The subcutaneous administration of biopharmaceuticals is advantageous over intravenous administration, particularly with regard to improved patient compliance. However, in highly concentrated protein formulations lower viscosity of the formulation and stability of the protein is difficult to achieve. One approach involves using the viscosity-reducing excipients to diminish the interactions between protein molecules. In this context, the main objective of the study was to develop an optimal formulation for a model monoclonal antibody (mAb) and to evaluate new test compounds as viscosity-reducing agents. The test compounds were investigated both individually at increasing concentrations up to 200 mM and in combinations for their viscosity-reducing effect. Our results showed that all individual test compounds reduced the viscosity of the mAb formulation by more than 30 %, with reduction achieved by the six test compounds exceeding that achieved by proline (Pro). A reduction in the viscosity of the formulation below the 20 mPas threshold was achieved either by combining two test compounds or by increasing the concentration of a single compound above 25 mM. An accelerated stability study showed similar stabilization effects regardless of whether the test compounds were used alone or in combination. The percentage of aggregates was below 5 % in most formulations. These viscosity-reducing and stabilization effects corresponded to the dynamic light scattering results, which indicated that the test compounds reduced the attractive forces between the mAb molecules.

Monoclonal antibodies (mAb) have transformed modern medicine, offering targeted therapies for cancer, autoimmune disorders, and infectious diseases. To enhance patient convenience, subcutaneous administration is increasingly prioritized, requiring highly concentrated formulations. However, high viscosity of these formulations hinders manufacturability, injectability, and stability. Viscosity-reducing additives are a promising solution, disrupting protein-protein interactions and thereby reducing the viscosity of mAb formulations. Most promising additives are small organic electrolytes, where key properties influencing their efficacy include charge, size, and salt form. Relatively small molecules with three or more charges are most efficient, while the counter-ion provides opportunity for increased solubility and additional interactions. Our data analysis further highlights topological polar surface area and heteroatoms as significant predictors of viscosity reduction, while the role of aromatic moieties remains questionable. Another promising strategy is combining viscosity-reducing additives, which can achieve synergistic viscosity reduction while enhancing stability. Polyelectrolytes also demonstrate promising efficacy via complex formation with mAbs. Conversely, other common excipients of mAb formulations, such as sugars and surfactants, often increase viscosity of the formulation. This review provides a foundation for advancing viscosity-reducing strategies and offers actionable insights to guide future research and development of effective, patient-oriented mAb formulations.

At present, society has embraced the fact apropos population aging and climate changes, that demand, amongst others, innovative pharmaceutical technologies, emphasising the development of patient-specific delivery systems and thus the provision of efficient and sustainable drugs. Protein drugs for subcutaneous administration, by allowing less frequent application, represent one of the most important parts of the pharmaceutical field, but their development is inevitably faced with obstacles in providing protein stability and suitable formulation viscosity. To gain further knowledge and fill the gaps in the already constructed data platform for the development of monoclonal antibody formulations, we designed a study that examines small model proteins, i.e., bovine serum albumin. The main aim of the presented work is to evaluate the effect of protein concentrations on critical quality attributes of both, pre-lyophilised liquid formulations, and lyophilised products. Through the study, the hypothesis that increasing protein concentration leads to higher viscosity and higher reconstitution time without affecting the stability of the protein was confirmed. The most important finding is that sucrose plays a key role in the lyophilisation of investigated protein, nevertheless, it can be predicted that, to ensure the beneficial effect of mannitol, its amount has to prevail over the amount of sucrose.

Razvija se vse več bioloških zdravil, med katerimi prevladujejo zdravila z monoklonskimi protitelesi (angl. monoclonal antibodies, mAb), ki omogočajo specifično in učinkovito zdravljenje z manj neželenimi učinki. Zaradi kompleksne in občutljive proteinske zgradbe mAb je treba takšna zdravila vnašati parenteralno. Intravensko apliciranje, ki prevladuje, je lahko boleče, dolgotrajno in zahteva celo hospitalizacijo. Zato je vse več zdravil z mAb v razvoju namenjenih subkutani aplikaciji, ki je hitrejša, omogoča nižje stroške zdravljenja in boljše sodelovanje bolnikov. Subkutano pa lahko vbrizgamo le majhen volumen raztopine, zato so pogosto potrebne visoke koncentracije mAb, ki lahko povečajo viskoznost raztopin in povzročijo fizikalno nestabilnost molekul mAb v njih. Zdravila z mAb zato vsebujejo pufre, stabilizatorje in pomožne snovi za znižanje viskoznosti, ki prispevajo tudi k fizikalni stabilnosti mAb. Poleg teh sestavin članek predstavi tudi pomožne snovi, ki omogočajo vbrizgati večji volumen zdravila v podkožje in kombinacije terapevtskih mAb. Opisani so tudi inovativni sistemi za subkutano apliciranje mAb, kot so kompleksi, nanoklastri, suspenzije, mikrodelci in hidrogeli, ki so še v fazi razvijanja.

