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This article is the second of a series of two articles. In the first article of the series, a new K v distribution model and an experimental methodology to measure the K v distribution were introduced. In this second part, the K v distribution is integrated into a lyo-simulation tool, to more accurately predict the variability of the product temperature, primary drying time, total sublimation mass flow and Pirani signal. The K v distribution is also integrated into the graphical design space. The impact of incorporating the R p distribution is briefly discussed. The comparison of the simulation tool with actual product temperature monitoring, Pirani signal or overall sublimation flow shows very good agreement in the case studies presented. Overall, the lyo-simulation incorporating the K v distribution is a very useful tool to support industrial development, i.e. process optimization, scale assessment, technology transfer, and troubleshooting of the lyophilization process., Competing Interests: Declaration of Competing Interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All the authors worked for Sanofi at the time where the research work was done, except one working for Astek - IT&M STATS (contracting company for Sanofi). All the work was Sanofi financed., (Copyright © 2024. Published by Elsevier Inc.)

As lyophilization continues to be a critical step in the manufacturing of sensitive biopharmaceuticals, challenges often arise during the scale up to commercial scale or the transfer from one manufacturing site to another. While data from the small-scale development of the lyophilization cycle is abundant it is typically much more difficult to extract important information from commercial scale cycles, due to the lack of process analytical technologies available on the commercial line. There is often a reluctance to include wireless temperature or pressure probes during GMP operations due to the additional contamination risk, and retrofitting equipment such as the TDLAS can be prohibitively expensive. Further, as products become more advanced, the cost of consuming the product or even the availability of material may limit the opportunities to run commercial scale trials. This paper presents two novel methods to garner critical cycle information to allow for the evaluation of cycle performance without the need for expensive analytical equipment, costly revalidation and line downtime. Critically, this can be achieved using commonly available temperature and capacitance probes on existing commercial scale equipment. The first method is a calorimetric method, based on quantifying the differences in heat transfer liquid temperature between the shelf inlet and shelf outlet. This change in temperature results from the on-going sublimation, an endo-thermic reaction occurring during lyophilization. The second method uses the differential pressure between the chamber and condenser resulting from the vapor flow from vial to condenser during primary drying. As stated by the authors both methods align well and provide valuable cycle characterization data., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Spray drying is increasingly being applied to process biopharmaceuticals, particularly monoclonal antibodies (mAbs). However, due to their protein nature, mAbs are susceptible to degradation when subjected to various stresses during a drying process. Despite extensive research in this domain, identifying the appropriate formulation composition and spray drying conditions remains a complex challenge, requiring further studies to enhance the understanding on how process and formulation parameters impact mAb stability in reconstituted solutions. This research aims to explore spray drying as technique for producing pharmaceutical mAbs-based powders intended for reconstitution and subcutaneous injection. In the initial phase of this study, using a model mAb (mAb-A), the influence of dissociated and coupled process stresses on protein stability after solution reconstitution was investigated. The findings revealed a detrimental interplay of mechanical, interfacial, and thermal/dehydration stresses on mAb-A stability, notably characterized by an increase in protein aggregation. Subsequently, in a second phase, the study delved into the impact of spray drying processing conditions, the level of excipients, and protein concentration on mAb-A aggregation in reconstituted solutions. The obtained results highlighted the critical role of formulation composition as a parameter deserving further study, specifically concerning the selection of type and concentration of stabilizers to be added in the liquid mAb-A solution to be dried., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Despite extensive research in spray drying of biopharmaceuticals, identifying the optimal formulation composition and process conditions to minimize the various stresses a biopharmaceutical undergoes during this drying process. The current study extends previous research on investigating how spray drying processing and solution composition can affect the stability of monoclonal antibodies (mAbs) in reconstituted solutions for subcutaneous injections. The decoupling process stresses on a model mAb (mAb-A) compared to the effect of coupled spray-drying stresses revealed that excipients and protein concentration had a more pronounced effect on stabilizing mAb-A against shear and thermal/dehydration stresses than spray drying operating conditions. These results prompted the continuation of the study, with the aim to investigate in greater depth the effect of mAb-A concentration in the formulation designated to spray-drying and then the effect of type and the concentration of individual excipients (sugars, amino acids and surfactants). The outcomes of this investigation suggest that a general increase in the concentration of excipients, particularly