drug substance itself. Differences in the analytical procedures used during development and those proposed for the commercial product should be discussed in the registration appli-cation. Analytical results should be provided in an application for all batches of a new drug substance used for clinical, safety, and stability testing, as well as for batches representative of the proposed commercial process. The application should also contain a table that links the specifi c new drug substance batch to each safety study and each clinical study in which the new drug substance has been used. Any impurity at a level greater than the reporting threshold (Table 30.1 ) and total impu-rities observed in these batches of the new drug substance should be reported with the analytical procedures indicated. Table 30.2 is an illustration of reporting impurity results for identifi cation and qualifi cation in an application.

The guidance also states that when analytical procedures change, results provided in the application should be linked to the procedure used, with appropriate valida-

TABLE 30.2 Reporting Impurity Results in an Application

Raw Result (%) Reported Result (%)

Action

Identifi cation (Threshold 0.10%)

Qualifi cation (Threshold 0.15%)

0.066 0.07 None None 0.0963 0.10 None None 0.12 0.12 a Yes None a 0.1649 0.16 a Yes Yes a

a After identifi cation, if the response factor is determined to differ signifi cantly from the original assump-tions, it may be appropriate to remeasure the actual amount of the impurity present and reevaluate against the qualifi cation threshold.


tion information provided, including representative chromatograms of representa-tive batches. The applicant should ensure that complete impurity profi les (e.g., chromatograms) of individual batches are available, if requested.

The ICH Q3A guidance also states that the specifi cation for a new drug substance should include a list of impurities. Individual impurities with specifi c acceptance cri-teria included in the specifi cation for a new drug substance are referred to as speci-fi ed impurities. Specifi ed impurities can be identifi ed or unidentifi ed. A rationale for the inclusion or exclusion of impurities in a specifi cation should be presented.

“ Acceptance criteria should be set no higher than the level that can be justifi ed by safety data and should be consistent with the level achievable by the manufactur-ing process and the analytical capability. Where there is no safety concern, impurity acceptance criteria should be based on data generated on batches of a new drug substance manufactured by the proposed commercial process, allowing suffi cient latitude to deal with normal manufacturing and analytical variation and the stability characteristics of the new drug substance. Although normal manufacturing varia-tions are expected, signifi cant variation in batch - to - batch impurity levels can indi-cate that the manufacturing process of the new drug substance is not adequately controlled and validated ” [21] .

Q 3 B ICH Guidance for Industry, Q3B(R) Impurities in New Drug Products , Revi-sion 1, was published November 2003 [12] . It is intended to provide guidance for registration applications on the content and qualifi cation of impurities in new drug products produced from chemically synthesized new drug substances not previously registered in a region or member state. A new drug product is a fi nished dosage form, for example, a tablet, capsule, or solution, that contains a drug substance, generally, but not necessarily, in association with one or more other ingredients [22] . The Q3B(R) complements the ICH guidance Q3A guidance Impurities in New Drug Substances , which should be consulted for basic principles along with ICH Q3C Impurities: Residual Solvents , when appropriate.

This guidance addresses only those impurities in new drug products classifi ed as degradation products of the drug substance, or reaction products of the drug sub-stance with an excipient and/or immediate container closure system (collectively referred to as degradation products ). Generally, impurities present in a new drug substance need not be monitored or specifi ed in new drug product unless they are also degradation products [21] . This guidance does not address impurities arising from excipients present in a new drug product or extracted or leached from the container closure system. This guidance also does not apply to new drug products used during the clinical research stages of development. It also does not cover the same types of products as in 3QA(R): biological/biotechnological, peptides, oligo-nucleotides, radiopharmaceuticals, fermentation products and associated semisyn-thetic products, herbal products, and crude products of animal or plant origin. Also excluded from this guidance are extraneous contaminants that should not occur in new drug products and are more appropriately addressed as GMP issues, polymor-phic forms, and enantiomeric impurities.

The rationale for the reporting and identifi cation of degradation products is similar to that in Q3A; however, identifi cation thresholds are different (Table 30.3 ). The impurity profi les of the batches representative of the proposed commercial process should be compared with the profi les of batches used in development, and any differences should be discussed in the submission.

REGULATIONS 1027

TABLE 30.3 Thresholds for Degradation Products in New Drug Products

Maximum Daily Dose a Threshold b,c

Reporting Thresholds

≤ 1 g 0.1% > 1 g 0.05%

Identifi cation Thresholds

< 1 mg 1.0% or 5 μ g TDI, whichever is lower 1 – 10 mg 0.5% or 20 μ g TDI, whichever is lower > 10 mg – 2 g 0.2% or 2 mg TDI, whichever is lower > 2 g 0.10%

Qualifi cation Thresholds

< 10 mg 1.0% or 50 μ g TDI, whichever is lower 10 – 100 mg 0.5% or 200 μ g TDI, whichever is lower > 100 mg – 2 g 0.2% or 3 mg TDI, whichever is lower > 2 g 0.15%

a The amount of drug substance administered per day. b Thresholds for degradation products are expressed either as a percentage of the drug substance or as total daily intake (TDI) of the degradation product. Lower thresholds can be appropriate if the degrada-tion product is unusually toxic. c Higher thresholds should be scientifi cally justifi ed.

As expected, qualifi cation of an impurity has similar concerns as Q3A. The main differences are the reporting, identifi cation, and qualifi cation thresholds (Table 30.3 ). The thresholds are basically higher than they were in Q3A; however, there are more categories for dosages. If the qualifi cation thresholds given in Table 30.3 are exceeded and data are unavailable to qualify the proposed acceptance criterion of a degradation product, additional studies to obtain such data may be appropriate (Fig. 30.2 ).

The analytical procedures and reporting in 3QB(R) are similar to those in Q3A. Differences between the analytical procedures used during development and those proposed for the commercial product should also be discussed in the registration application. Reported results should be rounded using conventional rules (see Table 30.4 ). In Table 30.4 , the doses have been divided into more categories than in 3QA, and, therefore, the action thresholds have changed.

