15 ROLE OF HPLC DURING FORMULA TION DEVELOPMENT Tarun S. Patel and Rosario LoBrutto 15.1 INTRODUCTION What is the definition of a formulation? Why is it needed? What is the impor- tance of a formulation? These are some important questions that need to be addressed during the development of a potential drug product. The strict def- inition of the word is to specify a formula or to express a formula in system- atic terms or concepts. The formula, in the current case, is a pharmaceutical dosage form. A formulation is needed to deliver the drug or the active phar- maceutical ingredient (API) to its targeted site. In order to overcome some of the physiochemical limitations of an API, it must be combined with inert ingre- dients (excipients) to safely and reproducibly deliver the API by the selected route of administration. Whether the delivery of a drug is oral (capsules, tablets, suspensions), by injection (intravenous and intramuscular injections), transdermal (patches), or by inhalation (metered dose inhaler, dry powder inhalation, nebulizer), the drug (active pharmaceutical ingredient) cannot be delivered by itself. Formulation development is a complex process involving the physiochem- ical characterization of the API, identifying compatible excipients, developing a reliable manufacturing process, and thorough analytical characterization of the dosage form. The entire process starts early in the preformulation stage (covered in Chapter 12) and continues until the final market image is devel- oped and launched (after approval from health authorities). The goal of any 679 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. formulation development is to ensure that each batch manufactured meets the specifications for identity , strength, quality, and purity. Formulation development has four generic phases that are somewhat aligned to the clinical phases: preclinical, clinical, registration, and commer- cial. During the preclinical stage, the first dosage form is developed to evalu- ate the safety and pharmacokinetic profile in humans. Generally due to limitations in the availability of an API and to minimize development time, this formulation is not optimized from a manufacturing perspective. For oral delivery, formulations such as powder in a bottle or a capsule are often selected.The information derived from preformulation studies is used to select the excipients for this first formulation. Depending upon the drug, its target, and its effect, a clinical trial may last anywhere from days to months. During this time, clinical programs are being updated and the product moves from one clinical phase to another (Preclinical, Phase I, Phase IIA, Phase IIB, Phase III, and Phase IV). Preclinical studies are initial studies that are conducted to determine poten- tial new treatments for specific disease indications.Animal models and in vitro assessments are used to help determine a treatment’s safety prior to intro- ducing it to humans. Phase I clinical trials are conducted to evaluate the safety of a drug, study absorption, and metabolism and how the drug should be administered (e.g., injection, oral), and different dose levels may also be eval- uated. Phase 1 trials typically involve a small group ranging from 5 to 80 patients which depends on the disease indication. Phase II clinical trials are conducted to further evaluate the safety of a drug and to evaluate its efficacy, and further studies are performed to determine optimal dose levels. Phase II trials typically involve approximately 100 to 300 patients, depending on the disease indication. They usually contain a control and treatment group. Phase II is usually broken up into two subphases, Phase IIa (Proof of concept) and Phase IIb (dose ranging). Phase III clinical trials are conducted to confirm the safety and efficacy of a new drug and determine the labeling information.Trial participants are usually included in one of two study groups such that one will receive the new drug being evaluated, and the other group receives the already approved, current standard of treatment. Phase III trials can enroll anywhere from 50 to 15,000 patients. In the early clinical phases, the safe and efficacious dose range is identified. Often based upon this new clinical information and marketing input, a new formulation must be developed, generally during Phase II. Before the pivotal Phase III clinical trials, the final market image should be available.This dosage form must be optimized, scaled-up, and validated. During initial clinical trials, only a small number of units of dosage forms are manufactured and put on stability. When additional and longer clinical trials are taking place, the scale-up process of making the dosage form is further investigated. The scale-up procedure is not a single-fold scale-up from few units to commercial scale (millions of units) but is rather compromised of several iterative steps. The scale-up is generally performed in stages: few units 680 ROLE OF HPLC DURING FORMULATION DEVELOPMENT (bench scale) to thousands (lab scale) to hundred thousands (pilot scale) to