surfactants, correlates with a reduction in aggregation and turbidity observed in the reconstituted spray-dried mAb-A powders. These results, contribute to the identification of a suitable composition for a spray-dried mAb-A powder that ensures robust stability of the protein in reconstituted solutions intended for subcutaneous injection. This valuable insight has important implications for advancing the development of pharmaceutical formulations with enhanced stability and efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This article is presenting completely new observations linked to Polysorbate 80 (PS80) oxidation in biologics drug product. Indeed, we observed that, in the drug product exposed to long contact time (∼ 1 h) in platinum-cured silicon tubing during the filling, the oxidation of PS80 is dramatically accelerated compared to short contact time. The phenomenon was observed in presence of iron traces (20 ppb), but not in absence of iron (< 2 ppb) or in presence of a chelator like EDTA. Electron Paramagnetic Resonance (EPR) measurements demonstrated the presence of radicals formed during the oxidation. It was deduced that platinum-cured silicon tubing is leaching some radical initiators, most probably peroxides decomposed by the iron. Alternative filling sets made of ThermoPlastic Elastomer (TPE) were investigated, both for the impact on PS80 stability and the filling performance using a peristaltic pump. The results showed that these filling sets were indeed not causing accelerated PS80 degradation but the process was not robust enough; these filling sets being too rigid for the constraints of the peristaltic pump rollers. These results show that there is no practical tubing alternative to platinum silicone cured tubing. To avoid the impact on PS80 oxidation the potential remediations presented in the article are to avoid any trace of iron or to add a chelating agent, or to discard the vials having experimented a filling stop (> 5 min)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A commonly encountered challenge with freeze-dried drug products is glass vial fogging. Fogging is characterized by a thin layer of product deposited upon the inner surface of the vial above the lyophilized cake. While considered to be a routine cosmetic defect in many instances, fogging around the shoulder and neck of the vial may potentially impact container closure integrity and reject rates during inspection. In this work, the influence of processing conditions i.e. vial pre-treatment, lyophilization cycle modifications and filling conditions on fogging was evaluated. A battery of analytical techniques was employed to investigate factors affecting glass vial fogging. A fogging score was used to quantify its severity in freeze-dried products. Additionally, a dye-based method was used to study solution upcreep (Marangoni flow) following product filling. Our lab-scale results indicate measurable improvement in fogging following the addition of an annealing step in the lyophilization cycle. Pre-freeze isothermal holding of the vials (at 5°C on the lyophilizer shelf) for an extended duration indicated a reduction in fogging whereas an increase in the freezing time exhibited no effect on fogging. Vial pre-treatment conditions were critical determinants of fogging for Type 1 vials whereas they had no impact on fogging in TopLyo® vials. The headspace relative humidity (RH) investigation also indicated sufficient increase in the water vapor pressure inside the vial to be conducive to the formulation of a hydration film - the precursor to Marangoni flow., Competing Interests: Declaration of competing interest All authors were employees of Sanofi during the work described in this manuscript and may hold shares and/or stock options in the company. The authors declare no other conflicts of interest., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Measurement of heat transfer coefficients (K v ) is an important part of freeze-dryers characterization and as well a necessary step for executing any modelling. In most cases only an average value of K v is calculated, or an average value of center and edge vials is provided. Our aim is to go a step further and to describe the overall K v distribution various vial/ freeze drier combinations, whatever the pressure. From an experimental point of view, in this article we propose three methods to calculate the K v value for individual vials based on the ice sublimation gravimetric method. The first method we use is the most usual one, where the K v value is calculated based on the mass of sublimated ice and the product temperature measured in selected vias. In the second method, the average product temperature is estimated for each vial, based on the mass difference before and after sublimation and the K v value is calculated accordingly. In the third method, the K v is estimated by comparison to sublimation results from a simulation. Results from methods 2 and 3 are very similar results and are slightly different from those of method 1. Method 1 was shown to exhibit a systematic bias due to the fact that it is based on the temperature of recording of selected vials only, which are not representative for all positions. Once the individual values of K v have been calculated, it is possible to establish a distribution for each method. It was observed that an overlay of two normal distributions describing the center and the edge vials provides a good representation of the empirical distribution. Furthermore, we propose a holistic model aiming to calculate the K v distribution for any specified pressure., Competing Interests: Declaration of Competing Interest All the authors worked for Sanofi at the time where the research work was done. All the work was Sanofi financed., (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage. In this work, we measured the aggregation rates of two antibodies in a broad temperature range (from 60 °C to -25 °C), using an isochoric cooling method to prevent freezing of the formulations below 0 °C. Both antibodies evidenced increasing aggregation rates when approaching extreme temperatures, because of hot and cold denaturation. This behavior was modelled using Arrhenius and Gibbs-Helmholtz equations, which enabled to deconvolute the contribution of unfolding from the protein association kinetics. This approach made possible to model the aggregation rates at refrigeration temperature (5 °C) in a relatively short timeframe (1-2 weeks) and using standard characterization techniques (SEC-HPLC and DLS)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Fill & finish manufacturing processes of biologics drug product involve multiple unit operations. In particular they often include a mixing step to reduce non-uniformities in fluids by eliminating gradients of concentration and pH may occur during freezing. This step should be conducted carefully to avoid any degradation of the protein under mechanical stress. This study was aimed at characterizing disposable vessels of square cross-section such as Levmixer® from Sartorius Stedim in terms of fluid dynamics and mixing in turbulent regime. The investigation included two tree large vessels (50, 200 & 650-l) and one 4-l vessel designed in house. For that purpose, the impact of stirrer speed, filling volume and duration of mixing on product quality attributes were studied, using a surrogate. Moreover, a scale-up rule, based on first principle, was established and allows prediction of the mixing time as a function of stirring speed and filling volume. A lab-scale test, using drug product, was performed at the same stress intensity but for a much longer duration than the commercial operation and did not reveal any trend to aggregation. Finally, based on the correlation, lab scale stress test and a single verification test at large scale, a design space within which the product can be processed without altering product quality was proposed., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Lipid nanoparticles (LNPs) for RNA and DNA delivery have attracted considerable attention for their ability to treat a broad range of diseases and to vectorize mRNA for COVID vaccines. LNPs are produced by mixing biomolecules and lipids, which self-assemble to form the desired structure. In this domain, microfluidics shows clear advantages: high mixing quality, low-stress conditions, and fast preparation. Studies of LNPs produced in micromixers have revealed, in certain ranges of flow rates, a degradation in performance in terms of size, monodispersity and encapsulation efficiency. In this study, we focus on the ring micromixer, which is well adapted to high throughput. We reveal three regimes, side-by-side, transitional and highly mixed, that control the mixing performance of the device. Furthermore, using cryo-TEM and biochemical analysis, we show that the mixing performances are strongly correlated to the characteristics of the LNPs we produce. We emphasize the importance of the flow-rate ratio and propose a physical criterion based on the onset of temporal instabilities for producing LNPs with optimal characteristics in terms of geometry, monodispersity and encapsulation yield. These criteria are generally applicable., (© 2022. The Author(s).)

The freezing of biologics has been widely studied from the physical chemistry point of view, for instance in terms of cryo-concentration, excipient crystallization, pH swing, potential protein denaturation, etc. In contrast, considerations on the processing aspects are very limited. For instance, the impact of freezer temperature, container size, freezer load, and freeze chilling capacity on the freezing rate in the most frequent case of freezing in a bottle have not been reported. In this study, the freezing time of either water or buffer solution was measured in various processing conditions. Experimental trials were conducted using containers ranging from 1 to 20 L in two types of freezers: a normal freezer (-30°C set point) and an ultra freezer (-70°C set point). These trials showed that both the container size and the freezer load influenced the freezing times. The current study demonstrated that the use of the well-established Plank model for freezing, coupled with freezer performance characterization, allows the description of the actual freezing kinetics in a very simple and accurate manner. The kinetics can then be modeled to accurately predict both the actual freezer temperature (possibly above the set point) and the freezing time based on freezer load., (© PDA, Inc. 2021.)

Jet milling is frequently used in pharmaceutical industry to achieve different objectives. It can be used as enabling technology to overcome poor water solubility linked to hydrophobic active of pharmaceutical ingredient (API) by reducing the particle size and therefore increasing the dissolution rate. Alternatively, jet milling can be used either to enhance blending efficiency of API with excipient in case of formulation at low dosage strength or to achieve the required particle size for inhalation therapy. In this study, development of commercial manufacturing process of sticky API and its industrialization are described. The methodology used is based on quality-by-design approach to deliver safe, effective and robust manufacturing process. The study showed that the specific energy is a key factor that drives particle size during jet milling and the scale-up from lab to industrial scale. After understanding the process, a design space was built where different zones such as operating point, operating space (where the product is compliant to specification despite variability of process parameters), and the knowledge space were outlined. Finally, an industrial installation was proposed to deliver product with high productivity yield, compliant with safety regulation, and cleanable in place.

