The use of a process analytical technology has been demonstrated using near-infrared spectroscopy for continuous manufacturing of pharmaceutical formulations and is within the scope of the U.S. Food and Drug Administration's real-time release testing initiative. While effective for simple formulations, this preliminary study investigates whether such a spectroscopic surrogate application can replace pharmaceutical dissolution testing for extended-release formulations. In this study, we will assess the use of process Raman spectroscopy for real-time dissolution testing. Extended-release tablet formulations often accomplish the release rate delay through the addition of gelling agents. In this work, hydroxypropyl methylcellulose (HPMC) polymers were used to formulate extended-release niacin tablets. Process Raman spectroscopy was evaluated as a tool to effectively model dissolution profiles to determine if the optical technique has the ability to differentiate HPMC polymers from the background and be selective for the polymer type employed. Our preliminary work indicates that while Raman can effectively detect and monitor the niacin response of the tablet formulations, there are not enough unique spectral features between the different HPMC polymers to selectively resolve their responses. Additional measurements and chemometric analysis might suggest otherwise. Thus, for extended-release tablet applications with continuous manufacturing, further dissolution surrogate development is needed.

Monoclonal antibody-drug conjugates (ADCs) are an expanding therapeutic class of biomolecules for which relatively few analytical and preparative separation options exist. Purification of ADCs with a specific drug antibody ratio is even more challenging. We report the first application of countercurrent separation (CCS) to this problem. An ADC mimic was successfully chromatographed using an aqueous two-phase system (ATPS) consisting of PEG 1000/sodium citrate pH 7.5/water, 17.75/17.75/64.50 (w/w/w). Notably, different partition coefficients (K) in this ATPS for the ADC mimic (0.09 < K < 0.16) and its monoclonal antibody backbone, IgG (0.16 < K < 0.27), were observed using CCS. Differential elution behavior of such high-molecular-weight biomolecules, 146,441 vs. ∼150,000 Da, using CCS has no precedent. The results provide a proof of concept for further exploration of the application of ATPSs and CCS to the separation of ADCs.

The use of mass spectrometry imaging for crystallinity detection offers improved matrix selectivity and sensitivity over the techniques, such as X-ray microscopy and Raman spectrometry, that are traditionally used with this work. Crystallinity is observed in electrospray laser desorption ionization mass spectrometry imaging (ELDI-MSI) as a high-intensity agglomeration of the analyte in a spatially resolved image. As this is an indirect method of crystallinity detection, confirmation of this method’s ability to detect crystallization in amorphous formulations is needed by directly correlating observations of tablet crystallinity by ELDI-MSI to those of an established detection technique. Micro-computed tomography (micro-CT) has the necessary sensitivity for this investigation and is ideal for use in evaluating the correlation with crystallinity detection by ELDI-MSI. In this work, micro-CT followed by ELDI-MSI, in the same location on tablets of amorphous formulations of miconazole spiked with trace levels of crystalline miconazole, were analyzed. Crystals detected by ELDI-MSI as an agglomeration spatially matched with the detected crystals but were chemically unidentified by micro-CT. The results of this correlation study and a conclusion about the effectiveness of ELDI-MSI as a complimentary technique to indirectly detect crystallinity in enabling formations of an amorphous API are presented.

Aims: The aim of this project was to develop a QC friendly and efficient method of protein species of origin identification to replace more costly mass spectrometric based methods currently being used for this testing. Background: NMR relaxation measurements with proteins in aqueous solutions exploit the fast chemical exchange between water and exposed NH and OH protons of amino acid side chains in the folded protein structure unique to each biologic drug. Implementation of this technique has led to routine testing for authentication and forensics of biopharmaceuticals, determination of moisture content in lyophilized protein formulations and aggregation of proteins in solution. For small molecule applications, TD-NMR can detect if solvents are received neat or tainted with moisture, impurities, or denaturants. Objective: The objective of this study was to evaluate the ability of NMR Relaxation measurements to differentiate between sources of Albumin proteins as a rapid QC test. Evaluation of differences in molecular mobility between components in a solution as reflected in the longitudinal (T1) and transverse (T2) relaxation times of protons demonstrate that NMR relaxation techniques can distinguish between different albumin sources of origin. Methods: Representative albumin proteins from differing sources of origin were studied. Using bovine serum albumin (BSA) as the target species of origin, NMR relaxation techniques as well as chemometric modeling were used to evaluate the use of this technique for protein source of origin identification. Results: NMR Relaxation using benchtop instrumentation showed that the bovine albumin species of origin can be distinguished from porcine, chicken egg white and sheep sources of origin. Goat albumin selectivity remained questionable and BSA cannot be distinguished from human or rabbit sources of origin within the representative variability. : T2 transverse relaxation results were significantly more discriminating for protein source identification than the T1 longitudinal relaxation result by itself. The T1 longitudinal relaxation result did not contribute significantly to this investigation. However, fusing the T1 data with the T2 transverse relaxation results and using larger data sets merit further evaluation in the hope of achieving additional selectivity. Conclusion: While additional lots are needed for more definitive results, this preliminary evaluation of using NMR Relaxation demonstrated the capability for the source of origin species discrimination and identification using benchtop NMR instrumentation.