The specifi cation for a new drug product should include a list of degradation products expected to occur during manufacture of the commercial product and under recommended storage conditions. This section, again, is similar to Q3A, with the exception of the threshold levels being different. In addition, a fi nal listing of impurities in the specifi cations should include the same items as in Q3A.

Q 3 C ICH Guidance for Industry, Q3C Impurities: Residual Solvents was published December 1997 [9] . It is intended to provide guidance for recommending acceptable amounts for residual solvents in pharmaceuticals for the safety of the patient. The guidance recommends use of less toxic solvents and describes levels considered to be toxicologically acceptable for some residual solvents. A complete list of the sol-vents included in this guidance is provided in a companion document entitled


TABLE 30.4 Illustration of Reporting Degradation Product Results for Identifi cation and Qualifi cation in an Application

Raw Result (%)

Reported Result (%) (Reporting

Threshold = 0.1%)

Total Daily Intake (TDI) of the Degradation

Product (rounded result in μ g)

Action

Identifi cation Threshold

0.2%

Qualifi cation Threshold 200 μ g TDI (equivalent

to 0.4%)

50 mg Maximum Daily Dose

0.04 Not reported 20 None None 0.2143 0.2 100 None None 0.349 0.3 a 150 Yes None a 0.550 0.6 a 300 Yes Yes a

Raw Result (%)

Reported Result (%) (Reporting

Threshold = 0.05%)

Total Daily Intake (TDI) of the Degradation

Product (rounded result in mg)

Action

Identifi cation Threshold 2 mg TDI

(equivalent to 0.11%)

Qualifi cation Threshold 3 mg TDI (equivalent

to 0.16%)

1.9 g Maximum Daily Dose

0.049 Not reported 1 None None 0.079 0.08 2 None None 0.183 0.18 a 3 Yes None a,b 0.192 0.19 a 4 Yes Yes a

a After identifi cation, if the response factor is determined to differ signifi cantly from the original assump-tions, it can be appropriate to remeasure the actual amount of the degradation product present and reevaluate against the qualifi cation threshold (see Attachment 1). b Although the reported result of 0.18% exceeds the calculated threshold value of 0.16%, in this case the action is acceptable since the TDI (when rounded) does not exceed 3 mg. Chromatograms with peaks labeled (or equivalent data if other analytical procedures are used) from representative batches, includ-ing chromatograms from analytical procedure validation studies and from long - term and accelerated stability studies, should be provided. The applicant should ensure that complete degradation product profi les (e.g., chromatograms) of individual batches are available, if requested.

Q3C — Tables and List [23] . These tables are not included in this chapter but may be found in the ICH or FDA website. The list is not exhaustive, and other solvents may be used and later added to the list.

Residual solvents in pharmaceuticals are defi ned here as organic volatile chemi-cals that are used or produced in the manufacture of drug substances or excipients, or in the preparation of drug products. The solvents are not completely removed by practical manufacturing techniques. Appropriate selection of the solvent for the synthesis of drug substance may enhance the yield, or determine characteristics such as crystal form, purity, and solubility. Therefore, the solvent may sometimes be a critical parameter in the synthetic process. This guidance does not address solvents deliberately used as excipients nor does it address solvates. However, the content of solvents in such products should be evaluated and justifi ed.

As there are no therapeutic benefi ts from residual solvents, all residual solvents should be removed to the extent possible to meet product specifi cations, good

REGULATIONS 1029

manufacturing practices, or other quality - based requirements. Drug products should contain no higher levels of residual solvents than can be supported by safety data. Some solvents that are known to cause unacceptable toxicities (carcinogens), such as benzene and carbon tetrachloride (Class 1, see Table 1 in Ref. 23 ), should be avoided in the production of drug substances, excipients, or drug products unless their use can be strongly justifi ed in a risk – benefi t assessment. Some solvents associ-ated with less severe toxicity (nongenotoxic animal carcinogens or possible caus-ative agents of other irreversible toxicity such as neurotoxicity or teratogenicity), such as acetonitrile and chlorobenzene (Class 2, see Table 2 in Ref. 23 ), should be limited in order to protect patients from potential adverse effects. Ideally, less toxic solvents, such as acetic acid and acetone (Class 3, see Table 3 in Ref. 23 ), should be used where practical.

Residual solvents in drug substances, excipients, and drug products are within the scope of this guidance. Therefore, testing should be performed for residual solvents when production or purifi cation processes are known to result in the presence of such solvents. It is only necessary to test for solvents that are used or produced in the manufacture or purifi cation of drug substances, excipients, or drug products. Although manufacturers may choose to test the drug product, a cumulative method may be used to calculate the residual solvent levels in the drug product from the levels in the ingredients used to produce the drug product. If the calculation results in a level equal to or below that recommended in this guidance, no testing of the drug product for residual solvents need be considered. If, however, the calculated level is above the recommended level, the drug product should be tested to ascertain whether the formulation process has reduced the relevant solvent level to within the acceptable amount. Drug product should also be tested if a solvent is used during its manufacture.

This guidance does not apply to potential new drug substances, excipients, or drug products used during the clinical research stages of development, nor does it apply to existing marketed drug products.

The guidance applies to all dosage forms and routes of administration. Higher levels of residual solvents may be acceptable in certain cases such as short - term (30 days or less) or topical application. Justifi cation for these levels should be made on a case - by - case basis and discussed with the appropriate FDA division.

The methods for establishing exposure limits in compounds are discussed, as are reporting levels. Some analytical procedures for the determination of exposure typi-cally include chromatographic techniques such as gas chromatography for solvents. Any harmonized procedures for determining levels of residual solvents as described in the pharmacopoeias should be used, if feasible. If only Class 3 solvents are present, a nonspecifi c method such as loss on drying may be used.

The limits of residual solvents may include a value for the Permitted Daily Expo-sure (PDE), which is the maximum acceptable intake per day of residual solvent in pharmaceutical products. These limits vary depending on the class.