Monoclonal antibodies (mAbs) are an evolving class of biopharmaceuticals, with advancements evident across various stages of their development. While discovery, mAb chemical optimization, production and purification processes have been thoroughly reviewed, this paper aims to offer a summary of novel strategies in administration of mAbs. At present, systemic delivery of mAbs is available through parenteral administration routes with focus on subcutaneous administration. In addition, oriented toward patient-friendly therapy, other less invasive administration routes of mAbs, such as inhalation, nasal, transdermal, and oral administration, are explored. Literature data reveals the potential for local delivery of mAbs via inhalation, nasal, transdermal, intratumoral, intravitreal and vaginal administration, offering high efficacy with fewer systemic adverse effects. However, to date, only mAb medicines are available for intravitreal administration, mainly due to higher bioavailability, and an intranasal spray is authorised as a medical device. The review highlights the promising data in approval of novel administration routes, likely through inhalation, but further intensive research considering the current obstacles, is essential.

Administration of monoclonal antibodies (mAbs) is currently focused on subcutaneous injection associated with increased patient adherence and reduced treatment cost, leading to sustainable healthcare. The main bottleneck is low volume that can be injected, requiring highly concentrated mAb solutions. The latter results in increased solution viscosity with pronounced mAb aggregation propensity because of intensive protein-protein interactions. Small molecule excipients have been proposed to restrict the protein-protein interactions, contributing to reduced viscosity. The aim of the study was to discover novel compounds that reduce the viscosity of highly concentrated mAb solution. First, the chemical space of proline analogs was explored and 35 compounds were determined. Viscosity measurements revealed that 18 proline analogs reduced the mAb solution viscosity similar to or more than proline. The compounds forming both electrostatic and hydrophobic interactions with mAb reduced the viscosity of the formulation more efficiently without detrimentally effecting mAb physical stability. A correlation between the level of interaction and viscosity-reducing effect was confirmed with molecular dynamic simulations. Structure rigidity of the compounds and aromaticity contributed to their viscosity-reducing effect, dependent on molecule size. The study results highlight the novel proline analogs as an effective approach in viscosity reduction in development of biopharmaceuticals for subcutaneous administration.

Orodispersible drug formulations are a current trend in the pharmaceutical industry, mostly intended for pediatric and geriatric patients. Oral lyophilizates are solid forms, intended either to be placed in the mouth or to be dispersed (or dissolved) in water before administration. The correct excipient composition is a prerequisite to provide lyophilizates with the appropriate visual appearance and disintegration time. Typically, they are composed of binders, such as gelatin and polyvinylpyrrolidone, fillers such as sucrose, mannitol, or sorbitol, taste modifiers, colorants, sweeteners, and preservatives. The main purpose of this study was to determine the optimal excipient scaffold to ensure the proper appearance of lyophilizates that have undergone aggressive drying conditions and a disintegration time of less than 3 min. In addition to mannitol and gelatin, the most frequently used binders, PVP K25, PVP K90, glycine, croscarmellose, and hydrolyzed gelatin were investigated. The results obtained revealed that lyophilizates with only mannitol and gelatin have a disintegration time that is too long, and that replacement of gelatin with PVP K25 led to friable and cracked lyophilizates. Considering disintegration time and visual appearance, lyophilizates with a mixture of gelatin, PVP K25, and mannitol (1:2:5) formed from liquid formulations with 6% (w/w) excipients were determined to be the most suitable. As a binder, PVP K25 expresses more appropriate characteristics relating to PVP K90. Addition of croscarmellose provided lyophilizates with a shorter disintegration time, whereas glycine only had a positive effect on the elegant appearance of lyophilizate cakes. Hydrolyzed gelatin was introduced with the aim of obtaining an even shorter disintegration time and at the same time an acceptable visual appearance of lyophilizates. This was achieved by lyophilization of solutions with 15% (w/w) of excipients with a hydrolyzed gelatin:PVP K25:glycine/croscarmellose:mannitol ratio of 4:2:0.5:4.5. Such lyophilizates show the highest potential for incorporation of poorly soluble and low-dose drugs.