millions (validation and commercial). During the scale-up , many attributes of a dosage form are defined. In addi- tion, excipients may need to be changed since the original formulation may not be scalable. Ratios and physical attributes (appearance, color, embossing, size, etc.) of all material within the dosage form are defined at this point. Each time the process is scaled-up, the dosage form must be carefully characterized to ensure that the physiochemical properties and quality attributes have not changed. During formulation development, these changes in the quality attributes are monitored employing several analytical methods (identification, assay, dis- solution rate, related substances, content uniformity, etc.). HPLC plays a vital role in these analyses. Similar to formulation development, the health author- ities recognize that the methods may change as one learns more about the drug product or changes occur in the formulation during the development of a given formulation. 15.2 PREREQUISITE FOR ANALYTICAL CHEMISTS DURING FORMULATION DEVELOPMENT One of the strengths that an analytical chemist in the pharmaceutical indus- try has is the ability to propose degradation pathways without doing an exper- iment. Based on a given API structure and the environment that the API is going to reside in (solid state and/or solution state over time), degradation pathways are postulated based on known organic chemistry. An exact struc- ture of degradation product is not necessary during the paper exercise, but impact of chemical stability must be addressed. In other words, which degra- dation pathway is most to least likely to occur, at certain conditions, must be discussed in order to direct formulation development from its inception. An addendum at the end of this chapter highlights some common degra- dation pathways, along with reactions of some common functional groups [1] present in the literature. For an analytical chemist who works in a pharma- ceutical research and development group, it is necessary to postulate degra- dation products and is essential that stability-indicating methods (SIMs) for new molecular entities (NMEs) be developed that can separate these poten- tial degradation products from the active pharmaceutical ingredient. 15.2.1 Major Degradation Pathways in Pharmaceuticals There are four major degradation pathways that an analytical chemists must focus on for pharmaceutical analysis: thermolytic (heat), hydrolytic (water), oxidative (oxygen, light, peroxide), and photolytic (UV and Vis light). Many typical organic reactions that are potential degradation pathways are discussed in the addendum to this chapter as they pertain to an analytical PREREQUISITE FOR ANALYTICAL CHEMISTS 681 chemist in drug substance and drug product sectors of pharmaceutical devel- opment. References 2–7 provide a more in-depth analysis of the major degra- dation pathways for pharmaceuticals. All of the background information discussed in the addendum (Common Functional Groups) will help an analytical chemist with development of a stability-indicating method (SIM) that will assist in the development of an optimized formulation that will become a drug product in the market- place. Remember, an analytical chemist must be able to provide knowledge, not just data. 15.3 PROPERTIES OF DRUG SUBSTANCE By the time an active pharmaceutical ingredient (API) is made available to an analytical chemist in the formulation development group, most or all of the physical characteristics of an API has already been studied and the informa- tion should be available in some sort of a report from the drug substance group or preformulation group. Some of the key parameters that an analytical chemist in formulation development requires from such a report are the sol- ubility and solution stability. 15.3.1 Solubility of Drug Substance in Presence of Formulation Many times, depending upon the active-to-excipient ratio, the solubility of active in presence of excipients in certain solvents will be different. When an API is mixed with excipients, its solubility is usually lower when compared to its solubility in the same solvent by itself. If the HPLC mode for the analysis of a given drug product is reversed- phase HPLC (RP-HPLC), then the solubility of an ionizable API in aqueous solutions (pH from 1.0 to 10), methanol, acetonitrile and in aqueous/organic mixtures should be determined by an analytical chemist during formulation development. On the other hand, if alternative modes of HPLC analysis for a given drug product such as normal phase is employed, then the solubility of API in IPA, and hexane, is also very important. The main reason that an analytical chemist supporting formulation development must know the solubility before the commencement of method development is that this information will provide an initial assessment of the type of mobile phase (separation or a potency method) and/or sample preparation solvent (sample preparation procedure) that will be used for drug product method development. However, in both of the cases, the solubility listed on a drug