For solvents to be avoided, solvents in Class 1 should not be employed in the manufacture of drug substances, excipients, and drug products because of their unacceptable toxicity or their deleterious environmental effect. However, if their use is unavoidable in order to produce a drug product with a signifi cant therapeutic advance, then their levels should be restricted as shown in Table 1 of Ref. 23 of the companion document, unless otherwise justifi ed. The solvent 1,1,1 - trichloroethane


is included (Table 1 in Ref. 23 ) because it is an environmental hazard. The stated limit of 1500 ppm is based on a review of the safety data.

For solvents to be limited, solvents in Class 2 (see Table 2 in Ref. 23 ) should be limited in pharmaceutical products because of their inherent toxicity. PDEs are given to the nearest 0.1 mg/day, and concentrations are given to the nearest 10 ppm. The stated values do not refl ect the necessary analytical precision of determination. Precision should be determined as part of the validation of the method.

For solvents with low toxic potential, solvents in Class 3 (see Table 3 in Ref. 23 ) may be regarded as less toxic and of lower risk to human health. Class 3 includes no solvent known as a human health hazard at levels normally accepted in pharma-ceuticals. However, there are no long - term toxicity or carcinogenicity studies for many of the solvents in Class 3. Available data indicate that they are less toxic in acute or short - term studies and negative in genotoxicity studies. It is considered that amounts of these residual solvents of 50 mg/day or less (corresponding to 5000 ppm or 0.5% under Option 1) would be acceptable without justifi cation. Higher amounts may also be acceptable provided they are realistic in relation to manufacturing capability and GMPs.

For solvents for which no adequate toxicological data were found, the solvents listed (see Table 4 in Ref. 23 ) may also be of interest to manufacturers of excipients, drug substances, or drug products. However, no adequate toxicological data on which to base a PDE were found. Manufacturers should supply justifi cation for residual levels of these solvents in pharmaceutical products.

30.3.2 U . S . FDA ( CDER )

U.S. FDA (CDER) Guidance for Industry, NDAs: Impurities in Drug Substances was published February 2000 [24] . The guidance refers applicants to ICH Q3A Impurities in New Drug Substances when seeking guidance on identifi cation, qualifi cation, and reporting of impurities in drug substances that are not considered new drug sub-stances. Q3A was developed by the ICH to provide guidance on the information that should be provided in a new drug application (NDA) in support of impurities in new drug substances that are produced by chemical syntheses. The FDA believes that the guidance provided there on identifi cation, qualifi cation, and reporting of impurities should also be considered when evaluating impurities in drug substances produced by chemical syntheses that are not considered new drug substances. ICH Q3A defi nes a new drug substance (also referred to as a new molecular entity or new chemical entity) as a designated therapeutic moiety that has not been previ-ously registered in a region or member state. The defi nition also states that a new drug substance may be a complex, a simple ester, or a salt of a previously approved drug substance.

This recommendation applies to applicants planning to submit NDAs and supple-ments for changes in drug substance synthesis or process. It also applies to holders of Type II Drug Master Files (DMFs) that support such applications. Applicants should note that this recommendation would not apply to DMFs cited in an NDA or supplement if the DMF information has been deemed acceptable for that dosage form, route of administration, and daily intake prior to the publication of the fi nal version of this guidance. Examples of NDAs affected by the recommendation include those submitted for new dosage forms of already approved drug products, or drug products containing two or more active moieties that are individually used

REGULATIONS 1031

in already approved drug products but have not previously been approved or mar-keted together in a drug product.

This guidance does not apply to applications for biological, biotechnological, peptide, oligonucleotide, radiopharmaceutical, fermentation, and semisynthetic products derived from herbal products or crude products of animal or plant origin, nor does it apply to Abbreviated New Drug Applications (ANDAs). Guidance on drug substances used for ANDA products is available [25] .

In February 2005, the FDA published the Guidance for Industry, ANDAs: Impuri-ties in Drug Substances [25] . This guidance provides revised recommendations on what chemistry, manufacturing, and controls (CMC) information to include regard-ing the reporting, identifi cation, and qualifi cation of impurities in drug substances produced by chemical synthesis when submitting original Abbreviated New Drug Applications (ANDAs), Drug Master Files (DMFs) including Type II DMFs, and ANDA supplements for changes in drug substance synthesis or process. The guid-ance also provides recommendations for establishing acceptance criteria for degra-dation products (specifi cally, degradation products of the active ingredient or reaction products of the active ingredient with an excipient(s) and/or immediate container/closure system) in generic drug products. The ANDA document refers to the ICH toxicology test scheme of up to 90 days in two species (rodent and nonro-dent). The guidance does not apply to an ANDA or ANDA supplement that has been reviewed prior to the publication of the fi nal guidance.

The submitter is referred to the ICH Q3B(R) guideline [12] that was developed to provide guidance on impurities in drug products for New Drug Applications (NDAs). However, the FDA believes that many of the recommendations provided on impurities in drug products also apply to ANDAs, including:

• Section I, Introduction • Section II, Rationale for the Reporting and Control of Degradation Products • Section III, Analytical Procedures • Section IV, Reporting Degradation Products, Content of Batches • Attachment 1, Thresholds for Degradation Products

The FDA recommends that the specifi cation for a drug product include a list of degradation products and include in the submission a rationale for the inclusion or exclusion of degradation products in the drug product specifi cation. It is important that the rationale include a discussion of the degradation profi les observed in stabil-ity studies and in the degradation profi les observed in the batch(es) under consid-eration together with a consideration of the degradation profi le of the batch(es) manufactured by the proposed commercial process.

The FDA recommends the inclusion of general acceptance criteria of not more than the identifi cation threshold (Fig. 30.2 ) for any unspecifi ed degradation product and acceptance criteria for total degradation products. In addition, the drug product specifi cation needs to include, where applicable, the following types of degradation products: each specifi ed identifi ed degradation product, each specifi ed unidentifi ed degradation product, any unspecifi ed degradation product with an acceptance cri-terion of not more than the fi gure in the identifi cation threshold in Q3B(R), and total degradation products.

