Whilst an alcohol can be forced to react with a sulfonic acid, this reaction produces minimal ester conversion even under extreme conditions (anhydrous, very low pH) that bear no resemblance to the mild synthetic procedures typically used for the formation of sulfonate salts of basic drugs. The latter involve the addition of a molar equivalent of pharma-grade sulfonic acid to the base form of a drug substance (pKa ≥3.5), dissolved or suspended in an alcohol solvent, normally ethanol (pKa -2). All added acid is neutralized, and so there is no potential for ester formation. Many drug-substance base forms are polyamines, thus preventing the generation of acidic reaction conditions even in the presence of excess of sulfonic acid. Despite the experimental evidence, the perception that short-chain mutagenic alkyl sulfonates are "potential impurities" in sulfonate salts is widely held within regulatory bodies. This stance implies that a mechanistically-impossible reaction can occur: nucleophilic displacement by sulfonate anion of the hydroxyl group from a short-chain alcohol under non-acidic conditions. The European Pharmacopoeia (Ph.Eur.) and the British Pharmacopoeia (BP) include "production statements" in monographs for sulfonate-salt drug substances requiring a "risk assessment" of the production process. Neither body has provided supporting evidence. Information obtained from the BP via Freedom of Information requests showed that expert-group discussions were characterised by a range of ad-hoc opinions rather than an evidence-based evaluation of mechanism, kinetics and experimental data. Alternative sources of alkyl-sulfonate impurities such as methyl methanesulfonate (MMS) arising from the use of impure, reagent-grade methanesulfonic acid (MSA) were not considered. Both BP and Ph.Eur. production statements appear to be based on policy rather than scientific evidence and so should be discontinued.

This work revisits the changing release behavior of indomethacin(IND)-copovidone amorphous solid dispersions (ASDs) when increasing their drug load (DL). While showing congruent release behavior at DL 0.1, ASDs with DLs of 0.3 and higher show incongruent release finally resulting in a complete loss of release. To study and explain this phenomenon, we modeled the release kinetics of these ASDs and looked into their phase behavior both experimentally and theoretically. We applied a diffusion model to accurately describe experimental release profiles for congruent release, incongruent release as well as for loss of release. Predicted concentration profiles for IND, copovidone, and water within the ASD revealed the formation of an ASD layer that almost exclusively contains amorphous IND. Our phase-diagram predictions and experimental data explain this phenomenon by water-induced phase separation in those parts of the ASD which did absorb water from the dissolution medium. Whereas the evolving copovidone-rich phase dissolved, the IND-rich phase remained undissolved and formed a super-hydrophobic cover of the remaining inner core of the ASD, thus finally completely preventing its dissolution. Higher DLs promote phase separation. This leads to the counterintuitive effect that the higher the DL, the lower the absolute amount of IND released. While the ASD containing 6 mg IND (DL 0.1) released 6 mg IND, the one containing 42 mg IND (DL 0.7) released only 1 mg IND. The theoretical approach applied in this work is for the first time able to quantitatively predict that reducing DL or tablet size could be used to overcome this problem.

We investigated the role of individual radical species during Fe-catalyzed oxidation of PS80. Solutions containing 1 gL PS80 (0.1% w/v) in 10 mM acetate buffer (pH 6) were exposed to various amounts of either Fe(II) or Fe(III), hydrogen peroxide (HO), and various enzymes or antioxidants. PS80 oxidation was measured using a fluorescence micelle assay (FMA) alongside LC-MS. Hydrogen peroxide inhibited PS80 oxidation in the presence of Fe(II) but promoted oxidation in the presence of Fe(III). Furthermore, Ferrostatin-1 (Fer-1), an antioxidant which is known to preferentially react with alkoxy radicals, inhibited PS80 oxidation in the presence of Fe(II). Superoxide dismutase (SOD) partially inhibited PS80 oxidation in the presence of either Fe(II) or Fe(III), suggesting that superoxide plays a role in both cases. Ferryl species (Fe=O) or hydroxyl radicals (HO•), produced by the Fenton reaction, do not play a major role in the oxidation of PS80. Rather, oxidation was initiated by the reaction of both Fe(II) and Fe(III) with pre-existing lipid hydroperoxides on PS80, as well as via superoxide.