Agglomeration of active pharmaceutical ingredients (API) in tablets can lead to decreased bioavailability in some enabling formulations. In a previous study, we determined that crystalline APIs can be detected as agglomeration in tablets formulated with amorphous acetaminophen tablets. Multiple method advancements are presented to better resolve agglomeration caused by crystallinity in standard tablets. In this study, we also evaluate three "budget" over-the-counter headache medications (subsequently labeled as brands A, B, and C) for agglomeration of the three APIs in the formulation: Acetaminophen, aspirin, and caffeine. Electrospray laser desorption ionization mass spectrometry imaging (ELDI-MSI) was used to diagnose agglomeration in the tablets by creating molecular images and observing the spatial distributions of the APIs. Brand A had virtually no agglomeration or clustering of the active ingredients. Brand B had extensive clustering of aspirin and caffeine, but acetaminophen was observed in near equal abundance across the tablet. Brand C also had extensive clustering of aspirin and caffeine, and minor clustering of acetaminophen. These results show that agglomeration with active ingredients in over-the-counter tablets can be simultaneously detected using ELDI-MS imaging.

Polysorbates (also known as "Tween") are common components of protein formulations used to minimize protein adsorption and stabilize the protein. These nonionic surfactants are heterogenous mixtures of fatty acids with a complex reversed-phase profile due to the inhomogeneity of the polymers present. Polysorbates can be oxidized, which can be hard to detect in the complex polymer profile. Further adding to the analytical challenge is the lack of a chromophore for the detection of these polymers. The routine analysis of polysorbates in protein formulations was greatly improved through the introduction of online solid-phase extraction (SPE) to simplify the polysorbate profile for quantification. However, this method combines many of the polysorbate polymers into a single peak for detection, thus limiting its effectiveness for detecting degradation. To address the need for a stability indicating method without the complexity of the reversed-phase profile, an optimized online SPE method was developed and investigated. Using polysorbate 80, this investigation shows that further expanding the step gradient can yield a profile that is stability indicating and available for routine testing of protein formulation.

Excipients play an important role in the formulation of dosage forms and can be used to improve the bioavailability of a drug through physical interactions that alter the rate of dissolution of a drug. The objective of this study was to predict the effect of formulation on the dissolution rate of a poorly soluble drug using computer simulations. Solid dispersion of ritonavir was prepared. Dissolution test results of direct compressed tablets with and without disintegrant in various media with physiologically relevant pH were compared with simulations. Solubilizer and disintegrant effect were evaluated on the Dose, Disintegration, and Dissolution Plus (DDDPlus) simulation software (version 5.0.0011, Simulations Plus, Inc., Lancaster, CA, USA) using previously published solubility data on ritonavir. Observed and predicted dissolution profiles similarity tests and drug release mechanisms were assessed. Optimization of the solubilizer effect coefficient (SEC) on the program gives good estimations of the effect of copovidone in the solid dispersion in the dissolution profiles of all tablets. The SEC is dependent on the solubility of the active pharmaceutical ingredient (API) at the local pH and the dissolved concentration of the solubilizer. Disintegrant concentration in the program has no effect on simulations, rather the disintegration time was the predictive factor. Drug release was formulation controlled in the tablets without disintegrant and in the tablets with disintegrant was via drug diffusion and polymer surface erosion. DDDPlus has the potential to estimate the effect of excipients in a formulation on in vitro dissolution at an early stage in the drug development process. This could be useful in decisions on formulation strategies to enhance bioavailability in poorly soluble drugs.

Dissolution testing is a major tool used to assess a drug product's performance and as a quality control test for solid oral dosage forms. However, compendial equipment and methods may lack discriminatory power and the ability to simulate aspects of in vivo dissolution. Using low buffer capacity media combined with an absorptive phase (biphasic dissolution) increases the physiologic relevance of in vitro testing. The purpose of this study was to use non-compendial and compendial dissolution test conditions to evaluate the in vitro performance of different formulations. The United States Pharmacopeia (USP)-recommended dissolution method greatly lacked discriminatory power, whereas low buffer capacity media discriminated between manufacturing methods. The use of an absorptive phase in the biphasic dissolution test assisted in controlling the medium pH due to the drug removal from the aqueous medium. Hence, the applied non-compendial methods were more discriminative to drug formulation differences and manufacturing methods than conventional dissolution conditions. In this study, it was demonstrated how biphasic dissolution and a low buffer capacity can be used to assess in vitro drug product performance differences. This can be a valuable approach during the early stages of drug product development for investigating in vitro drug release with improved physiological relevance.

