Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control, including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.

The effect of transporters and enzymes on drug pharmacokinetics is increasingly evaluated using genetically modified animals that have these proteins either knocked-out or their human orthologues transgenically expressed. Analysis of pharmacokinetic data obtained in such experiments is typically performed using non-compartmental analysis (NCA), which has limitations such as not being able to identify the PK parameter that is affected by the genetic modification of the enzymes or transporters and the requirement of intense and homogeneous sampling of all subjects. Here we used a compartmental population pharmacokinetic modeling approach using PK data from a series of genetically modified mouse experiments with lorlatinib to extend the results and conclusions from previously reported NCA analyses. A compartmental population pharmacokinetic model was built and physiologically plausible covariates were evaluated for the different mouse strains. With the model, similar effects of the strains on the area under the concentration-time curve (AUC) from 0 to 8 hours were found as for the NCA. Additionally, the differences in AUC between the strains were explained by specific effects on clearance and bioavailability for the strain with human expressing CYP3A4. Finally, effects of multidrug efflux transporters ATP-binding cassette (ABC) sub-family B member 1 (ABCB1) and G member 2 (ABCG2) on brain efflux were quantified. Use of compartmental population PK modeling yielded additional insight into the role of drug-metabolizing enzymes and drug transporters in mouse experiments compared to the NCA. Furthermore, these models allowed analysis of heterogeneous pooled datasets and the sparse organ concentration data in contrast to classical NCA analyses.

Microplate-based formulation screening is a powerful approach to identify stabilizing excipients for therapeutic proteins while reducing material requirements. However, this approach is sometimes not representative of studies conducted in relevant container closures. The present study aimed to identify critical parameters for a microplate-based orbital shaking method to screen biotherapeutic formulations by agitation-induced aggregation. For this purpose, an in-depth methodological study was conducted using different shakers, microplates, and plate seals. Aggregation was monitored by size exclusion chromatography, turbidity, and backgrounded membrane imaging. Both shaker quality and liquid-seal contact had substantial impacts on aggregation during shaking and resulted in non-uniform sample treatment when parameters were not suitably selected. The well volume to fill volume ratio (V_well/V_fill) was identified as an useful parameter for achieving comparable aggregation levels between different microplate formats. An optimized method (2400 rpm [a_c 95 m/s²], V_fill 60-100 µL [V_well/V_fill 6-3.6], 24 h, RT, heat-sealed) allowed for uniform sample treatment independent of surface tension and good agreement with vial shaking results. This study provides valuable guidance for miniaturization of shaking stress studies in biopharmaceutical drug development, facilitating method transfer and comparability between laboratories.

We developed a composite system combining self-targeted carbon dots and thermosensitive in situ hydrogels for ocular drug delivery of diclofenac sodium (DS). DS-CD_C-HP nanoparticles were prepared by loading DS on the surface of CD_C-HP via electrostatic interactions. An orthogonal experimental design was selected to screen the optimal thermosensitive hydrogel matrices and then DS-CD_C-HP nanoparticles were embedded to form the composite system. The physicochemical properties and release behavior of this system were characterized, and in vivo fluorescence imaging was carried out. Corneal penetrability and in vitro cellular studies (cytotoxicity, cell imaging and cell uptake) were performed to test the feasibility and potential of this ocular delivery system. Finally, the optimal gel matrix consisting of Poloxamer 407: Poloxamer 188: HPMC E50 was 21:1:1 (w/v %), and the gelation temperature before adding artificial tear fluid was 26.67°C and 34.29°C, respectively. This system has the characteristics of biphasic drug release. In addition, the corneal penetrability and in vivo fluorescence study indicated that corneal transmittance was enhanced and drug retention time was extended. Cellular studies revealed that the DS-CD_C-HP-Gel has good cytocompatibility and CD44 targeting. In summary, this composite system combines carbon dots with hydrogels, offering new potential for ocular drug delivery.

A mitochondrial electron transport chain member complex I inhibitor, amobarbital, can reduce oxidative damage and chondrocyte death, eventually preventing post-traumatic osteoarthritis (PTOA). Viscosupplementation using a crosslinked hyaluronic acid (HA) hydrogel is currently applied clinically for knee OA pain relief. In this work, we utilized the HA hydrogel as a drug delivery vehicle to improve the long-term efficacy of amobarbital. Here we evaluated the pharmaceutic stability of amobarbital when dispersed in a crosslinked HA hydrogel formulated in proportions intended for clinical use. We validated a high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) method following International Conference for Harmonization Q2(R1) guidelines to ensure its suitability for amobarbital detection. The feasibility of this formulation's drug delivery capability was proven by measuring the release, solubility, and drug uniformity. The amobarbital/HA hydrogel showed comparable amobarbital stability in different biological fluids compared to amobarbital solution. In addition, the amobarbital/HA hydrogel imparted significantly greater drug stability when stored at 70°C for 24 hours. In conclusion, we confirmed the pharmaceutical stability of the amobarbital/HA hydrogel in various conditions and biological fluids using a validated HPLC-UV method. This data provides essential evidence in support of the use of this amobarbital/HA formulation in future clinical trials for PTOA treatment.