An innovative approach was developed to identify the optimal crystalline form, usually the thermodynamically most stable form. This method involves using virtual polymorph screening and targeted crystallization based on in silico solid-state modeling. By utilizing advanced crystal structure prediction (CSP) technology, the virtual polymorph screening method helps confirm whether the most stable crystalline form has been identified in actual crystallization experiments. If the predicted most stable form is not observed in experiments, predictions based on the method of COnductor like Screening MOdel for Real Solvents (COSMO-RS) are used to highlight solvent systems that can increase the likelihood of experimentally obtaining the desired form through a targeted crystallization process. In this work, such an approach has enabled the rapid discovery of the most stable polymorphic form and the development of a crystallization process of an adenosine receptor antagonist using minimal amounts of the sample within a shortened timeframe. Additionally, it provides a scientific rationale for ensuring the selection of the most stable form in the early stages of drug discovery, thereby reducing risks in future pharmaceutical development.

Adeno-Associated Virus (AAV) is often selected as the vector of choice for gene therapy due to its superior clinical performance compared to other gene delivery systems. Currently the characterization of AAV degradation, especially the chemical degradation of capsid, has been limited due to lack of suitable methods. Our study using AAV9 as a model molecule shows that anion exchange chromatography (AEX) as a charge-based separation method has limitations in monitoring the chemical degradation of AAV9 capsid due to a confounding effect from DNA cargo ejection. We developed a hydrophobic interaction chromatography (HIC) method, free from DNA interference, that could serve as a quick and reliable alternative to resource-demanding peptide mapping method for monitoring AAV capsid chemical degradation. Compared with brief thermal stress at 75 °C, AAV9 capsid exhibited much higher levels of chemical degradation but slower capsid titer loss upon extended exposure for 4 weeks at 40 °C.

Polysorbate 80 (PS80), a widely used polymeric surfactant in biotherapeutic formulation, possesses a unique structural composition that effectively prevents protein aggregation in highly concentrated protein drug formulations. However, PS80 is susceptible to hydrolysis, due to the presence of fatty acid esters that can be enzymatically hydrolyzed, The unsaturated bonds in the fatty acids are prone to oxidative degradation when exposed to air, especially in the presence of transition metals such as iron and copper, which may be introduced during production and purification processes or from contamination in raw materials used in drug formulation. The degradation of PS80, particularly through metal-mediated oxidative degradation, poses a significant challenge for the industry. Among the identified trace metals, iron plays a crucial role as the redox reaction between ferrous ion (Fe(II)) and ferric ion (Fe(III)) generates radicals that initiate the degradation process. In order to investigate the impact of iron on PS80 degradation and understand the mechanism of iron-catalyzed oxidation, we utilized charge-reduction mass spectrometry and two-dimensional ion density mapping technologies to characterize the degradation of PS80. This method has proven to be a convenient and effective tool for the quick and detailed profiling of PS80, allowing for visual monitoring and examination of the changes that reflect the difficult-to-identify and easy-to-miss oxidized species of PS80. Additionally, a high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry method was developed for the separation and measurement of Fe(II) and Fe(III). Through this investigation, we determined that the involvement of Fe(II)/Fe(III) in PS80 degradation is a temperature dependent process. Furthermore, we found citrate not only promotes the conversion of Fe(II) to Fe(III), but it also chelates Fe(III) and prevents its reduction to Fe(II), thus inhibiting the initiation of the PS80 degradation. Therefore, the addition of citrate can be a crucial ingredient for controlling the degradation of PS80 in biologic drug substances and products. Overall, this investigation has provided valuable insights to enhance product stability, optimize processes, and ensure the quality of formulations containing PS80.