substance report will be higher, when compared to the API with excipient diluted in the same solvent. This may not be true in all cases since it will mainly depend on the type and the amount of excipients present in the formulation. Some excipients may change the final pH of the solution after addition and/or some 682 ROLE OF HPLC DURING FORMULATION DEVELOPMENT excipients will act as solubilizing agents for a given API. In both of these cases , an analytical chemist must know the properties of excipients in the formulation. For analytical sample preparation, measurement of the final pH of the sample solution (excipients, API(s), and sample solvent mixed together) will be helpful in the development of any analytical procedure. If an API is known to be stable in acidic pH (pH 1–2), then an analytical chemist will try to utilize a certain sample solvent that has a pH in the required range. However, when a dosage form is dissolved in a sample solvent, the excipients present in the formulation (and even the API) will change the pH of the solution. The final pH of the solution must be measured in order to determine the optimal pH of sample solution to achieve longest solution stability. This is particularly important for a long sequence of injections on autosamplers for analysis, so solutions do not need to be made daily. 15.3.2 Solution Stability The objective is to determine the degradation products in the solution, not in the dosage form. It is very important to determine how the active will behave in solution. Is an API going to degrade in the solution, and to what extent? These questions can be answered with solution stability studies. The goal of solution stability for different tests can vary. For assay and degradation product test, no new degradation products (over time and above LOQ) should be formed in solution. Certain acceptance criteria (max % increase) may be set for impurities at particular levels during the solution sta- bility study. For assay determination (assay, CU, and dissolution), the content of assay over time should not change more than 2.0%. Also, refer to Chapter 9, which discusses method validation. 15.4 PROPERTIES OF EXCIPIENTS Excipients are the so-called inert ingredients contained in a pharmaceutical dosage form. Excipients are chosen based on their compatibility with the API and the function of a given excipient. Analytical chemist must spend as much time studying the properties of API as to study the properties of excip- ients. Even though the excipients are said to be inert,they are chemicals having functional groups (refer to Table 15-1). Some of the functional groups may react with your API to form degradation products that could not have been foreseen by just focusing on the structure of the API. A special consideration must be given to the effect of water on a given drug product (or a formulation). Water presents a real problem for a formulation when the drug substance or the excipients are sensitive or susceptible to hydrolysis.Whether the excipients will dissolve in certain solvents or not, their interactions with the active (in solution and solid state) are very important. PROPERTIES OF EXCIPIENTS 683 15.5 IMPACT OF EXCIPIENTS ON DEGRADATION OF API(S) Excipients are chemicals and they have functional groups . The functional group on the excipients may react with the API under certain conditions.These conditions could be under normal storage conditions or accelerated condi- tions. For these reasons, excipients must not be considered inert. For RP-HPLC, if an API is interacting with any of the excipients, then the resulting degradation product could be of a higher molecular weight than the API. However, this does not necessarily mean that it will elute after API, since the retention of components in RPLC is dependent on their relative hydrophobicity and interactions with the stationary phase. If a more hydrophobic degradation product is formed, then such a degradation product will elute after API peak (usually true for RP-HPLC when neutral species are involved, but may not be true when ionizable species are involved). In the example, shown in Figure 15-1, a development compound A has a carboxylic acid functional group (pK a ∼4.5). Three known synthetic by- products of the carboxylic acid active pharmaceutical ingredient were the alcohol, ethyl ester, and an aldehyde, which are all neutral. Three degradation products were observed in excipient compatibility stability and/or when dif- ferent formulations were put on accelerated stability: neutral degradation product and two acidic degradation products (A and B). Note that the drug substance stored under similar conditions did not produce these three degra- dation products, indicating that some excipient(s) may be inducing degrada- tion on the API. Using RP-HPLC and performing a pH study, by varying the mobile-phase pH, the ionogenic nature of these degradation products was elucidated. The retention of degradation products A and B (acidic compounds) was dependent on the mobile-phase pH, while the retention time of the neutrals 684 ROLE OF HPLC