The acceptance criterion for degradants should be set as in Q3B(R). In establish-ing degradation product acceptance criteria, the fi rst critical consideration is whether


a degradation product is specifi ed in the United States Pharmacopoeia (USP). If there is a monograph in the USP that includes a limit for a specifi ed identifi ed degradation product, the FDA recommends that the acceptance criterion be set no higher than the offi cial compendial limit. If the level of the degradation product is above the level specifi ed in the USP, the FDA recommends qualifi cation. Then, if appropriate qualifi cation has been achieved, an applicant may wish to petition the USP for revision of the degradation product ’ s acceptance criterion.

If the acceptance criterion for a specifi ed degradation product does not exist in the USP and this degradation product can be qualifi ed by comparison to an FDA - approved human drug product, the acceptance criterion should be consistent with the level observed in the approved human drug product. In other circumstances, the acceptance criterion may need to be set lower than the qualifi ed level to ensure drug product quality. For example, if the level of the metabolite impurity is too high, other quality attributes, like potency, could be seriously affected. In this case, the FDA recommends that the degradation product acceptance criterion be set lower than the qualifi ed level.

Recommended qualifi cation thresholds for degradation products based on the maximum daily dose of the drug are provided in ICH Q3B(R). The decision tree (Fig. 30.2 ) describes considerations for the qualifi cation of degradation products when the usual qualifi cation threshold recommended in ICH Q3B(R) is exceeded.

30.4 TECHNIQUES

30.4.1 General

For safety and effi cacy, it is important to know the purity of the drug substances and drug products. Useful information can often be obtained by carrying out a simple thin layer chromatographic analysis, and the ultraviolet spectrum can also be valu-able. It is also possible to measure the absorbance of a solution of the drug and compare the result with tabulated specifi c absorbance values (the absorbance of a 1% (w/v) solution in a cell of 1 cm path length). For example, the specifi c absor-bances for the drug colchicine in ethanol are 730 and 350 at 243 nm and 425 nm, respectively. Thus, a 10 mg/L solution in ethanol should give absorbance readings of 0.73 and 0.35 at 243 nm and 425 nm, respectively, in a cell of 1 cm path length. However, this procedure, along with other methods, does not take into account the presence of impurities with similar relative molecular masses and specifi c absor-bance values or other impurities. Biologics have their own unique analytical chal-lenges and may need both chemical assays and bioactivity assays (microbial, cellular, metabolic, enzymatic, gene expression). This section discusses chemical assays.

There are many different methods for the detection and quantitation of impuri-ties in materials. Some of these are discussed next. Data on analytical methodology for specifi c chemicals is readily available on the Web. OSHA has an index of sam-pling and analytical methods for hundreds of chemicals at http://www.osha.gov/dts/sltc/methods/toc.html .

The National Institute of Standards and Technology (NIST) Chemistry WebBook provides access to data compiled and distributed by NIST under the Standard Ref-erence Data Program and can be found at http://webbook.nist.gov/ . The WebBook

contains thermochemical data for over 7000 organic and small inorganic com-pounds, reaction thermochemistry data for over 8000 reactions, IR spectra for over 16,000 compounds, mass spectra for over 15,000 compounds, UV/Vis spectra for over 1600 compounds, gas chromatography data for over 27,000 compounds, elec-tronic and vibrational spectra for over 5000 compounds, constants of diatomic mol-ecules (spectroscopic data) for over 600 compounds, ion energetics data for over 16,000 compounds, and thermophysical property data for 74 fl uids.

Method Validation Method validation is an analytical procedure used to demon-strate that the analytical procedure in question is suitable for its intended purpose and is reproducible. A GMP requirement in 21 CFR 211.165(e) states that the accuracy, sensitivity, specifi city, and reproducibility of test methods employed shall be established and documented [26] . The U.S. FDA (CDER) has a draft guidance that discusses analytical procedures and methods validation, chemistry, manufactur-ing, and controls documentation [27] .

This draft guidance provides recommendations to applicants on submitting data for the validation study, including analytical procedures, validation data, and samples to support the documentation of the identity, strength, quality, purity, and potency of drug substances and drug products. The recommendations apply to drug sub-stances and drug products covered in New Drug Applications (NDAs), ANDAs, Biologics License Applications (BLAs), Product License Applications (PLAs), and supplements to these applications. The principles also apply to drug substances and drug products covered in Type II Drug Master Files (DMFs). If a different approach is chosen, the applicant is encouraged to discuss the matter in advance with the center with product jurisdiction to prevent the expenditure of resources on prepar-ing a submission that may later be determined to be unacceptable. Although this guidance does not specifi cally address the submission of analytical procedures and validation data for raw materials, intermediates, excipients, container closure com-ponents, and other materials used in the production of drug substances and drug products, validated analytical procedures should be used to analyze these materials.

The principles of methods validation presented apply to all types of analytical procedures. However, the specifi c recommendations in this guidance may not be applicable to certain unique analytical procedures for products such as biological, biotechnological, botanical, or radiopharmaceutical drugs. For example, many bioas-says are based on animal challenge models, immunogenicity assessments, or other immunoassays that have unique features that should be considered when submitting analytical procedure and methods validation information. For questions on appro-priate validation approaches for analytical procedures or submission of information not addressed in this guidance, applicants should consult with the appropriate chem-istry review staff at the FDA.

FDA investigators inspect the analytical laboratory testing sites to ensure that the analytical procedures used for release and stability testing comply with GMPs (21 CFR part 211) [26] or good laboratory practices (GLPs; 21 CFR part 58) [28] , as appropriate. These assure that proper guidelines and documentation are followed throughout the studies. All analytical procedures are of equal importance from a validation perspective. In general, validated analytical procedures should be used, irrespective of whether they are for in - process, release, acceptance, or stability

TECHNIQUES 1033


testing. Each quantitative analytical procedure should be designed to minimize assay variation. Reference standards must be used. A list of information that should typically be included in a description of an analytical procedure is included in this guidance. Impurities and degradants are addressed in the method validation infor-mation and the following should be reported:

• Data to demonstrate the stability of all analytical sample preparations through the time required to complete the analysis.

