Development and Qualification of Visible Particle Load Analysis Methods for Injectable Drug Product Primary Packaging Components.

In the biopharmaceutical industry, the observation of a single particle in a vial or syringe may result in entire lots of drug product recalls. U.S. Pharmacopeia <787> and <788> describe light obscuration methods and particle collection (membrane filtration) followed by light microscopy for particle counting of filled drug products. However, there are no corresponding pharmacopeial methods for determining the particle levels of unfilled primary packaging components or their packaging materials (tubs, nests, bags, etc.). This article describes a quantification method to accurately assess the number of particles in primary containers and corresponding closures. As a microscopic method, the size ranges can be set by the user and are limited only by the optical properties of the microscope and analysis time. Particle load is a critical quality attribute that has a direct impact on product safety. Applying a standardized method to compare the effect of process changes on particle load can aid manufacturers in refining their processes to minimize particulates. Described herein are the critical parameters to develop physical rinse methods and the subsequent qualification results to measure the visible particle load of nonsiliconized and siliconized primary packaging systems.

Characterization of a Novel Particle in a Pharmaceutical Drug Product.

A previously unreported particle type was observed during routine visual vial inspection of a liquid drug product and suspected to be the result of vial delamination. Delamination is the corrosive attack on the interior surface of a glass container resulting in the release of thin flake-like glass particles, lamellae, into solution. It is a major concern for pharmaceutical companies, especially for parenteral solutions, and drug programs with a high risk for delamination are typically monitored for lamellae formation through long-term stability studies. Although these particles observed resembled lamellae (i.e., thin, reflecting light, buoyant) they were not the result of glass delamination. In this study, the authors describe a previously unreported particle type and provide a detailed comparison with known lamellae exposed to the same drug formulation. The chemical, elemental, and morphological characteristics of the particles and respective vials are described in detail. Overall, the particles' high organic and low silica elemental signature, along with no signs of delamination on the glass vial inner surface demonstrate that this lamellae-like observation is a novel particle form that can be distinguished from lamellae formed from vial glass delamination.

Critical Aspects of pH Measurement for Bacteriostatic Water for Injection.

Bacteriostatic water for injection (bWFI) is a common diluent for parenteral pharmaceutical products. bWFI is sterile water for injection containing one or more suitable antimicrobial agents to suppress the growth of microbial contaminants. United States Pharmacopeia (USP) monograph describes bWFI with pH ranging from pH 4.5 to 7.0. Lacking buffering reagents, bWFI has very low ionic strength, no buffering capacity and is prone to sample contamination. These characteristics pose a challenge for accurate bWFI pH measurements which are characterized by long response times and noisy signals, resulting in inconsistent results. The challenging nature of bWFI pH measurement, however, is not fully recognized as pH is generally considered a routine analytical technique. Even with the addition of KCl to increase ionic strength as recommended by the USP bWFI monograph, variability in pH results is still observed without careful consideration of other critical measurement factors. To bring awareness to the challenges associated with bWFI pH measurement, we present a comprehensive characterization of the bWFI pH measurement process that includes an evaluation of probe suitability, measurement stabilization time, and pH meter settings. While these factors may be non-critical and sometimes overlooked when developing pH methods for buffered samples, they can have a significant impact on bWFI pH measurement. We present recommendations that can help reliable bWFI pH measurements for routine execution in a controlled environment. These recommendations also apply to other pharmaceutical solutions or water samples with low ionic strength.

Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer.

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.

Recommendation of Single Time Point Leachables Testing for Lyophilized Biotechnology Products Stored in Rubber Stoppered Glass Vial Systems.