DURING FORMULATION DEVELOPMENT TABLE 15-1. Common Excipients and Their Functional Groups [8] Excipient Functional Group(s) Functional Category a Talc ––OH Anticaking agent, glidant, tablet and capsule diluent, and lubricant Mg- or Ca-stearate ––COO–– Tablet and capsule lubricant Lactose, cellulose ––O––, ––OH Diluent for dry-powder inhalers, tablets, and capsules PEG ––O––, ––OH Plasticizer, solvent, suppository base, tablet and capsule lubricant Polymethacrylates ––CO––O–– Film former, tablet binder and diluent, sustained release polymer in right combinations Triethyl citrate ––CO––O––, ––OH Plasticizer a R. C. Rowe, P. J. Sheskey, and P. J. Weller (eds.), Handbook of Pharmaceutical Excipients, 4th edition, 2003 [8]. (neutral degradation product) did not change (refer to Figure 15-1). The optimal pH of the aqueous phase was chosen to be pH 6.5 to avoid any sig- nificant changes in retention with minor variations in the pH of the aqueous portion of the mobile phase . Using this pH, the API elutes prior to all the neutral species (ethyl ester, alcohol, aldehyde, and the neutral degradation product). The neutral degradation product eluted prior to the aldehyde peak at all pH conditions, which indicated that this peak is less hydrophobic than the aldehyde and is a neutral compound (varying mobile-phase pH did not change its retention time; refer to Figure 15-1). This degradation product was later identified by LC-MS as a cyclic product. The two known neutral degra- dation products (alcohol and aldehyde) have lower molecular weight than the active, but both of them still elute after the active peak. The main reason is the retention in HPLC is dependent on the hydrophobic nature of the com- pound and not the molecular weight. However, all three neutral species elute in the order of their hydrophobicity (alcohol elutes first, then the aldehyde and then the ethyl ester, Figure 15-1). The acidic degradation products, A and B, show the greatest retention at low pH (analyzed in their predominately neutral form) and the least retention at high pH (analyzed in their predomi- nately ionized form), which is typical retention behavior for acidic compounds in RP-HPLC. Another example in which the excipient reacts with the API is the reaction between lactose and fluoxetine hydrochloride (refer to Figure A15-6 in the Addendum of this chapter). This is a typical example of the Maillard reaction [9]. Another example of the Maillard reaction is presented where Prozac (fluoxetine hydrochloride) and two generic drug products of Prozac were compared at accelerated conditions (40°C/75% RH), and the amount of degradation products (analyzed by HPLC) were found to be very different between the studied formulations (refer to Table 15-2) [10]. The authors IMPACT OF EXCIPIENTS ON DEGRADATION OF API(S) 685 Figure 15-1. Dependence of retention volume on pH of an aqueous mobile phase in RP-HPLC for drug product A on a 150 × 3.0-mm column using a 0.7-mL/min flow rate. indicated that the difference in amounts of degradation products is not due to different dosage form (capsule or the tablet), but rather due to the type and the amount of excipients used in the formulation (in particular lactose). It is also interesting to note that the formulation containing starch instead of lactose gave the lowest amount of degradation products. One of the lessons learned here must be that if an API contains an amine functional group (primary or a secondary), avoid lactose (or a similar carbohydrate) in the formulation. Also, if an API is prone to hydrolytic degradation, then the formulations in both capsules and tablets should be investigated. Capsules are made of gelatin, sugar, and water and contain about 10–15% moisture, and gelatin can absorb additional moisture. If gelatin capsules are placed in areas of high humidity, they may become malformed as they absorb moisture; and if capsules are placed in low humidity, they may become dry and brittle and may crack. The amount of degradation products formed could be influenced by the type of dosage form (tablets versus capsules) and the respective storage conditions for each of the dosage forms. 15.6 TEST METHODS FOR MOST COMMON DOSAGE FORMS IN WHICH HPLC IS THE PRIMARY TECHNIQUE The list below is a summary of test methods that are most likely to be required during the testing of most common dosage forms (tablets, capsules, and solu- tions). The list is by no means an exhaustive (since test methods to determine moisture content, pH, sterility, particulate matter, and microbial testing are not listed) and the selection of test methods depends on an evaluation of a dosage form and also on the phase of drug development. For each type of test, most common techniques utilized are listed in square brackets. As can be seen, HPLC can be employed for all of the following test methods. • Batch release and stability testing Identification [HPLC, UV, IR, NIR] (for release only) Assay [HPLC, NIR, CE, GC] Degradation products [HPLC, CE] 686 ROLE OF HPLC DURING FORMULATION DEVELOPMENT TABLE 15-2. Stability of Fluoxetine HCl Products at 40°C/75% RH [10] % Total Degradation Products by HPLC Product Major Excipient Initial 1 month 3 months 6 months 9 months Prozac Starch 0.17 0.19 0.21 0.23 0.23 Generic A Lactose 0.43 0.47 0.60 0.90 1.10 Generic Z Lactose 0.30 0.35 0.45 0.63 0.74 Content uniformity (not needed for parenterals and on stability) [UV, NIR, HPLC, CE] Dissolution (not needed for parenterals) [UV, HPLC] • Cleaning verification for active [HPLC, UV, ion mobility spectrometry, MS [11–13]] Very sensitive and specific methods are needed • Process validation Analysis of blends [HPLC, NIR] Cores and coated tablets [HPLC, NIR] • Extractables/Leachables (for parenterals) [HPLC] • Structure determination, trouble shooting [hyphenated HPLC techniques] The most common HPLC test methods from the above list are selected and thoroughly described below to ensure that the dosage form for which these test methods are selected will meet the criteria for identity, quality, potency, and purity for a given dosage form. 15.6.1 Assay and Related Substances The two most fundamental issues of importance in drug therapy are safety and efficacy. The impurities found in the bulk and dosage form may cause adverse effects by their pharmacological–toxicological profile, which in turn deter- mines the safety of a drug.The impurities found in a drug product may possess unwanted pharmacological and/or toxicological effects [14]. Both the quality and safety of a drug are said to be assured when the related substances found in the drug product are controlled and monitored effectively. Therefore, the most important topic (from the view of an analytical chemist) during formu- lation development is the activities around a test method for related substances [15–17]. The related substances found in an API could have originated during the synthetic steps, from the original starting materials/intermediates or from impurities from the starting materials that reacted in the downstream chem- istry (all of these are known as synthetic by-products). When a given API is utilized to manufacture a drug product, the degradation products found in the drug product must be identified, characterized, and/or qualified based on ICH guidelines [18].The most important reason for this is to have a quality product, which is the basis of having a safe and an efficacious drug to begin with. The relationship between synthetic by-products, degradation products, and related substances is that related substances contain the sum of synthetic by-products (originating from chemical synthesis which do not change with time and con- ditions) and degradation products (increases with time and varies under dif- ferent storage conditions). However, sometimes the synthetic by-products of the API can also be degradation products of the drug product. TEST METHODS FOR MOST COMMON DOSAGE FORMS 687 A test method for related substances must be able to separate all known and unidentified components from a given drug product. The phrase that defines this process is called “stability-indicating.” 15.6.2 Stability-Indicating Method (SIM) What is a “stability-indicating method” (SIM)? The answer to this question is found in the FDA guidance document [19] as follows: “Validated quantitative analytical methods that can detect the changes with time in the chemical, phys- ical, or microbiological properties of the drug substance and drug product, and that are specific so that the contents of active ingredient, degradation prod- ucts, and other components of interest can be accurately measured without interference.” The above statement has lot of details in reference to “what is a SIM?” The statement starts with method validation (refer to Chapter 9). Next, most methods need to be specific (specificity, resolution of active from related sub- stances, peak purity), reproducible (precision), quantitative (recovery, linear- ity, LOD, LOQ), and able to monitor a change in chemical, physical, and/or microbiological properties of drug products over time (refer to sections on stability testing and mass balance). All assay and purity methods during formulation development should start with an identical method that has been developed and validated for the par- ticular drug substance utilized to manufacture that drug product. The drug substance method should have demonstrated that the API is resolved from all potential degradation products, synthetic intermediates, and degradation products. When possible, the same drug substance method should be used for the drug product. This is especially helpful if the impurities (synthetic by- product) have been qualified (based on toxicological data and appropriate safety factor) in the drug substance.Therefore if a synthetic by-product is also a degradation product, the proposed specification limit of that degradation product in the drug product can be assigned. However, sometimes the same HPLC method cannot be used for the drug product due to potential interfer- ence of excipients and solubility of the excipients at a particular mobile-phase pH.Then a cross-correlation of the relative retention time (RRT) of the impu- rities in the drug substance which were qualified and the RRT of the impuri- ties in the drug product should be determined. The use of LC-MS and/or LC-NMR is strongly encouraged. Also, if authentic synthetic by-products of the API are available, then elution can be confirmed using both the API and drug product methods. Even if the same drug substance HPLC method is used for the drug product, forced decomposition studies must be performed again for the drug product to confirm the resolution of potential degradation products from the API. In addition, forced decomposition studies must also be performed for dif- ferent dosage forms (capsule, tablet, suspension, injectable, etc.) of the same drug substance. 