• Representative calculations using submitted raw data, to show how the impuri-ties in drug substance are calculated.

• Information from stress studies. • Impurities labeled with their names and location identifi ers (e.g., RRT for

chromatographic data) for the impurity analytical procedure. • For drug substances:

A discussion of the possible formation and control of polymorphic and enantiomeric substances.

Identifi cation and characterization of each organic impurity, as appropriate. This information may not be needed for all products (e.g., botanicals). Other impurities (e.g., inorganics, residual solvents) should be addressed and quantitated.

Recommendations on submitting information on impurities is provided in various FDA guidances such as the ICH guidance Q3A Impurities in New Drug Substances (January 1996).

A list of known impurities, with structure if available, including process impurities, degradants, and possible isomers.

• For drug products: A degradation pathway for the drug substance in the dosage form, where

possible. Data demonstrating recovery from the sample matrix as illustrated by the

accuracy studies. Data demonstrating that neither the freshly prepared nor the degraded

placebo interferes with the quantitation of the active ingredient.

The draft guideline lists examples of common problems that can delay successful validation.

• Failure to provide a sample of a critical impurity, degradation product, internal standard, or novel reagent.

• Failure to submit well - characterized reference standards for noncompendial drugs.

• Failure to provide suffi cient detail or use of unacceptable analytical procedures. For example, use of arbitrary arithmetic corrections, failure to provide system suitability tests, and differing content uniformity and assay analytical proce-dures without showing equivalence factors for defi ning corrections as required by the current USP Chapter 905 — Uniformity of Dosage Units.

• Failure to submit complete or legible data. For example, failure to label instru-ment output to indicate sample identity and failure to label the axes.

• Inappropriate shipping procedures. For example, failure to properly label samples, failure to package samples in accordance with product storage condi-tions, and inadequate shipping forms (e.g., missing customs form for samples from outside the United States).

• Failure to describe proper storage conditions on shipping containers.

Prior to the start of the validation study, conducting a feasibility study will help uncover issues early on so that the main study fl ows with ease. Feasibility studies address the setup method and system suitability, determine limits of detection (LOD) and quantitation (LOQ), perform forced degradation studies, check placebo for interferences, test related compounds, and check the label and specifi cation claim.

The protocol needs to address specifi city, linearity, accuracy, range, LOD/LOQ, precision, and robustness [29] .

Specifi city is the degree to which the measured response is due to the analyte of interest and not to other substances expected to be present in the sample matrix. For example, degradation products may be formed when the drug substance is exposed to environmental and/or chemical conditions, such as acid hydrolysis (HCl), base hydrolysis (NaOH), oxidation (peroxide), thermal degradation (heat), or pho-tolysis (irradiation).

Linearity is the ability to obtain results that are directly proportional. Standards may be prepared at concentration levels ranging from 50% to 150% of the theoreti-cal range of the analyte in a sample solution concentration prepared according to the method. The upper and lower limits of the linear range need to be reported. A graph of the standard curve and results of linear regression should also be deter-mined. Low - level linearity for impurities needs to be determined, but sometimes impurity reference standards are unavailable. Since impurities are typically deter-mined based on the active ingredient peak, demonstration of linearity at a low level is critical. A low - level linearity curve should include concentrations of 0.05%, 0.1%, 0.5%, 1.0%, 1.5%, and 2.0% of the active compound.

Accuracy is the measure of the total error of a method, including both systematic and random errors. For impurities assays, the accuracy should be assessed on samples (drug substance/drug product) spiked with known amounts of impurities. If impuri-ties or degradation products are not available, it is acceptable to compare results obtained by a second, well - characterized method.

Range is the interval between the upper and lower concentration of analyte for which a suitable level of precision, accuracy, and linearity has been demonstrated.

Limit of Detection ( LOD ) The LOD is the lowest concentration of analyte that can be detected, but not necessarily quantitated, by the analytical method. It is usually expressed as concentration of analyte generating an instrument response equivalent to three times the noise. The LOQ is the lowest concentration of analyte that can be determined with acceptable accuracy and precision by the analytical method. It is usually expressed as concentration of analyte generating an instrument response equivalent to ten times the noise.

Precision is the measure of how close the data values are to each other for a number of determinations under the same analytical conditions. ICH has defi ned precision to contain three components: repeatability (six replicate measurements of

TECHNIQUES 1035


a single sample preparation), intermediate precision (ruggedness; second analyst repeats method precision on different instrument, column, day, etc.), and reproduc-ibility (method precision, duplicate measurements of six sample preparations). Precision is expressed as the percent relative standard deviation (%RSD).

Robustness is similar to ruggedness. It is a measure of how the results are affected by variations of different factors internal to the method, as in the mobile phase pH, mobile phase composition, temperature, fl ow rate, injector/detector temperatures, and oven ramp rates. According to ICH Q2A, the robustness of an analytical pro-cedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Metabolism of Impurities and Degradants Although they are not true impurities in the drug substance or the drug product, metabolites might be produced in vivo . The drug substance or product is metabolized in a manner so that the animal becomes exposed to the new metabolite. These metabolites may be detected from bioanalysis samples (whole blood, plasma, serum, urine). Due to exposure in vivo , ICH Q3A states that impurities that are also signifi cant metabolites present in animal and/or human studies are generally considered qualifi ed.