Currently, the two technologies primarily used for the manufacturing of nano-crystalline suspensions using top down process (i.e. wet milling) are high pressure homogenization (HPH) and stirred bead milling (SBM). These two technologies are based upon different mechanisms, i.e., cavitation forces for HPH and shear forces for stirred bead milling. In this article, the HPH and SBM technologies are compared in terms of the impact of the suspension composition the process parameters and the technological configuration on milling performances and physical quality of the suspensions produced. The data suggested that both HPH and SBM are suitable for producing nano-crystalline suspensions, although SBM appeared more efficient than HPH, since the limit of milling (d 50 ) for SBM was found to be lower than that obtained with HPH (100 nm vs 200 nm). For both these technologies, regardless of the process parameters used for milling and the scale of manufacturing, the relationship of d 90 versus d 50 could be described by a unique master curve (technology signature of milling pathway) outlining that the HPH leads to more uniform particle size distribution as compared to SBM., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Stirred media milling is the main technology for producing colloidal nanocrystalline suspensions. A number of studies have been reported on the effect of different operating parameters for lab, pilot, and industrial scales. However, typical milling tool box that can be used to support candidate from selection up to phase III clinical supplies can involve different mill configurations. This article describes a parametric study and milling kinetic modelling of the different mills. The impact of active pharmaceutical ingredient (API) type and process parameters on milling kinetics was determined. The milling kinetics were modeled using an empirical model which allows for predicting and simulation of milling kinetics of stirred annular and pin mills. The proposed model was found to accurately fit milling kinetics whatever the API considered, technology employed, and the process parameters used for milling. Moreover, the model was found to be able to ensure the process transfer from one mill to another., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The small particle size of nano-crystalline suspensions can be responsible for their physical instability during drug product preparation (downstream processing), storage and administration. For that purpose, the commercial formulation needs to be sufficiently robust to various triggering conditions, such as ionic strength, shear rate, wetting/dispersing agent desorption by dilution, temperature and pH variation. In our previous work we described a systematic approach to select the suitable wetting/dispersant agent for the stabilization of nano-crystalline suspension. In this paper, we described the assessment of the formulation robustness (stabilized using a mixture of sodium dodecyl sulfate (SDS) and polyvinylpyrrolidone (PVP) and) by measuring the rate of perikinetic (diffusion-controlled) and orthokinetic (shear-induced) aggregation as a function of ionic strength, temperature, pH and dilution. The results showed that, using the SDS/PVP system, the critical coagulation concentration is about five times higher than that observed in the literature for suspension colloidaly stable at high concentration. The nano-suspension was also found to be very stable at ambient temperature and at different pH conditions. Desorption test confirmed the high affinity between API and wetting/dispersing agent. However, the suspension undergoes aggregation at high temperature due to the desorption of the wetting/dispersing agent and disaggregation of SDS micelles. Furthermore, aggregation occurs at very high shear rate (orhokinetic aggregation) by overcoming the energy barrier responsible for colloidal stability of the system., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This paper describes a systematic approach to select optimum stabilizer for the preparation of nano-crystalline suspensions of an active pharmaceutical ingredient (API). The stabilizer can be either a dispersant or a combination of dispersant and wetting agent. The proposed screening method is a quick and efficient way to investigate a large number of stabilizers based on the principles of physical-chemistry and employs a stepwise approach. The methodology has been divided in two main parts; the first part being focused on the qualitative screening with the objective of selecting the best candidate(s) for further investigation, the second part has been focused on quantitative screening with the objective to optimize the ratio and amount of wetting and dispersing agents, based on wettability, surface charges measurement, adsorption evaluation, process-ability evaluation and storage stability. The results showed clearly that SDS/PVP 40/60% (w/w) (sodium dodecyl sulfate/poly(vinyl pyrrolidone)) at a total concentration of 1.2% was the optimum stabilizer composition, at which the resulting nanosuspensions were stable for more than 50 days at room temperature., (Copyright © 2014 Elsevier B.V. All rights reserved.)