Recently, nanofiber-based wound dressings are currently a viable strategy to expedite the healing of wounds by providing a suitable microenvironment for tissue growth with active ingredients. This research study subjects the development of electrospun cellulose acetate (CA) nanofibers loaded with the XLAsp-P2, an antimicrobial peptide (AMP) that holds great potential for enhanced wound healing as a therapeutic agent. The synthesized XLAsp-P2-loaded CA nanofibers were fabricated via three loading percentages, 0.1 %, 0.2 %, and 0.3 % w/w, and characterized and evaluated their antimicrobial potential with MTT assay and Agar overlay methods as an alternative strategy. FT-IR analysis confirmed the compatibility of the peptide loaded CA nanocomposite, showing distinct peaks corresponding to the constituent materials. Scanning electron microscopy (SEM) analysis was employed to characterize the morphology of electrospun peptide CA nanocomposites and illustrate the fiber's size at the nanoscale. The in vitro release study during the 24 hrs, 87 % of the peptide was released which was approximately 5.2 mg; which was closer matched to the square root model of Higuchi at room temperature. MTT assay presented sensitive results towards Gram-positive bacteria compared to Gram Negative bacteria; which corresponded to the inhibition zones of the Agar overlay method proving that Escherichia coli (ATCC 25922) 17.66 ± 0.38 mm and Pseudomonas aeruginosa (ATCC 27853) 17.44 ± 0.38 mm exhibited moderate susceptibility, while Staphylococcus aureus (ATCC 25923)19.89 ± 0.69 mm and Bacillus cereus (ATCC 11778) 23.00 ± 0.33 mm showed promising responses. Collectively, The study's findings indicate that the XLAsp-P2 incorporated CA mat possesses an opportunity to function as an efficient platform for delivering therapeutic peptides.

Nitrosamine impurities have been classified as probable human carcinogens for decades. These impurities were reported in water, food, tobacco, pesticides, and plastics but received attention in mid-2018 when N-nitrosodimethylamine (NDMA) was reported in valsartan drug products. Subsequently, it was revealed that several small molecule and complex nitrosamine impurities can form in any active pharmaceutical ingredient (API) or drug product in which secondary or tertiary amines are present (as API or as impurities) along with a nitrosating agent. Consequently, regulators have provided several guidelines for the risk assessment of nitrosamine formation during manufacturing, storage, or from contaminated supply chains. This has led to a demand for validated analytical methods that quantify N-nitrosamine impurities in pharmaceutical products. In this study, a highly sensitive and robust analytical method was developed and validated for quantitatively determining up to 15 small nitrosamines at low levels (0.01 ppm) in sartan drug substances. The study also suggests that this method can be extended not only to corresponding sartan drug products but could also be used as a generic screening method to test a variety of drug substances, and drug products with the minimum required optimization of method conditions.

According to the ICH M9 Guideline, the triazole antifungal voriconazole is a Biopharmaceutics Classification System (BCS) class II drug, being highly soluble at the highest dose strength but not at the highest single dose. Although the ICH M9 allows for consideration of BCS-based biowaivers in such cases, voriconazole does not meet the additional requirement of dose proportional pharmacokinetics (PK) over the therapeutic dose range. By contrast, if the classification were based on the FDA solubility criteria that were in place prior to ICH M9 (based on the highest dose strength), voriconazole would belong to BCS class I and thus qualify for the BCS-based biowaiver. Since the highest oral dose strength of voriconazole dissolves very rapidly under all BCS conditions, and comparative in vitro dissolution of different tablet formulations aligns with the demonstration of BE in clinical studies, it seems that the ICH Guideline may be unnecessarily restrictive in the case of voriconazole. Therefore, this review discusses potential revisions of eligibility criteria and the extension of biowaiver approvals to encompass a wider range of appropriate drugs. Specifically, a classification system that is more relevant to in vivo conditions, the refined Developability Classification System (rDCS), coupled with biorelevant dissolution testing, may be more applicable to compounds like voriconazole.