As a popular format of primary container closure systems, rubber stoppered glass vials are often used in storing and delivering lyophilized and liquid formulated therapeutic protein products. Assessing extractables and leachables from rubber stoppered glass vial systems is required to ensure drug product quality and patient safety. Lyophilized biopharmaceutical drug products are generally considered as less impacted by leachables during storage and transportation than the liquid formulated drug products. Single time point leachables testing for lyophilized biopharmaceutic drug products is recommended. The recommendation is based on our published comprehensive leachable data collected at multiple time points for five lyophilized drug products stored in different rubber stoppered glass vial systems with additional supporting comprehensive leachable data collected for nineteen liquid formulated drug products stored in different syringe and vial systems, which is statistically and scientifically sound. The leachable data evaluated herein were generated based on a holistic approach which ensured successful qualification of different vial systems as primary containers and delivery systems for various biotherapeutic products. The organic and elemental impurities of the leachable profiles of all the twenty-four drug product samples were below the limit of detection at all the time points. For lyophilized drug products, product surface interaction during storage time and shipping is unlikely. Timing of single time point leachables testing can be flexible. Performing leachables testing at one-year time point is recommended as it allows for enough time for chemicals to leach out from product contact surfaces into drug products and thus provides the earliest opportunity for mitigation of unpredicted leachables of concern, if any. However, testing at other stability time points can also be considered depending on the development strategy of the sponsor. Therefore, recommendation of single time point leachables testing for lyophilized drug products stored in rubber stopped glass vials at an appropriate time point is a scientifically sound approach.

Non-targeted characterization of attributes affecting antibody-FcγRIIIa V158 (CD16a) binding via online affinity chromatography-mass spectrometry.

Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5< G0F < G0 afucosylated ≅ G1F < G2F). Sialylated species bound weaker to their asialylated counterparts as reported previously. High mannose glycoforms bound weaker than G0F, contrary to previously published studies using glycosylated receptors. Also, increased receptor binding affinity associated with afucosylated antibodies was not observed with the aglycosylated FcγRIIIa. This apparent difference from previous findings highlighted the importance of the glycans on the receptors for mediating stronger binding interactions. Characterization of temperature-stressed samples by LC-MS peptide mapping revealed over 200 chemical and post-translational modifications, but only the Fc glycans, deamidation of EU N325, and an unknown modification to either proline or cysteine residues of the hinge region were found to have a statistically significant impact on binding.Abbreviations: Antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antigen receptor (CAR), Chinese hamster ovary (CHO), dithiothreitol (DTT), electrospray ionization (ESI), hydrogen-deuterium exchange (HDX), filter aided-sample preparation (FASP), Fcγ receptor (FcγR), fragment crystallizable (Fc), high-pressure liquid chromatography (HPLC), immunoglobulin G (IgG), liquid chromatography (LC), monoclonal antibody (mAb), mass spectrometry (MS), natural killer (NK), N-glycolylneuraminic acid (NGNA), N-acetylneuraminic acid (NANA), principal component analysis (PCA), surface plasmon resonance (SPR), trifluoroacetic acid (TFA), and extracted mass chromatogram (XMC).

State-of-the-art and emerging trends in analytical approaches to pharmaceutical-product commercialization.

The biopharmaceutical landscape continues to evolve rapidly, and associated modality complexity and the need to improve molecular understanding require concomitant advances in analytical approaches used to characterize and release the product. The Product Quality Attribute Assessment (PQAA) and Quality Target Product Profile (QTPP) frameworks help catalog and translate molecular understanding to process and product-design targets, thereby enabling reliable manufacturing of high-quality product. The analytical target profile forms the basis of identifying best-fit analytical methods for attribute measurement and continues to be successfully used to develop robust analytical methods for detailed product characterization as well as release and stability testing. Despite maturity across multiple testing platforms, advances continue to be made, several with the potential to alter testing paradigms. There is an increasing role for mass spectrometry beyond product characterization and into routine release testing as seen by the progress in multi-attribute methods and technologies, applications to aggregate measurement, the development of capillary zone electrophoresis (CZE) coupled with mass spectrometry (MS) and capillary isoelectric focusing (CIEF) with MS for measurement of glycans and charged species, respectively, and increased application to host cell protein measurement. Multitarget engaging multispecific modalities will drive advances in bioassay platforms and recent advances both in 1- and 2-D NMR approaches could make it the method of choice for characterizing higher-order structures. Additionally, rigorous understanding of raw material and container attributes is necessary to complement product understanding, and these collectively can enable robust supply of high-quality product to patients.

Observation and Mitigation of Lamellar Silica Particles Formed in Pharmaceutical Products Packaged in Glass Vials.