30.4.2 Ultraviolet and Visible ( UV / V is ) Spectroscopy

A number of the quantitative methods use ultraviolet (UV) (200 – 400 nm) or visible (400 – 800 nm) spectrophotometry. Quantitative analysis of solutions is commonly performed by ultraviolet spectroscopy [30] . The major problem encountered with this technique is interference, and some form of sample purifi cation is needed, such as solvent extraction. The spectrophotometer may be of the single - beam or double - beam type. With a single - beam instrument, light passes from the source through a monochromator and then via a sample cell to the detector. With double - beam instruments, light from the monochromator passes through a beam - splitting device and then via separate sample and reference cells to the detector. Double - beam instruments with automated wavelength scanning and a variety of other features are also available.

30.4.3 Atomic Absorption Spectroscopy

Atoms can absorb energy of a specifi c wavelength that corresponds to the wave-length they would emit when eroded by a high energy source. In atomic absorption, the energy needed for the atomic emission is supplied by a source other than a fl ame. The fl ame is actually the cuvette that holds the sample. The decrease in the emission intensity of the source is proportional to the concentration of atoms in the sample. Atomic absorption spectroscopy is important for the analysis of trace metals in biological fl uids [31] .

30.4.4 Thin Layer Chromatography ( TLC )

Thin layer chromatography (TLC) is a technique for determining the composition of a mixture. It involves the movement by capillary action of a liquid phase (usually

an organic solvent) through a thin, uniform layer of stationary phase (usually silica gel, SiO 2 ) held on a rigid or semirigid support, normally a glass, aluminum, or plastic sheet or plate. Compounds are separated by partition between the mobile and sta-tionary phases. It may be used to establish the extent of a reaction, the purity of a compound, or the presence or absence of materials in fractions from column chro-matography. TLC is relatively inexpensive and simple to perform and can be a powerful qualitative technique when used together with some form of sample pre-treatment, such as solvent extraction. However, some separations can be diffi cult to reproduce. The interpretation of results can also be very diffi cult, especially if a number of drugs or metabolites are present.

30.4.5 High Pressure Liquid Chromatography ( HPLC )

In general, chromatography is used to separate mixtures of chemicals into individual components. Once separated, the components can be individually evaluated. With chromatography, separation occurs when the sample mixture is introduced (injected) into a mobile phase. In liquid chromatography (LC), the mobile phase is a solvent. The mobile phase transports the mixture through a stationary phase. The stationary phase is a chemical that can selectively draw components from a sample mixture. The stationary phase is usually contained in a tube (column). Columns can be glass or stainless steel of various dimensions.

The mixture of compounds in the mobile phase will interact with the stationary phase. Individual compounds in the mixture tend to interact at various rates. Those that interact the fastest will exit (elute from) the column fi rst. Those that interact slowest will exit the column last. By changing characteristics of the mobile phase and the stationary phase, different mixtures of chemicals can be separated.

HPLC is commonly used for the separation, identifi cation, purifi cation, and quan-tifi cation of chemical compounds (Fig. 30.3 ) . Chemical separations can be accom-plished since certain compounds have different migration rates given a particular column and mobile phase. Therefore, separation of compounds from each other can occur; the extent or degree of separation is mostly determined by the choice of the stationary phase and the mobile phase. The chromatographer may choose the condi-tions, such as the proper mobile phase, to allow adequate separation or purifi cation in order to collect or extract the desired compound as it elutes from the stationary phase. Each compound should have a characteristic peak under certain chromato-graphic conditions. The migration of the compounds and contaminants through the column need to differ enough so that the pure desired compound can be collected or extracted without bringing on any other undesired compound.

Preparative HPLC is the process of isolation and purifi cation of compounds. Analytical HPLC determines information about the sample compound, including identifi cation, quantifi cation, and resolution.

Identifi cation of compounds by HPLC is a critical part of any HPLC assay and is accomplished by researching the literature and by trial and error. After a few steps, a separation assay must be developed. The parameters of this assay should be such that a clean, well - separated peak of the known sample is observed from the chromatograph.

Quantifi cation of compounds by HPLC is the process of determining the unknown concentration of a compound in a known solution. It involves injecting a series of

TECHNIQUES 1037


known concentrations of the standard compound solution onto the HPLC for detec-tion. The chromatograph of these known concentrations will give a series of peaks that correlate to the concentration of the compound injected.

30.4.6 Mass Spectrometry ( MS )

Mass spectrometry is a powerful analytical tool because it can provide valuable structural information with a high degree of specifi city. Compounds enter an elec-tron ionization (mass spectrometer) detector and are bombarded with a stream of electrons causing them to break apart into fragments. These fragments can be large or small pieces of the original molecules and are charged ions with a certain mass. A group of four electromagnets (quadrupole) focuses each of the fragments through a slit and into the detector. The quadrupoles are programmed by the computer to direct only certain fragments through the slit. The rest bounce away. This happens repeatedly for a specifi c range of fragments. A graph, the mass spectrum, is then produced.

FIGURE 30.3 HPLC. Compliments of Midwest BioResearch, Evanston, IL.

The production of a characteristic mass spectrum or fragmentation pattern acquired for each molecule makes it a defi nitive and effective tool for identifying unknown impurities or degradation products. This characteristic pattern is a “ fi n-gerprint. ” Comparing the MS “ fi ngerprint ” with large mass spectral databases further facilitates identifi cation.

30.4.7 Gas Chromatography ( GC ) and Combination of GC and MS

As discussed previously, chromatography is used to separate mixtures of chemicals into individual components. The procedure for separation is similar for LC and GC with the exception of the mobile phase. In GC, the mobile phase is an inert gas such as helium.

With the combined GC/MS, the GC separates the components of a mixture. After the individual compounds elute from the GC column, they enter the electron ioniza-tion (mass spectrometer) detector. The fragments go through the same procedure as discussed for MS. MS characterizes each of the components individually. A 3D graph is produced, which provides qualitative and quantitative data. By combining the two techniques, an analytical chemist can take an organic solution, inject it into the instrument, separate the individual components, and identify each of them. Furthermore, the researcher can determine the quantities (concentrations) of each of the components.