The identification and characterization of novel compounds with improved functionality and safety is of great importance. Ionic liquids are potential candidates for use in dermal formulation as multifunctional components with a large variability potential. The behavior of Ionic Liquids (ILs) in aqueous solutions has an impact on their functionality in the formulation as well as on their biological activity. Therefore, the solutions of selected ILs containing salicylate anions were investigated in the present work. The alkyl chain length of the cation determined most of the studied parameters. Thus, the surface activity, the antimicrobial activity, and cytotoxicity were directly proportional to the chain length. The salicylate anion did not affect the surface activity significantly, but had an important influence on the biological activity, especially for ILs with short chain lengths. It was found that the antimicrobial activity of benzalkonium-based ILs was mainly dependent on the cation, and the minimal inhibitory concentration (MIC) values were three order of magnitude lower than those of salicylic acid. Nevertheless, the slightly lower MIC values of benzalkonium salicylate, compared to benzalkonium chloride, might indicate a synergistic effect resulting from different modes of action of the two ions. N-hexyl nicotinamide salicylate also showed a higher antimicrobial activity than salicylic acid and, at the same time, a very good skin tolerance at concentrations up to 5% w/w. Based on our investigations N-hexyl nicotinamide salicylate was identified as potential emulsifiers / co-emulsifiers with antimicrobial properties for dermal formulations.

Roll compaction/ dry granulation is gaining importance. Numerous papers have been published and many attempts to model the process are available in the meantime. Johanson published a model in 1965, which is the basis for many further modifications until today. The aim of the paper is to improve process understanding in roll compaction, which can be used to setup a roll compaction process, to design a scale-up strategy or to help in process transfer between different types of roll compactors. Based on some assumptions, simple considerations help to estimate a required densification factor and to visualize the relations between roll diameter, gap width and nip angle. Two recently published papers based on simplified Johansen models are used to visualize the relations between specific compaction force and the maximal pressure experienced by the material. The influence of roll diameter, gap width and compressibility constant are discussed. This helps to estimate, if a scale-up or process transfer is reasonable. The recently introduced dimensionless Midoux-number can also be used to design scale-up or process transfer of roll compaction without knowledge about the maximal pressure. Exploring the simple concepts can help to improve process understanding even without a background in engineering.

The co-crystals formation of etravirine with three carboxylic acids was investigated. New co-crystals of etravirine with adipic acid, benzoic acid, and 4-hydroxybenzoic acid have been synthesized by wet milling of ingredients for 120 minutes. The novelty of these solid forms was first evidenced by powder X-ray diffraction. Their different morphology was evidenced by SEM microscopy. Spectroscopic analyses (FT-IR, MAS-NMR, and XPS) highlighted the hydrogen bonds between etravirine and co-formers, as a result of the solid-state reaction of the ingredients by wet milling. Thermal analyses pointed out that the milling process caused in co-crystals a reduction in the fusion enthalpy and the melting temperature, compared to the values obtained for etravirine. These co-crystals are stable up to four months on storage under extreme conditions, excepting the co-crystal with benzoic acid which begins to transform into a polymorph of etravirine after 30 days. The UV absorption spectra of the samples tested in three simulated physiological media with pH values of 6, 6.3, and 7 have evidenced the conformation change of etravirine due to hydrogen bonds between etravirine and carboxylic acids.

Carfilzomib (CFZ) is an FDA-approved proteasome inhibitor with antineoplastic properties against various cancers, yet its short blood retention time after intravenous injection (< 30 min) makes clinical applications limited to multiple myeloma. We previously developed ternary polypeptide nanoparticles (tPNPs) as a new nanoparticle formulation of CFZ to overcome these limitations. The formulation was prepared by polyion complexation between poly(ethylene glycol)-poly(L-glutamate) block copolymers (PEG-PLE) and CFZ-cyclodextrin (CD) inclusion complexes, where CDs were positively charged with 7 primary amines attached while PEG-PLE carried 100 carboxyl groups per polymer chain. Although tPNPs greatly improved biostability of CFZ, CFZ-loaded tPNPs (CFZ-tPNPs) still showed burst drug release and mediocre drug retention under physiological conditions. To address these issues, organic acids are tested as stabilizers in this study to improve particle stability and drug retention for tPNPs. Charge densities in the core of CFZ-tPNPs were optimized with selected organic acids such as citric acid (CA) and lactic acid (LA) at varying mixing ratios. Organic acids successfully maintained small particle size suitable for intravenous injection and drug delivery (diameters < 60 nm), improved CFZ solubility (> 1 mg/mL), allowed for lyophilization and easy reconstitution in various buffers, enhanced drug retention (> 60% post 24 h incubation), and suppressed burst drug release in the first 6 h following solubilization. These results demonstrate that organic acid stabilized tPNPs are useful as an injection formulation of CFZ, which may expand the utility of the proteasome inhibitor.

The fluidized bed is an essential and standard equipment in the field of process development. It has a wide application in various areas and has been extensively studied. This review paper aims to discuss computational modeling of a fluidized bed with a focus on pharmaceutical applications. Eulerian, Lagrangian, and combined Eulerian-Lagrangian models have been studied for fluid bed applications with the rise of modeling capabilities. Such models assist in optimizing the process parameters and expedite the process development cycle. This paper discusses the background of modeling and then summarizes research papers relevant to pharmaceutical unit operations.