A new type of lamellae-like particles was observed in protein based liquid therapeutic protein drug product (DP) packaged in standard (STD) and delamination controlled (DC) Type IB glass vials stored at 2-8°C as early as two weeks after manufacture. These particles were determined to be remarkably different from lamellae in not only in their chemical composition, but in the mechanism by which these are formed. The lamellae-like particles were an ultra-thin (< 200 nm) film, appeared curled, sheet-like, folded with no defined edges identified as lamellar silica composed of silica and polysorbate 80 (PS 80). It was also observed that the lamellar silica particles, when formed in a given drug product lot, not only were observed in a small percentage of vials, but also remained at low (≤ 5) numbers in affected vials, often decreasing in number over time. This is in contrast to the large number of commonly reported glass lamellae (hundreds per vial) observed in vials prone to delamination with a glass vial interior showing a delaminated inner surface. In this case study, evidence from low Si leachable levels in solution and various surface analytical techniques supported the conclusion that there was neither delamination nor early signs of glass delamination like reaction zones occurring in those impacted vials, regardless. A mechanism for particle formation was hypothesized and experimentally confirmed. Lamellar silica particles are composed of an admixture of condensed silica and PS 80 deposited on the interior walls of glass vials, which form and may be released into solution over time. The root cause was determined to be conditions present during preparation of the vials for drug product filling, specifically the vial washing and depyrogenation steps. These conditions are known to make glass vials prone to delamination; in this case study, they resulted in interactions between the glass and PS 80 present in the formulation. Incomplete drying of the glass vials during depyrogenation in closed ovens was confirmed as the contributing factors that led to lamellar silica particle formation via the studies of silicate spiked into the DC Type IB glass vials filled with the mAb DP in which lamellar silica particles were observed. Prevention of lamellar silica particles formation was successfully achieved through optimization of the duration and pressure of air blow during the vial washing and drying process in a depyrogenation oven. This was evidenced by the lack of appearance of the lamellar silica particles over 48 months for the DP lots filled post optimization. Additionally, the formation of lamellar silica was also mitigated by changing the vial washing process from a closed oven process to a tunnel process, which allowed for improved air flow and hence better drying of the vial primary container.

Enabling development, manufacturing, and regulatory approval of biotherapeutics through advances in mass spectrometry.

Rapid technological advances have significantly improved the capability, versatility, and robustness of mass spectrometers which has led to them playing a central role in the development, characterization, and regulatory filings of biopharmaceuticals. Their application spans the entire continuum of drug development, starting with discovery research through product development, characterization, and marketing authorization and continues well into product life cycle management. The scope of application extends beyond traditional protein characterization and includes elements like clone selection, cell culture physiology and bioprocess optimization, investigation support, and process analytical technology. More recently, advances in the MS-based multi-attribute method are enabling the introduction of MS in a cGMP environment for routine release and stability testing. While most applications of MS to date have been for monoclonal antibodies, the successes and learnings should translate to the characterization of next-gen biotherapeutics where modalities like multispecifics could be more prevalent. In this review, we describe the most significant advances in MS and correlate them to the broad spectrum of applications to biotherapeutic development. We anticipate rapid technological improvements to continue that will further accelerate widespread deployment of MS, thereby elevating our overall understanding of product quality and enabling attribute-focused product development.

Observation and Mitigation of Leachables from Non-Product Contact Materials in Electromechanical Delivery Devices for Biotechnology Products.

