Forced Degradation of Pharmaceuticals

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Mass Balance
Assessments of bass balance in degradation studies checks to see if the amount of material detected in a stressed sample matches the amount present before stress was applied. The assay for the drug substance plus the amounts of impurities detected are added together to give the total mass present. Appropriate response factors should be applied to degradation products to give the most accurate sum. Approximate totals can be had by assuming the responses of the degradation products are the same as the drug substance. In the case of UV detection schemes, the UV spectra of the drug substance and degradation products can be compared to determine similarities and differences in response factors. Mass balance deficits in stressed samples can be investigated by checking response factors; checking for highly retained compounds by eluting with strong mobile phase or the detection of spots near the origin on TLC; using 3D photodiode array analysis for detecting peaks with different absorption wavelengths; looking for peaks without chromophores by LC/MS, GC, TLC (with I2 or acid/charring visualization), or ELSD; and looking in the void for poorly retained peaks. In practice, mass balance may not always be obtained. The FDA recognizes this and instead will look at the thoroughness of the degradation studies and elucidation of degradation pathways during review of the submission [7].
Stereochemical Stability
Chiral drugs consisting of disproportionate amounts of the possible stereoisomers should be assessed for their stereochemical stability during stress studies. Drugs with one or two chiral centers should be analyzed with a chiral method to assess stereoisomer content. Drugs with three of more chiral centers will most likely convert to diastereomers prior to racemization so achiral analysis should suffice. Stereoisomers should be treated like any other drug related impurity with respect to quantitation, identification and qualification thresholds, etc. [8]. Stereochemical degradation can be more rapid than any other pathway and should, for this reason, always be examined early. Peak purity studies using LC/MS and LC/DAD will typically not detect co-eluting stereoisomers, although LC/DAD may detect co-eluting geometric isomers of olefins. LC/NMR may detect co-eluting diastereomers. If the stereochemical stability of a molecule is demonstrated by stress studies, it may no longer be necessary to monitor stereoisomers during stability trials [8].
Solid State Reactions
Some reactions only occur in the solid state of some polymorphs [9]. For this reason, each polymorph that is advanced during a drug's development life should be stressed and analyzed for content, impurities, and physical form. Some solvates will lose solvent molecules more rapidly than they undergo solvolysis. For this reason, solvates and hydrates should be stressed in closed and open containers to insure the solvolysis pathway is observed. Experience has shown that a polymorph appearing late in development may require reformulation, new SIM development, and drug substance manufacturing changes.
Insoluble Drugs
For drugs that have poor water solubility, stress studies can be conducted on suspensions or solutions using organic co-solvents [9]. Ideally, organic co-solvents should be inert to the drug and the medium of stress. Alcohols are often a poor choice due to their reactivity. DMSO, acetic acid, and propionic acid are useful under acidic conditions. DMSO, NMP, and acetonitrile work under neutral or natural pH conditions. Glyme and 1,4-dioxane facilitate reactions in base. The medium can consist of even 80-90% organic co-solvent without preventing the desired hydrolytic reactions. Acetonitrile is the co-solvent of choice for photochemical reactions. The potential drawbacks to using organic co-solvents include impact on reaction rates (this may be undesirable if reaction rates as a function of pH are being examined) and obscuring early eluting peaks (mainly a problem with DMSO and NMP).
Stable Drugs
Some drugs are very stable, even under stress conditions. It is the opinion of this author that a drug need not degrade during stress testing but only be subjected to an amount of stress in excess of what can be expected in accelerated storage or storage to expiry. A rationale for setting limits on the stress storage will be discussed below.
Combination Products
Drug products that contain more than one active ingredient should be stressed and assessed for drug-drug and drug-excipient degradation products. The combination of two or more actives can lead not only to the formation of novel degradation products, but also to changes in physical properties of the formulation such as hygroscopicity.
Experimental Approach
Specific experimental conditions for stress studies will be discussed in this section. A recent survey revealed that a wide variety of conditions are used for forced degradation studies in the pharmaceutical industry [10].