ICH Q3C states that residual solvents are typically determined using chromato-graphic techniques such as gas chromatography. Any harmonized procedures for determining levels of residual solvents as described in the pharmacopoeias should be used, if feasible.

30.4.8 MS / MS

The tandem MS method has been used as an alternative to GC/MS. This method uses collision - activated dissociation on a triple quadruple mass spectrometer. This assists in the quick and direct qualitative and semiquantitative analysis. Other ben-efi ts of tandem MS include the elimination of most wet chemical and chromato-graphic separation steps, and the detection of both known and unknown compounds by molecular weight and functional group. A disadvantage is that tandem MS is somewhat less specifi c than GC/MS in the identifi cation of some isomeric compounds.

30.4.9 HPLC /Mass Spectrometry ( LC / MS or LC / MS / MS )

LC/MS data includes molecular weight and structural information that can help identify an impurity or a degradation product.

LC/MS/MS, also known as a triple - quad LC/MS, has become standard in the pharmaceutical analytical laboratory (Fig. 30.4 ) . For the quantitative analysis of many analytes, LC/MS/MS is a fast, universal, selective, and sensitive tool. The quantitation is typically performed with high selectivity, which practically eliminates matrix components. LC/MS/MS is also a very important technique in the bioanalyti-cal arena for characterizing large molecules such as botanicals, biologics, proteins, and peptides, which are typically present in challenging matrices.

TECHNIQUES 1039


30.4.10 Common Problems

Meeting GMP Standards for Maintenance and Recordkeeping The FDA has promulgated 21CFR Part 211, Current Good Manufacturing Practice for Finished Pharmaceuticals [26] . There are other sections for the FDA - issued Good Manufac-turing Practices (GMPs) and the manufacturer needs to look closely to ensure that the proper regulations are followed. The regulations in this part (21CFR Part 211) contain the minimum current GMP for preparation of drug products in general for administration to humans or animals: for drug products, in Parts 600 through 680, as they pertain to drugs that are also biological products for human use; and in Part 1271, as they are applicable to drugs that are also human cells, tissues, and cellular and tissue - based products (HCT/Ps) and that are drugs.

Maintenance and recordkeeping are addressed in many sections of 21CFR Part 211. Strict guidelines are in place, such as all equipment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals to prevent malfunctions or con-tamination that would alter the safety, identity, strength, quality, or purity of the drug product beyond the offi cial or other established requirements.

Virtually all actions need to be documented and stored. All records need to be maintained for specifi ed periods, including any production, control, or distribution records; records for all components, drug product containers, closures, and labeling; records for maintenance; and use logs. Therefore, everything needs to be docu-mented and explained so that the study can be reconstructed, if necessary.

For all impurities, approval of the drug product would be based on the applicant ’ s ability to demonstrate that the product is manufactured to ensure both effi cacy and safety for patients. This can only be achieved by paying detailed attention to identity, strength, quality, and purity of the products and their components during the entire manufacturing and distribution process.

Locating Suitable Standard and Reference Materials Occasionally, there may be problems locating suitable standards and reference materials. Some of these may be purchases through a reputable dealer or the USP. Some may be custom made in

FIGURE 30.4 LC/MS/MS. Compliments of Midwest BioResearch, Evanston, IL.

a laboratory. But no matter which route is selected, documentation of all procedures and steps is critical to be in compliance with GMPs.

Diffi culty of Separation of Chromatogram Background Noise If a chromato-graph has a lot of peaks, there is a good possibility that all of the nonactive peaks are due to impurities. It is best to look for a method that puts your peak in a clear area. Depending on the type of equipment used for analyses, changes may be made to an existing method to place the peak in a clear area.

30.5 TOXICITY ASSESSMENT

Once it has been determined that an impurity needs to be qualifi ed, research will need to be conducted to establish safety. The literature from peer - reviewed journals will need to be assessed. Toxicology and safety assessment studies may also need to be performed.

30.5.1 Literature - Based Approaches

Literature searches can be obtained in many different ways. There are many com-panies who offer subscriptions to their search programs, and Information Services professionals are also available to assist you in your search. For those who prefer to perform their own searches for no charge, many websites will provide you with results that include the citations and possibly abstracts. In many cases, there are fees for full text articles. Below is a brief introduction to some of the free databases on the Web. Links are current as of November 2007 .

The U.S. National Library of Medicine ( www.nlm.nih.gov ) has many links to other databases, including PubMed ® , MEDLINE ® , TOXNET ® , LNM Gateway, LNM Catalog, the Hazardous Substance Data Bank (HSDB) and meeting abstracts. Other resources exist and it is worth a visit to the website.

PubMed/MEDLINE ( http://www.ncbi.nlm.nih.gov/entrez/query.fcgi to register) includes over 15 million citations from MEDLINE and other life science journals for biomedical articles back to the 1950s. PubMed includes links to full text articles and other related resources.

TOXNET ( http://toxnet.nlm.nih.gov/ ) contains databases on toxicology, hazard-ous chemicals, environmental health, and toxic releases. Databases include HSDB, IRIS, CCRIS, GENETOX, TOXLINE, EMIC, DART/ETIC, TRI, and ChemID/ plus .

LNM Gateway ( http://gateway.nlm.nih.gov/gw/Cmd ) can be used to search multiple NLM retrieval systems.

LNM Catalog ( http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nlmcatalog ) provides access to NLM bibliographic data for journals, books, audiovisuals, computer software, electronic resources, and other materials. Links to the library ’ s holdings in LocatorPlus, NLM ’ s online public access catalog, are also provided.

TOXICITY ASSESSMENT 1041


Local, state, national, and international websites exist to provide information on impurities, chemicals, and regulations. For U.S. FDA information, including guidances and regulations, go to www.fda.gov .