Battery-powered drug delivery devices are widely used as primary containers for storing and delivering therapeutic protein products to improve patient compliance and quality of life. Compared to conventional delivery approaches such as pre-filled syringes, battery-powered devices are more complex in design requiring new materials/components for proper functionality, which could cause potential product safety and quality concerns from the extractable and leachables (E&L) of the new materials/components. In this study, E&L assessments were performed on a battery-powered delivery device during the development and qualification of the device, where novel compound 2­hydroxy-2-methylpropiophenone (HMPP) and related compounds were observed in both E&L. The source of the HMPP and related compounds was identified to be the nonproduct contact device batteries, in which HMPP photo-initiator was used as a curing agent in the battery sealant to prevent leakage of the battery electrolytes. Toxicology assessment was performed, which showed the levels of HMPP observed in the device lots were acceptable relative to the permitted daily exposure. A drug product HMPP spike study was also performed, where no product impact was observed. Based on these assessments, an action threshold and specification limits could be established as a control strategy, if needed, to mitigate the potential risks associate with the observed leachables. As a full resolution, seven battery candidates from different suppliers were screened and one new battery was successfully qualified for the delivery devices. Overall, the holistic E&L approach was fully successful in the development and qualification of the battery-powered devices for biotherapeutic products delivery ensuring product quality and patient safety. Non-product contact materials are commonly rated as low or no risk and typically considered as out of scope of E&L activities for delivery systems following industry benchmark and regulatory agency guidance. This case study is novel as it brings into attention the materials that might not normally be in consideration during the development process. It is highly recommended to understand materials in the context of intended use on a case-by-case basis and not to generalize to ensure successful development and qualification.

Identification of critical chemical modifications and paratope mapping by size exclusion chromatography of stressed antibody-target complexes.

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

A Risk-Based Approach to Evaluate and Control Elemental Impurities in Therapeutic Proteins.

Control of elemental impurities in the drug products evolved from the generic visual testing of heavy metals as their sulfides to specific elements of toxicological concern in the final drug products by instrumental analysis. The International Council for Harmonisation (ICH) Q3D (R1) guideline for elemental impurities describes a risk-based approach to identify, assess, and control the potential elemental impurities in drug products within the established permitted daily exposures (PDE). Challenges to this approach include how to assess the risks associated with contributing sources such as utilities, manufacturing equipment, container-closure systems, and excipients. Defining at what stage of development that such assessment should be performed to identify the risk levels can be equally challenging. In this article, we report an approach to control elemental impurities of toxicological concern, compliant to the Q3D (R1) guideline, and a summary of results obtained on multiple protein therapeutic products. This approach follows the elements of Process Validation, i.e., Design, Qualification, and Continuous Verification. The design includes the selection of excipients and their suppliers that meet the Option 1 requirement of Q3D (R1). It also comprises the selection of manufacturing equipment, container-closure systems, and utilities. The qualification includes the testing of the potential sources of elemental impurities, i.e., excipients, utilities, and leachables/extractables from the manufacturing equipment and container-closure systems. The Continuous Verification comes from the testing of representative batches at the initiation of stability studies of clinical or commercial drug product batches and at the end of shelf-life expiry of the drug product, and when changes are made to the manufacturing equipment, sources of excipients and container closure systems, and any formulation changes. Our experience shows that the risk associated with the impurity levels of the ten elements of toxicological concern in the therapeutic protein drug products, parenterally administered, is well below the control threshold (30% PDE) in the drug product recommended by the ICH Guideline. Although our focus is on the injectable therapeutic proteins, this approach can be applied to the products administered via other routes as well.

Development and Validation of a HPLC-UV Method for Urea and Related Impurities.

Urea is used in biopharmaceutical manufacturing processes for the purification of therapeutic proteins, for cleaning columns, and for refolding proteins after purification. The urea used for such purposes is typically USP grade material obtained from commercial sources and further characterization is required prior to use, such as determination of purity and identity. For this purpose, a robust analytical method is needed that can characterize the known organic impurities of urea. However, the existing methods show high assay variability and are not able to resolve all known organic impurities as desired for accurate quantification. In the present manuscript we developed a new high-performance liquid chromatography method with UV detection for the separation of urea and its impurities (biuret, cyanuric acid, and triuret). The method performance characteristics evaluated for urea and biuret were specificity, linearity, accuracy, identity, precision, and robustness and the newly developed method met all predefined performance acceptance criteria.

Determination of interactions between antibody biotherapeutics and copper by size exclusion chromatography (SEC) coupled with inductively coupled plasma mass spectrometry (ICP/MS).