688 ROLE OF HPLC DURING FORMULATION DEVELOPMENT [...]... technique to help in developing a robust formulation for a drug product However, the current section will discuss sample preparation solvent since it becomes an integral part of HPLC when we are discussing HPLC methods for a particular formulation Any sample preparation solvent that is chosen for any HPLC method must be compatible with the HPLC solvents utilized for that particular test method The current... utilized for a particular drug product The pros and cons of utilizing the information in hand for method development during formulation development are as follows for any HPLC method During the formulation development stage, much of the physical characterization of an API has already been performed and is known Many of the HPLC methods (identification, related substances, etc.) are also known for an API... time (compared to API) for a given HPLC method The HPLC method should be able to resolve all possible major degradation 692 ROLE OF HPLC DURING FORMULATION DEVELOPMENT Figure 15-4 Chromatogram of an overly stressed solid dosage form products generated from the forced degradation studies (heat-, light-, water-, acid-, base-, and peroxide-related) from the API Initial solution forced decomposition experiments... Excipient compatibility screening performed in early formulation development tion at 2 weeks and further degradation at 4 weeks, indicating that if these excipients are to be used for further formulations, the formulation should be protected from moisture The major degradation product was identified by HPLC- MS as the carboxylic acid impurity 698 ROLE OF HPLC DURING FORMULATION DEVELOPMENT This drug substance... acceptable This was the preliminary HPLC method and was further optimized during development for the combination drug product FORCED DECOMPOSITION 691 15.7 FORCED DECOMPOSITION When developing stability-indicating methods, forced degradation testing is performed to demonstrate specificity of any separation method as well as to gain some insight into the degradation pathways Forced degradation studies are... was repeated at 20% and 100% Metformin HCl in solution to confirm that arginine inhibits the interaction of Metformin HCl with croscarmellose because arginine is believed to be competing for the 702 ROLE OF HPLC DURING FORMULATION DEVELOPMENT Figure 15-14 Percent recovery of Metformin HCl in the presence of arginine and without arginine at different concentrations of Metformin HCl in solution same functional... product/retention of API) 15.11 UNIFORMITY OF DOSAGE UNITS The term uniformity of dosage units (UDUs) is defined as the degree of uniformity in the amount of the active substance among dosage units [35] The UDUs can be demonstrated by either of two methods: content uniformity (CU) or mass variation (MV) Mass variation can be utilized for drug 708 ROLE OF HPLC DURING FORMULATION DEVELOPMENT Figure 15-19... Chromatogram showing a separation of three APIs for a CU method by RP -HPLC for a combination product products containing 25% (w/w) active in a dosage form and has a label claim of 25 mg or higher The HPLC method (if a separation of two components is required) should be very simple because it is for the measurement of the API only However, during the HPLC method there should be no interference at the... per bath as well as multiple time points for profile testing, fast run times are preferred to quickly determine the results If a fast HPLC method for CU is available, then the identical HPLC method can be utilized for dissolution analysis 15.16 METHOD DEVELOPMENT HPLC method development has already been covered in Chapter 8 The focus of this chapter is to utilize HPLC and the data generated by this technique... [22], who surveyed 20 pharmaceutical companies on the practices of forced decomposition studies During formulation development, forced decomposition would be the first study that must be repeated (for the reasons stated above) in the presence of API + excipients of the potential formulation A drug product without the API is utilized as a control (placebo) The potential loss of API for each stress condition . specify a formula or to express a formula in system- atic terms or concepts. The formula, in the current case, is a pharmaceutical dosage form. A formulation. of any 679 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. formulation