Particle engineering aims to design particles with specific properties. A deeper understanding of how particle formation relates to material attributes and process conditions are critical to strengthen knowledge on powder properties and enhance modeling capabilities. New, alternative powder characterization techniques can offer novel and more accurate measures for particle properties, giving more advanced characterization information. In this context, a case study is presented in which spray dried amorphous solid dispersion powders produced by modifying process conditions were characterized by both well-established compendial methods (i.e., laser light diffraction, SEM image analysis, bulk and tapped density, and gas adsorption), as well as a new method combining synchrotron computed tomography (SyncCT) with AI-based image analysis. SyncCT was used to classify and quantify the spray dried particles as hollow spheres and solid particles, giving a more detailed quality measure of the particle shape, as they impact downstream processing differently. Moreover, hollow particle wall thicknesses, as well as internal and external particle surface areas were measured by SyncCT. Altogether, powder characterization data from SyncCT show similar trends to that obtained from compendial techniques and giving additional quality measure regarding particle shape, showing promise of this new and advanced characterization method.

Self-assembled peptide nanoparticles are unique stimuli responsive biodegradable materials with applications in biomedicines as delivery carriers and imaging agents. This study investigates the controlled self-assembly of chicken Angiogenin 4 derived immunomodulatory macrocyclic peptide (mCA4-5) in the presence of an inert amphipathic stabilizing peptide and as a function of pH, temperature and presence of ions to yield optically active, physiologically stable and biodegradable peptide nanoparticles. The photoluminescent peptide nanoparticles (PLPNs) produced were characterized for the size, surface charge, optical properties and crystallinity. The carvacrol loaded nanoparticles prepared by facile encapsulation of the drug during the self-assembly process were evaluated for the drug release efficacies, as a function of pH and in the presence of reducing agent. Carvacrol loaded, physiologically stable PLPNs obtained with high conversion efficacy were highly effective against planktonic bacteria and bacterial biofilms and efficiently eradicated intracellular bacteria in infected macrophages and fibroblast. Furthermore, the drug-loaded nanoparticles exhibited significant antioxidant activities and immunomodulatory effects, highlighting their multifunctional therapeutic potential.

Residual host cell proteins (HCPs) in drug products may impact product quality, stability, efficacy and safety. To support consistent and accurate quantitative analysis for low levels of HCPs (≥ 1 ppm), the data-independent sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS/MS-based method provides unique advantages over data dependent acquisition (DDA) or targeted methods for HCP identification and quantification. However, SWATH MS/MS-based methods can generate biased quantitative results that are highly dependent on the selected reference protein. In this study, we enhanced the accuracy of SWATH-based HCP quantitation relative to a spiked-in reference protein by selecting appropriate reference proteins based on their ranking values. We developed a reliable SWATH-based method for quantifying specific HCPs by adding sodium deoxycholate (SDC) during digestion to enhance both protein detection and quantitation consistency. By combining SWATH-based quantitation with standard addition, we showed its use in measuring HCP levels with good accuracy and reproducibility, confirmed by both targeted MRM-MS/MS and ELISA. Additionally, we demonstrated an automated Spectronaut data analysis workflow can efficiently generate SWATH quantitative results for HCPs in different in-process pools. Using SWATH-based quantitation, we were able to measure specific HCPs (e.g. Peroxiredoxin-1) and support process development with good throughput and quantitation consistency.

Amorphous solid dispersions (ASDs) have been extensively utilized to improve the bioavailability of drugs that have low aqueous solubility. The influence of different excipients on the conversion of amorphous drugs into their crystalline forms in ASDs has been extensively researched. However, there is limited knowledge examining the impact of film coating materials on the physical stability of oral tablet formulations containing ASDs. In this study, we demonstrate that plasticizers present in film coats can have a detrimental impact on the physical stability of ASDs. We systematically compared two frequently used plasticizers in film coats: triacetin and polyethylene glycol 3350 (PEG 3350). To gain mechanistic insights into the detrimental effects of plasticizers on the physical stability of ASDs, plasticizer leaching studies and physical stability studies of solvent-evaporated and spray-dried intermediates (SDI) using two BCS class II drugs were conducted. Triacetin was found to leach into the tablet core within one week when stressed at 40°C/75% RH, whereas no leaching was observed for PEG 3350, as discerned from spectroscopic studies. We also found that triacetin-containing ASDs exhibited greater amorphous to crystalline form conversion of the drug compared to PEG 3350-containing ASDs after stability testing. Moreover, the incorporation of triacetin into polymers was found to cause a significant depression of glass transition temperature and upon equilibration with moisture, a drop below room temperature. Overall, these observations underscore the importance of carefully selecting plasticizers to be present in film coatings when developing ASD pharmaceutical products.