The concept of coupling of size-exclusion HPLC with ICP/MS (SEC-ICP/MS) was first applied in this work as a novel approach in the biotechnology field to assess metal binding to Immunoglobulin G (IgG) mAbs. This method can be used to probe the mechanism and biophysical properties of metal-protein interactions to gain a deeper understanding of the potential impact of metals during drug product manufacturing. Two IgG1s and one IgG2 drugs were investigated. Cu2+ was selected as the metal of interest due to its known ability to form strong complexes with organic molecules and to bind and enhance the degradation of mAbs. Instrument and separation conditions (interface, columns, and mobile phase) were studied for the separation of the protein-metal bound and unbound fractions of a bovine superoxide dismutase (SOD) standard prior to on-line detection of the mAb-metal (Cu) binding. The SEC-ICP/MS method was used to show copper binding by biotherapeutics by comparing the retention times of the protein by SEC and the metal by ICP/MS, to see if they co-elute at the same time. The approach developed offers considerable advantages over methods based on ultrafiltration followed by off-line metal determination in terms of speed, simplicity, precision and selectivity regarding the molecular weight of the complexes involved. In conjunction with other techniques, this method may provide in-depth knowledge of metal-induced mAb degradation mechanisms in biologics process development, be used as an analytical tool for mAb manufacturing in the cell culture process, and be applied during various stages of drug product manufacturing to gain a deeper understanding of the potential impact of metals during biotherapeutic development.

Stability-indicating hydrophilic interaction liquid chromatography method for highly polar and basic compounds.

A hydrophilic interaction liquid chromatography (HILIC) method was developed for the analysis of very polar and basic 4-(aminomethyl)pyridine (4-AMP) and its related compounds. Separation parameters such as stationary phase, buffer pH, buffer ionic strength, organic modifier, and column temperature were evaluated. The retention mechanisms were explored through the evaluation of the common chromatographic parameters in the method development. The data indicated the existence of surface adsorption phenomena for 4-AMP and its positional isomers (2-AMP, 3-AMP). For two degradants, different retention mechanisms might be involved when compared to 4-AMP. The selectivity of two critical pairs 3-/4-AMP isomer and Degradant-1/-2 diastereomer changed through isoelution temperature with reversal of elution order. The validation results indicated that the HILIC method is a sensitive, reproducible, and robust method suitable for the analysis of 4-AMP and its related compounds.

Column temperature programming in enantioseparation of dihydropyrimidinone compounds using derivatized cellulose and amylose chiral stationary phases.

We report the application of column temperature programs as a tool to examine unusual temperature-induced behaviors of polysaccharide chiral stationary phases (CSPs). Using dihydropyrimidinone (DHP) compounds as probes we observed the heating (10-50 degrees C) and cooling (50-10 degrees C) van't Hoff plots of retention factors and/or selectivities of DHP compounds were not superimposable on AD, IA, and AS-H columns solvated with ethanol (EtOH)/n-hexane (n-Hex) mobile phases. The plots were not superimposable on AD, IB, and AS-H columns solvated with 2-propanol (2-PrOH)/n-Hex mobile phases. The thermally induced path-dependant behaviors were caused by slow equilibration as evidenced by the disappearance of the hysteresis in the second heating to cooling cycle and in a cooling to heating cycle. From the step-temperature program (10-50-10 degrees C), only EtOH solvated AD and AS-H phases showed the change of retention factors and/or selectivities with time while only 2-PrOH solvated AS-H phase showed similar behaviors.

Effect of column temperature on enantioseparation of dihydropyrimidinones using alcohol solvated Chiralpak AS and AS-H columns.

In this study we report the application of column temperature programs to compare the chromatographic behaviors of four commercially available dihydropyrimidinone (DHP) compounds on Chiralpak AS and AS-H phases under different alcohol solvation conditions such as 1-propanol (1-PrOH), 2-methyl-1-PrOH (2-Me-1-PrOH), 1-butanol (1-BuOH), 2-BuOH, and tert-BuOH (t-BuOH) in n-hexane (n-Hex). It was observed that the apparent retention factors of the DHP compounds on the AS phase in t-BuOH/n-Hex mobile phase had the largest reduction (>27%) among the mobile phases studied after a heating and cooling temperature cycle (10 to 50 back to 10 degrees C). However, the average reduction of the apparent retention factors of the compounds on t-BuOH solvated AS-H was less than 4%. No clear trend of changes in the apparent retention factors on AS and AS-H columns was observed in other alcohol modified mobile phases. Step-temperature programs showed that alcohol solvated AS phase had larger changes in the apparent retention factors with time than those of AS-H phase at the elevated temperature step (50 degrees C). Both t-BuOH solvated AS and AS-H phases showed kinetic behaviors in chromatographic processes at the elevated temperature.