Background: Enabling formulations have been implemented by the pharmaceutical industry as an effective tool for keeping Active Pharmaceutical Ingredient (API) in an amorphous state. Upon dosing in the amorphous state, many drugs which fail to demonstrate bioactivity due to the limited solubility and bioavailability of their crystalline form become bioavailable. Purpose: The analytical techniques use today for crystallinity detection are challenged by the sensitivity and robustness needed to achieve a 5% quantitation limit in low dose drug products. Our laboratory has developed a novel procedure capable of meeting this sensitivity and selectivity requirement. This is achieved by exploiting the differences in kinetic solubility of the formulated amorphous and free crystalline forms of API currently being used in dosage form platforms. Methods: Representative amorphous drug formulations were prepared and spiked with varying levels of crystalline drug substances to evaluate the selectivity and recovery of the crystalline drug substance from the product formulation. Kinetic solubility testing using a (i) Particle wetting phase, (ii) Particle suspending/erosion phase, (iii) Sampling time point and (iv) A total recovery determination for the drug substance. Results: The method selectively and quantitatively distinguishes crystalline drug substance from amorphous drug substance for samples spiked from 2.5% to 10% of the nominal label concentration of the API in the dosage form matrix. Conclusion: The kinetic solubility approach reported here achieves sensitive crystallinity quantitation for low drug level amorphous drug formulations at levels not yet achieved by complimentary analytical techniques.

Cleaning in any Good Manufacturing Practice (GMP) laboratory is an important aspect of the analytical experiment. The laboratory must ensure the equipment does not contain residual active pharmaceutical ingredients (APIs) or impurities that may affect the outcome of any current or future experiments. While this is standard practice for GMP manufacturing operations, common laboratory equipment is often held to less stringent standards. The potential manhours lost due to investigations for extraneous peaks and contamination can be significant and cause delays in releasing product. Potential compliance issues related to ineffective cleaning are particularly important for dissolution instrumentation. Our laboratory has modeled the challenges of cleaning automated dissolution systems using representative soluble and poorly soluble APIs. Poorly soluble drugs often entail the use of surfactants in the dissolution media which also have a potential carryover issue. Using a manufacturing cleaning validation based approach, the study discussion presented will address the cleaning effectiveness for both sample and media considerations.

Nonionic surfactants are used in the pharmaceutical industry in a variety of formulations.

In-vitro dissolution testing of pharmaceutical formulations has been used as a quality control test for many years. At early drug product development, in vivo predictive dissolution testing can be used for guidance in the rational selection of candidate formulations that best fit the desired in vivo dissolution characteristics. At present, the most widely applied dissolution media are phosphate-based buffers and, in some cases, the result of dissolution tests performed in such media have demonstrated reasonable/acceptable IVIVCs. However, the presence of phosphates in human GI luminal fluids is insignificant, which makes the use of such media poorly representative of the in vivo environment. The gastrointestinal lumen has long been shown to be buffered by bicarbonate. Hence, much interest in the development of suitable biorelevant in vitro dissolution media based on bicarbonate buffer systems has evolved. However, there are inherent difficulties associated with these buffers, such as maintaining the pH throughout the dissolution test, as CO2 tends to leave the system. Various mathematical models have been proposed to analyze bicarbonate buffers and they are discussed in this review. Approaches such as using simpler buffer systems instead of bicarbonate have been proposed as surrogate buffers to produce an equivalent buffer effect on drug dissolution on a case-by-case basis. There are many drawbacks related to simpler buffers systems including their poor in vivo predictability. Considerable discrepancies between phosphate and bicarbonate buffer dissolution results have been reported for certain dosage forms, e.g. enteric coated formulations. The role and need of bicarbonate-based buffers in quality control testing requires scientific analysis. This review also encompasses on the use of bicarbonate-based buffers as a potentially in vivo predictive dissolution medium for enteric coated dosage forms.

Adding an in-line mixer between the sample injector and column in a liquid chromatography system can be an effective way to resolve problems with peak shape caused by the sample diluent.

In early drug development, the selection of a formulation platform and decisions on formulation strategies have to be made within a short timeframe and often with minimal use of the active pharmaceutical ingredient (API). The current work evaluated the various physicochemical parameters required to improve the prediction accuracy of simulation software for immediate release tablets in early drug development. DDDPlus™ was used in simulating dissolution test profiles of immediate release tablets of ritonavir and all simulations were compared with experimental results. The minimum data requirements to make useful predictions were assessed using the ADMET predictor (part of DDDPlus) and Chemicalize (an online resource). A surfactant model was developed to estimate the solubility enhancement in media containing surfactant and the software’s transfer model based on the USP two-tiered dissolution test was assessed. One measured data point was shown to be sufficient to make predictive simulations in DDDPlus. At pH 2.0, the software overestimated drug release while at pH 1.0 and 6.8, simulations were close to the measured values. A surfactant solubility model established with measured data gave good dissolution predictions. The transfer model uses a single-vessel model and was unable to predict the two in vivo environments separately. For weak bases like ritonavir, a minimum of three solubility data points is recommended for in silico predictions in buffered media. A surfactant solubility model is useful when predicting dissolution behavior in surfactant media and in silico predictions need measured solubility data to be predictive.