Oral lyophilizates are intended for application to the oral cavity or for dispersing in water. The purposes of this research were: (i) to set up the quality by design approach in the development of oral lyophilizates for drug incorporation; and (ii) to evaluate the established approach by comparing its outcomes with experimentally obtained results. Within the knowledge space, properties about drugs, excipients, and the lyophilization process were acquired, followed by the determination of critical quality attributes via risk identification. Risks were assessed by failure mode and effective analysis, which recognized critical material attributes, i.e., type, concentration, particle size, solubility of drug and excipients, while as main critical process parameters, cooling rate, shelf temperature, and chamber pressure during drying were pointed out. Additionally, design space was established using the Minitab® 17 software and valued with an 88.69% coefficient of determination. A detailed comparison between the model and experimental results revealed that the proposed optimal compositions match in the total concentration of excipients (6%, w/w) in the pre-lyophilized liquid formulation, among which mannitol predominates. On the other hand, a discrepancy regarding the presence of gelatin was detected. The conclusion was that the set model represents a suitable onset toward optimization of drug-based oral lyophilizates development, preventing unnecessary investment of time and resources.

Recently, nanocrystal dispersions have been considered as a promising formulation strategy to improve the bioavailability of the deuterated pyrazoloquinolinone ligand DK-I-56-1 (7‑methoxy-2-(4‑methoxy-d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one). In the current study, the freeze-drying process (formulation and process parameters) was investigated to improve the storage stability of the previously developed formulation. Different combinations of lyoprotectant (sucrose or trehalose) and bulking agent (mannitol) were varied while formulations were freeze-dried under two conditions (primary drying at -10 or -45 °C). The obtained lyophilizates were characterized in terms of particle size, solid state properties and morphology, while the interactions within the samples were analyzed by Fourier transform infrared spectroscopy. In the preliminary study, three formulations were selected based on the high redispersibility index values (around 95%). The temperature of primary drying had no significant effect on particle size, but stability during storage was impaired for samples dried at -10 °C. Samples dried at lower temperature were more homogeneous and remained stable for three months. It was found that the optimal ratio of sucrose or trehalose to mannitol was 3:2 at a total concentration of 10% to achieve the best stability (particle size < 1.0 μm, polydispersity index < 0.250). The amorphous state of lyoprotectants probably provided a high degree of interaction with nanocrystals, while the crystalline mannitol provided an elegant cake structure. Sucrose was superior to trehalose in maintaining particle size during freeze-drying, while trehalose was more effective in keeping particle size within limits during storage. In conclusion, results demonstrated that the appropriate combination of sucrose/trehalose and mannitol together with the appropriate selection of lyophilization process parameters could yield nanocrystals with satisfactory stability.

Pharmaceutical industry is constantly focused on researching patient-centered dosage forms that can maximize the therapeutic potential of an active pharmaceutical ingredient, as for example orodispersible form. The main advantage of orodispersible dosage forms is that they are suitable for patients with swallowing problems, children, geriatric, and psychiatric patients, leading to improvement in patient compliance. Orodispersible drug formulations include orodisperisble tablets, oral lyophilizates, orodispersible granules, minitablets, orodispersible films, and some less common. Oral lyophilizates are solid forms, intended either to be placed in the mouth or to be dispersed (or dissolved) in water before administration. The composition of oral lyophilizates determines their quality attributes, and generally includes binders, fillers, taste modifiers, sweeteners, and preservatives (Bjelošević Žiberna et al., 2023; Slavkova & Breitkreutz, 2015). Our aim was to design formulations for preparation of oral lyophilizates by implementation of aggressive primary drying conditions, contributing to a smaller financial and environmental burden. The objectives were: (i) to examine the influence of glycine, and croscarmellose on the quality attributes of lyophilizates based on mannitol, gelatin, and polyvinylpyrrolidone K25; and (ii) to discover the potential of hydrolyzed gelatin in oral lyophilizates.

Biopharmaceuticals represent one of the fastest growing areas in the pharmaceutical industry. Previously, protein formulations for intravenous applications were most widespread, while now the subcutaneous route of administration is gaining importance. Subcutaneous administration requires high concentrations of protein drugs. Lyophilisation represents the method of choice for drying such formulations. In this study the effects of monoclonal antibody (mAb) 'drug' concentration on the thermal characteristics and viscosities of pre-lyophilised formulations were determined. Additionally, the effects of mAb concentration on reconstitution time, cake appearance and mAb stability were evaluated. The main aim of the study was to investigate the effects of amino acids as selected bulking agents and their ratios to sucrose on the critical quality attributes of lyophilisate formulations at low and high mAb concentrations, as a comparison with the most commonly used mannitol formulations. Harsher aggressive drying conditions did not have any major impact on the critical quality attributes of lyophilisates or on mAb stability, while primary drying time was shortened by up to 80%. The isoleucine:sucrose ratio of 1:4 provided a lyophilised cake with superior macroscopic appearance for low and high mAb concentrations despite aggressive drying, which greatly shortened lyophilisation time. However, the drawback of isoleucine in high protein concentration formulations is the long reconstitution time. In contrast, for low mAb concentrations, an isoleucine:sucrose ratio of 1:4 is promising for replacement of mannitol, as it provides suitable reconstitution time, cake appearance and mAb stability.