Ethylene vinyl acetate copolymers (EVA) have been extensively used in controlled drug delivery systems due to its good biocompatibility and tunable applicability based on simple variations in vinyl acetate (VA) content. We investigated impacts of material properties of EVA, including VA content and molecular weight, as well as extrusion process parameters, including draw down ratio and cooling rate, on permeability of etonogestrel in EVA films. Among all factors studied, the VA content was the most dominant factor that controls drug permeability by affecting crystallinity of EVA. MW, DDR, and cooling rate exhibited less significant effects. The impacts of these factors on crystallinity, crystallite size, and degree of crystalline orientation of EVA were characterized using polarized light microscope, differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS). Based on the solution-diffusion model, the mechanisms by which the crystalline properties controlled drug solubility and diffusivity in EVA were discussed. These results can be applied to investigate the effects of material properties of EVA and manufacturing process conditions on drug release properties of reservoir-type EVA-based drug delivery systems with a rate-controlling membrane.

Formulating active pharmaceutical ingredients (APIs) as co-crystals requires a thorough understanding of co-crystallization behavior under different process conditions. This study employs two forms of coherent Raman microscopy, narrowband coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) with spectral focusing, to study co-crystallization via liquid-assisted ball milling. Indomethacin and nicotinamide served as the model API and co-former, and the results were compared with established analytical methods. Narrowband CARS, with univariate peak position analysis, was useful to visualize co-crystal formation, but suffered some degree of signal mixing that affected component identification. Hyperspectral SRS imaging, combined with classical least squares multivariate analysis, separated the different components with high confidence and proved to be a robust and rapid tool to qualitatively and quantitatively image co-crystallization. The coherent Raman imaging results explained divergent co-crystallization endpoints obtained with the conventional solid-state analysis methods. CARS and SRS microscopies also revealed the presence of otherwise undetected trace forms. Finally, we also demonstrated the dramatic reversal of partial co-crystal formation during milling, depending on ethanol content. Overall, the study demonstrates the added value coherent Raman microscopy can provide for analysis of co-crystallization processes.

B-cell lymphoma has a poor prognosis due to difficulties in early diagnosis and the negative effects of systemic chemotherapy. Therefore, there is an urgent need to develop highly accurate and effective theranostic strategies for B-cell lymphoma. In this study, we designed a poly (lactic-co-glycolic acid) (PLGA)-based theranostic nanoplatform (denoted as TscNPs) to achieve ultrasound (US)/magnetic resonance (MR) bimodal imaging-guided photothermal (PTT)/chemo synergistic therapy of B-cell lymphoma. The nanoplatform was conjugated with a CD20 monoclonal antibody specifically targeting B-cell lymphoma to promote tumor accumulation. Encapsulated superparamagnetic iron oxide nanoparticles (SPIONs) as photothermal and MR imaging agents enabled thermal ablation of tumors and imaging-guided tumor therapy. When exposed to near-infrared (NIR) laser, TscNPs generate heat that induces optical droplet vaporization (ODV) of perfluoropentane (PFP), which transforms into microbubbles. This process not only enhanced ultrasound imaging, but also facilitated the release of celastrol (CST) from the nanoplatform, ultimately achieving a PTT/chemo synergistic therapy effect. In the tumor-bearing nude mice model, TscNPs were effectively accumulated in the tumor region. Furthermore, the combined treatment mode of TscNPs and NIR laser irradiation demonstrated a tumor inhibition rate of approximately 96.57%, which was significantly superior to the rates observed with PTT or chemotherapy alone. These results suggest that the multifunctional theranostic nanoplatform represents a promising new strategy for the therapy of B-cell lymphoma.