30.5.2 Common Testing Requirements for New Impurities

New impurities, depending on the level present in the drug product, will need to be tested for safety. Studies to be performed are based on ICH and FDA guidances and will vary on a case - by - case basis. A few studies may be needed, or the entire list. Discussions with the appropriate division of the FDA for future study strategy may be prudent. Mammalian studies most likely are done in a rodent and a nonro-dent species. Rat and dog, respectively, have previously been recommended, but recent tests have used the minipig in place of the dog. In certain specifi c cases, non-human primates may also be used.

The studies include but are not limited to:

• Pharmacodynamics • Pharmacokinetics • Safety pharmacology • Genetic toxicology battery • Acute dosing • Repeated - dose studies • Carcinogenicity • Reproductive and teratology studies • Multigeneration assessments • Special testing as appropriate (i.e., phototoxicity, dermal, inhalation, sensitiza-

tion, etc.)

If needed, a repeated - dose general toxicity study in rodents and nonrodents can range from a minimum duration of 14 days to 90 days. The clinical route of admin-istration is recommended; however, other routes (i.e., intravenous, intraperitoneal) may be necessary if exposure is an issue. A minimal genetic toxicology battery, consisting of two in vitro assays to detect point mutations and chromosomal aber-rations, should be conducted with the test article containing the contaminant, although more precision will be gained if the isolated impurity is tested. There may be a need for carcinogenicity studies if the drug product is administered daily for > 3 months. Embryo - fetal development assessment may be necessary when the parent drug is used in a population that includes women of childbearing potential. Other reproductive toxicity studies may be requested on a case - by - case basis depending on results of the general toxicity and embryo - fetal developmental studies.

30.5.3 Study Design Based on Exposure, Route, Dosage, and Other Factors

Details of the studies may also need to be discussed with the FDA or other regula-tory agencies. Details such as route, dosage, length of exposure, parameters

measured, species, and age of animals are just a few considerations in the study design. Study protocols developed by the FDA or the Organisation of Economic Cooperation and Development (OECD) have been used.

30.5.4 Documentation Content and Format

Content of regulatory submissions will vary between regulatory bodies and types of submissions. Within the FDA, for example, there are differences between what is needed for approval of pharmaceuticals, biologics, and medical devices. In addi-tion, each regulatory agency has a specifi c procedure for formatting. Many are moving toward the Common Technical Document (CTD). Virtually all agencies have guidelines posted on their websites to explain what is necessary for submission. More than one guideline for submissions may exist within an agency.

The FDA CDER Guidance page ( http://www.fda.gov/cder/guidance/ ) has a few links to submission information, including information in Chemistry, Drug Safety, Electronic Submissions, ICH (fi nal and draft), and IND (Investigational New Drug).

Providing the regulatory agency with the information that it needs for product approval is both a science and an art, and in many cases, expert assistance should be sought.

30.6 BRIDGING STUDIES

Changes are made many times during development of a drug substance or a drug product. When most of the preclinical safety testing has already been conducted, and changes are made, it may not be necessary to repeat all of the previous studies. However, a change in manufacturing techniques or a change in the impurity profi le would necessitate testing to ensure that the materials before and after the change are comparable in terms of quality, safety, and effi cacy. Comparability can often be deduced from available data alone but might sometimes need to be supported by comparability bridging studies.

Bridging studies may consist of toxicology studies in an appropriate species, either rodent and/or nonrodent. The nature of the bridging studies must be deter-mined on a case - by - case basis. Examples of bridging studies are:

• An acute study. • A repeated - dose study of 90 days ’ duration. Toxicokinetic samples should be

analyzed and blood levels compared to levels observed in animals prior to the change.

• Pharmacodynamic studies would also need to be conducted to detect any possible changes.

• Teratology (Segment II) study in the most sensitive species (rat or rabbit, primarily).

• Pre - and postnatal development including maternal functions (Segment III). • Other studies as needed.

BRIDGING STUDIES 1043


Chapter 31 , Bridging Studies in Pharmaceutical Safety Assessment, has more details of bridging studies.

30.7 DISCUSSION REGARDING TESTING THE DRUG WITH THE IMPURITY VERSUS ACTUAL IMPURITY

What exactly is tested for studies to determine the safety of impurities? As time goes on and the chemists have more experience with the drug substances, they fi nd more effi cient methods of producing the substance in hopes that the impurity profi le will decrease. Most of the time, especially early on in development, the impurity is already present in the drug substance that is administered to the animals. If the impurity is present in a high enough concentration to provide a relevant dosage to the test systems in repeated - dose studies, the impurity may be considered adequately tested.

ICH Q3A states that the level of any impurity present in a new drug substance that has been adequately tested in safety and/or clinical studies would be considered qualifi ed. ICH Q3B (R) describes considerations for the qualifi cation of degradation products when thresholds are exceeded. If additional safety testing is needed, such studies can be conducted on the new drug product or substance containing the degradation products to be controlled, although studies using isolated degradation products can sometimes be appropriate.

Testing the actual impurity is important in some cases, and it would be prudent for the industry, FDA, and other agencies to hold discussions on this topic. Some impurities are present in rather small amounts. Toxicology tests may not be sensitive enough to characterize their individual toxicities if impurities are tested within a drug substance or product. Testing the toxicity of an impurity present in a drug substance or product in toxicology studies or bridging studies may very well be a waste of time, money, and animals if no toxicity from the impurity is observed due to low levels. However, using the predictive power of several structure – activity relationship (SAR) programs, specifi c toxicities identifi ed for individual impurities could be assessed.

Testing the impurity at appropriate levels to produce appropriate safety factors for in vivo studies would elucidate specifi c toxicities from the impurity. But more crucial is testing the isolated impurity in genetic toxicology studies. If tested as a component of the drug substance, the exposure of the impurity to the target cells in these studies would be so low that only the most potent mutagens would be detected. To understand the toxicological implications of impurities, testing of the isolated impurity at appropriate concentrations is essential.

ACKNOWLEDGMENTS

I would like to thank Robert E. Osterberg, Offi ce of New Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, for his review of the draft manuscript for this chapter. I also thank Mike Schlosser, president of Midwest BioResearch, for allowing me to photograph the equipment .

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