Biopharmaceuticals are one of the fastest growing areas within the pharmaceutical industry. As protein drugs require parenteral administration, they are commonly formulated as aqueous solutions. However, this is not always feasible due to their general instability. In such cases, lyophilised powders for injection are the dosage form of choice, for the preparation of stable products. Lyophilisation is known to be highly time and energy consuming, and hence it is an expensive technological process. Thus, the pharmaceutical industry is increasingly focused on its optimisation. Implementation of aggressive conditions, together with optimisation of formulation parameters, represent the contemporary approach to reduction of the primary drying time. As such, incorporation of drug-specific excipients can contribute significantly to the stability of a biologically active ingredient, and indirectly they can also affect the time needed for lyophilisation. The addition of the most relevant protein stabilisers, surfactants, buffers and bulking agents is therefore crucial. The main aim of the present review is to define the most important groups of biopharmaceutical excipients, based on their roles in formulations and the mechanism(s) through which they support the lyophilisation process, to provide products with the required protein efficiency and product characteristics. The scope of the article is to critically discuss the suitability of novel stabilizers, with higher critical temperatures and bulking agents in terms of implementation of aggressive primary drying. For better assignment of the topic-related challenges, the stabilities of biopharmaceutical drugs and the fundamentals of the lyophilisation process are also briefly described.

Biopharmaceuticals are one of the fastest growing areas within the pharmaceutical industry. As protein drugs require parenteral administration, they are commonly formulated as aqueous solutions. However, this is not always feasible due to their general instability. In such cases, lyophilised powders for injection are the dosage form of choice, for the preparation of stable products. Lyophilisation is known to be highly time and energy consuming, and hence it is an expensive technological process. Thus, the pharmaceutical industry is increasingly focused on its optimisation. Implementation of aggressive conditions, together with optimisation of formulation parameters, represent the contemporary approach to reduction of the primary drying time. As such, incorporation of drug-specific excipients can contribute significantly to the stability of a biologically active ingredient, and indirectly they can also affect the time needed for lyophilisation. The addition of the most relevant protein stabilisers, surfactants, buffers and bulking agents is therefore crucial. The main aim of the present review is to define the most important groups of biopharmaceutical excipients, based on their roles in formulations and the mechanism(s) through which they support the lyophilisation process, to provide products with the required protein efficiency and product characteristics. The scope of the article is to critically discuss the suitability of novel stabilizers, with higher critical temperatures and bulking agents in terms of implementation of aggressive primary drying. For better assignment of the topic-related challenges, the stabilities of biopharmaceutical drugs and the fundamentals of the lyophilisation process are also briefly described.

Crystalline bulking agent in lyophilized biopharmaceutical formulations provides an elegant lyophilized cake structure and allows aggressive primary drying conditions. The interplay between amorphous and crystalline state of excipients heavily influence the stability of lyophilized biological products and should be carefully evaluated in the formulation and process development phase. This study focuses on: (1) elucidating the influence of formulation and lyophilization process variables on the formation of different states of mannitol and (2) its impact on model monoclonal antibody stability when compared to sucrose. The main aim of the present research work was to study the influence of different mannitol to sucrose ratios and monoclonal antibody concentrations on mannitol physical form established during lyophilization. In addition, also the effect of process variables on mannitol hemihydrate (MHH) formation was under investigation. Thermal analysis and powder X-ray diffraction results revealed that the ratio between sucrose and mannitol and mAb concentration have a decisive impact on mannitol crystallization. Namely, increasing amount of mannitol and monoclonal antibody resulted in decreasing formation of MHH. From the process parameters investigated, a higher secondary drying temperature has the biggest impact on the complete dehydration of MHH. Specifically, higher secondary drying temperature reflected in complete dehydration of MHH. Annealing temperature was shown to affect the MHH content in the final product, wherein the higher annealing temperature was preferential for formation of anhydrous mannitol. Temperature stress stability study revealed that the most important parameter influencing monoclonal antibody stability is the ratio of protein to sucrose. Contrary to widespread assumption, we did not detect any impact of MHH on the stability of the investigated monoclonal antibody.