Cyclodextrin complexation has a potential to modulate the physicochemical properties of peptide drugs. The ability of peptides to form an inclusion complex can be influenced by factors such as size, amino acid sequence of peptide, and the size and charge of the cyclodextrin cavity. In this study, the inclusion complexes of the cyclic peptide drug lanreotide acetate with two common β-cyclodextrin derivatives, Sulfobutyl ether β-CD (SBEβ-CD) and hydroxypropyl β-CD (HPβ-CD) were investigated. NMR spectroscopy was used to examine the interaction between β-cyclodextrin derivatives and specific residues of lanreotide. It was observed that the hydrophobic side chain of aromatic residues in the lanreotide sequence can fit into the cavities of both β-cyclodextrin derivatives. Additionally, NMR revealed a lower diffusion coefficient and higher hydrodynamic radius of complex, indicative of binding to the cavities. Each aromatic residue was individually studied by substituting alanine in lanreotide to measure its association binding with both β-cyclodextrin derivatives. The alanine-substitute study indicated a stronger binding of SBEβ-CD to Lanreotide compared to HPβ-CD. Docking studies suggested that the 1:1 inclusion complex is more favorable than higher-order complexes due to the steric hindrance and size considerations. Docking analysis indicated the stable conformation of all three aromatic side chains with both β-cyclodextrin derivatives, SBEβ-CD and HPβ-CD.

The development time of therapeutic monoclonal antibodies (mAbs) has been shortened by formulation platforms and the assessment of 'protein stability' using 'developability' assays. A range of assays are used to measure stability to a variety of stresses, including forces induced by hydrodynamic flow. We have previously developed a low-volume Extensional Flow Device (EFD) which subjects proteins to defined fluid flow fields in the presence of glass interfaces and used it to identify robust candidate sequences. Here, we study the aggregation of mAbs and Fc-fusion proteins using the EFD and orbital shaking under different formulations, investigating the relationship between these assays and evaluating their potential in formulation optimisation. EFD experiments identified the least aggregation-prone molecule using a fraction of the material and time involved in traditional screening. We also show that the EFD can differentiate between different formulations and that protective formulations containing polysorbate 80 stabilised poorly developable Fc-fusion proteins against EFD-induced aggregation up to two-fold. Our work highlights common platform formulation additives that affect the extent of aggregation under EFD-stress, as well as identifying factors that modulate the underlying aggregation mechanism. Together, our data could aid the choice of platform formulations early in development for next-generation therapeutics including fusion proteins.

Pharmacokinetics (PK) is the result of a complex interplay between compound properties and physiology, and a detailed characterization of a molecule's PK during preclinical research is key to understanding the relationship between applied dose, exposure, and pharmacological effect. Predictions of human PK based on the chemical structure of a compound are highly desirable to avoid advancing compounds with unfavorable properties early on and to reduce animal testing, but data to train such models are scarce. To address this problem, we combine well-established physiologically based pharmacokinetic models with Deep Learning models for molecular property prediction into a hybrid model to predict PK parameters for small molecules directly from chemical structure. Our model predicts exposure after oral and intravenous administration with fold change errors of 1.87 and 1.86, respectively, in healthy subjects and 2.32 and 2.23, respectively, in patients with various diseases. Unlike pure Deep Learning models, the hybrid model can predict endpoints on which it was not trained. We validate this extrapolation capability by predicting full concentration-time profiles for compounds with published PK data. Our model enables early selection and prioritization of the most promising drug candidates, which can lead to a reduction in animal testing during drug discovery and development.

Polysorbate degradation in biotherapeutics formulations is an industry-wide problem and mainly caused by residual host cell-derived enzymes. We present a proof-of-concept study of a control strategy which takes advantage of lower thermal stability of such enzymes relative to therapeutic proteins. We profiled heat sensitivity of host cell-derived enzyme activity with chemical proteomics and observed that PLA2G7 became inactive after brief heating. Further biophysical studies indicated that these enzymes were less thermally stable than a monoclonal antibody. Importantly, brief heat treatment had minimal impact on the stability of the antibody. Consequently, heat inactivation of polysorbate-spiked protein-A pool decelerated polysorbate degradation. This study suggests that heat inactivation of host cell-derived enzymes could be a control stragy for polysorbate degradation.