Purification method development for chiral separation in supercritical fluid chromatography with the solubilities in supercritical fluid chromatographic mobile phases.

A comprehensive approach was applied to develop a chiral purification method for an analyte that was found to be unusually difficult to scale-up in supercritical fluid chromatography (SFC). This was performed by studying major factors such as the solubility of an analyte in SFC mobile phases, impurity profiles, and cycle time. For this case study, the solubility in SFC mobile phase was measured by a packed column technique, coupled with a novel trapping mechanism to enhance measurement precision in SFC conditions. The solubility studies in SFC mobile phases suggested a couple of possible SFC mobile phases, in which the analyte would potentially be most soluble. The SFC methods were developed to purify a sample containing 15% of an impurity, after considering impurity profiles and cycle times of several potential methods in addition to SFC mobile phase solubility. An equal volume mixture of acetonitrile and ethanol was chosen for the final purification method, since this mixture demonstrated the relatively high SFC solubility among all solvent combinations with enhanced resolution between the analyte and the impurity as well as the shortest run time. The solubility of the compound was also determined in various organic solvents using a high throughput solubility screening system to better understand relative change of solubility from neat solution to SFC mobile phases.

Determination of residual isobutylene oxide--a genotoxic starting material in a drug substance by static headspace gas chromatography.

A sensitive static headspace gas chromatography (sHS-GC) method is developed and validated for the determination of residual isobutylene oxide (IBO)-a genotoxic starting material in a drug substance for Phase I clinical trial studies. The experimental parameters, such as headspace vial pressure, headspace oven temperature, vial equilibration time, column flow rate, and GC oven temperature programs are optimized. Under the optimal conditions, the recovery is between 89.3% and 102.4% for spiked samples at three levels of IBO concentration in triplicates in sample preparations. Limits of quantitation (LOQ) and detection (LOD) of the standard solutions are 0.048 and 0.018 microg/mL, respectively. Linear range from 0.018 to 6 microg/mL is obtained with a correlation coefficient of 0.9999. The method is applied to determine residual IBO in drug substance samples from different batches.

Analysis of 4-bromo-3-fluorobenzaldehyde and separation of its regioisomers by one-dimensional and two-dimensional gas chromatography.

A starting material, 4-bromo-3-fluorobenzaldehyde, was used for active drug substance (API) AMG 369 production. The presence of the regioisomer impurities in the starting material 4-bromo-3-fluorobenzaldehyde presented significant challenges for the API synthetic route development due to the physical-chemical similarities of the impurities. These impurities significantly impact on the purity of the starting-material and final drug substance. Control of these impurities is important due to the potential genotoxicity of these impurities (p-GTI). Analytical development was carried out to develop GC methods with high resolving power and high sensitivity to quantify the regioisomers presented in starting material and therefore to control the purity of the starting material and the final drug substance. In the study, complete resolution of the ten regioisomers by 1D-GC and heart-cutting two-dimensional GC (2D-GC) was achieved. A sensitive GC/micro electron capture detection (µ-ECD) method with high resolving power and sensitivity to fully resolve all the ten regioisomers of 4-bromo-3-fluorobenzaldehyde was obtained by using a CHIRALDEX GC column (1D- GC). To facilitate the systematic GC method development, heart-cutting two-dimensional gas chromatography (2D-GC) using a Deans switch was exploited for the separation of the ten regioisomers. The resulting heart-cutting 2D-GC method successfully separated all the ten regioisomers with better sensitivity and resolution. Regioisomer impurities in the starting material were identified and quantified by these GC methods. The sensitivity for the methods is in the range of 0.004ng to 0.02ng for the regioisomers. Linearity for the methods is: R(2)=0.999 to 1.000. The methods were suitable for control of the regioisomer impurities, p-GTIs, in the starting material and final drug substance.