General Strategy
One of the goals of forced degradation studies is to efficiently produce samples that contain the degradation products likely to be formed during manufacture, handling, and normal storage of the drug substance and drug product. Efficiency and severity/duration of stress conditions must be balanced with generation of realistic and representative impurity profiles. Overstressing can destroy compounds that are relevant or generate irrelevant compounds. Understressing, on the other hand, may fail to generate important degradation products. The extent of degradation targeted should be approximately 5-10%. However, there is a limit to how much stress should be applied, even if the drug does not degrade 10% or at all.
Experience has shown that storage of drug substances and products at 50-80?C under the various stress conditions usually affords representative samples without overstressing. The maximum duration can be estimated by making rational kinetic assumptions. The energy of activation (Ea) for most pharmaceutical degradation reactions is 12-24 kcal/mole [11]. Assuming Arrhenius kinetics and an Ea = 12 kcal/mole, the reaction rate should double for every 10?C temperature increase. Hence, a reaction that takes 6 months at 40?C would take 3 months at 50?C, 6 weeks at 60?C, 3 weeks at 70?C, and just under 2 weeks at 80?C. Notice for reactions with Ea >12 kcal/mole, the reaction rate more than doubles for every 10?C increase; hence, assuming an Ea of 12 kcal/mole is conservative and insures against understressing. For most compounds and products, storage at 80?C for up to 2 weeks or at 60?C for 6 weeks provides realistic degradation profiles. Storage can cease after these periods, even if little or no degradation occurs.
Initial stress conditions can often be selected by considering the conditions used in previous studies of related compounds. For this reason, it is advisable to compile the quantitative and qualitative results of previous degradation studies in a substructure searchable database. In the absence of previous studies, a good rule of thumb is to start gentle and intensify the stress as required, but only to a point (e.g. 80?C). Storage at two temperatures (e.g. 60?C and 80?C) allows for more rapid degradation at the higher temperature with a backup at the lower, should the more intense condition cause too much degradation.
Flint glass serum vials are ideal for degradation work. These vials come in clear and amber glass in various sizes and are sealed with septa making them air tight. Teflon closures are available. These vials facilitate: stress studies of drug substance and products in solution and the solid state, light exposure control, headspace gas control and analysis (useful for studying oxidation reactions), sample retention, quantitative experiments/mass balance investigations, sample lyophilization, quick sample cooling, and observation of visual changes.
Accurately thermostated ovens make good stability chambers. Relative humidity can be controlled by using desiccators containing various saturated salt solutions and a platform above the solutions for samples [12].
Drug Substance
The drug substance must be stressed hydrolytically under a broad range of pH. A drug substance concentration of 1-10 mg/mL usually works well. Conditions of low pH are provided by 0.1N HCl. Natural pH experiments can be conducted in water under nitrogen, air, and oxygen. These conditions allow the study of hydrolysis and oxidation. Neutral conditions can be provided by pH 7 phosphate buffer. Basic conditions are provided by 0.1N NaOH. More dilute acid or base may be desirable if the drug has especially labile functional groups [13]. Organic co-solvents may be used as appropriate. Heated storage vessels can be quickly cooled in tap water. Acid and base reactions should be neutralized. Trial workups from the various stress media should be conducted before stress studies begin to insure drug solubility throughout the storage and analysis process. Initial samples (prior to applying heat) from each stress condition should be worked up for analysis immediately after dissolving the drug.
Oxidation can be carried out under an oxygen atmosphere or in the presence of peroxides. Experience has shown that use of oxygen will provide samples containing the relevant oxidation products. Peroxides tend to be too reactive and may not always reveal all potential oxidation pathways, especially where molecular oxygen can react directly with the drug [14].
Solid state stress studies can be conducted at 80?C for 2 weeks, 80?C/75%RH for 2 weeks, and in excess of ICH light exposure. For photolysis, one can use option 1, option 2, or separate storage. The latter affords insight into which wavelengths do the most damage.
Drug Product
Appropriate stress conditions for solid dosage forms include 80?C for up to 2 weeks (or 60?C and 60?C/75%RH for up to 6 weeks), 80?C/75%RH for up to 2 weeks (or 60?C alternatives), and