The influence of different phospholipid types (pure phospholipids 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine, POPC, 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine, DPPC, and one commercial phospholipid mixture, Lipoid H100), sterol types (cholesterol vs. β‐sitosterol), and various sterol concentrations (5–50 mol%) on liposomal membrane fluidity, thermotropic properties, liposome size, zeta potential, and lipid oxidation kinetics using fluorescent lipid probe BODIPY 581/591 C11 (4,4‐difluoro‐5‐[4‐phenyl‐1,3‐butadienyl]‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐undecanoic acid) are investigated. DPPC bilayer is more rigid than POPC and phospholipids mixture membranes. Pure DPPC gives the smallest liposomes, while liposomes of Lipoid H100 have the largest diameter. Both sterols reduce membrane fluidity of all liposomes, increase absolute zeta potential, cause significant changes in particle size, and decrease phase transition temperature (Tm) and enthalpy of DPPC. POPC/β‐sitosterol liposomes exhibit the most significant lipid oxidation of the lipophilic probe. Along with beneficial effects of phytosterols on human health, better membrane fluidity, more favorable and stabilizing interactions with phospholipids, smaller vesicle size, and enhanced physical stability in comparison to cholesterol are some of the encouraging results for the use of β‐sitosterol in liposome formulations for potential application in foods, pharmaceutics, and cosmetics.Practical Applications: Adjusting the composition of liposomal membrane (lipid type, sterol type, and concentration) can be used as a tool to control membrane fluidity, permeability, and thermotropic properties, and thus predict release properties, physical, thermal, and oxidative stability. A commercial phospholipid mixture of different natural phospholipids with impurities creates less uniform liposomal membrane that is characterized by higher fluidity in comparison to DPPC. The type of phospholipid has huge influence on MLVs size. β‐sitosterol, which is a phytosterol with beneficial effects on human health can be used as a replacement for cholesterol in liposomal formulations, but with the following in mind: β‐sitosterol reduces fluidity of the phospholipid bilayer to a lesser extent than cholesterol, β‐sitosterol gives smaller MLVs than cholesterol, DPPC/β‐sitosterol SUVs are bigger than 100 nm in diameter (relevant for intravenous administration), MLVs with ≥30 mol% of β‐sitosterol can be considered as physically stable (unlike those with cholesterol), irrespective to the phospholipid type.The influence of different phospholipid types, sterol types, and various sterol concentrations on liposomal membrane fluidity, thermo tropic properties, liposomesize, zeta potential, and lipid oxidation kinetics are investigated.

Freeze-drying is the method of choice to dry formulations with biopharmaceutical drugs, to enhance protein stability. This is usually done below the glass transition temperature of maximally freeze-concentrated solutions (T-g'), to avoid protein aggregation, preserve protein activity, and obtain pharmaceutically 'elegant' cakes. Unfortunately, this is a lengthy and energy-consuming process. However, it was recently shown that drying above T-g' or even above the collapse temperature (T-c) is not necessarily detrimental for stability of biopharmaceuticals, and hence provides an attractive option for freeze-drying cycle optimisation. The goal of the present study was to optimise the freeze-drying cycle for a model IgG monoclonal antibody (20 mg/mL) in sucrose and sucrose/glycine formulations, by reducing primary drying time. To study the impact of shelf temperature (T-s) and chamber pressure on product temperature (T-p), one conventional and five aggressive cycles were tested. Aggressive conditions during primary drying were achieved by increasing T-s from -20 degrees C (conventional cycle) to 30 degrees C, with chamber pressure set to 0.1 mbar, 0.2 mbar or 0.3 mbar. These combinations of Ts and chamber pressure resulted in T-p well above T-g', and in some cases, even above Tc, without causing macrocollapse. Other critical quality attributes of the products were also within the expected ranges, such as reconstitution time and residual water content. Physical stability was tested using size exclusion chromatography, dynamic light scattering, and micro-flow imaging. All of the lyophilised samples were exposed to stress and the intended storage conditions, with no impacts on the product seen. These data show that implementation of aggressive conditions for the investigated formulations is possible and can significantly contribute to the reduction of primary drying times by up to 54% (from 48 to 22 h) in comparison to conventional freeze-drying.

We investigated the influence of carrier systems for different commercially available water-soluble formulations for coenzyme Q10 on structural changes of model lipid membranes formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and by a mixture of phosphatidylcholine and sphingomyelin (2.4:1). Structural changes in the membranes were measured using fluorescence anisotropy, electron paramagnetic resonance, and differential scanning calorimetry. Two fluorophores and two spin probes were used to monitor membrane characteristics close to the water-lipid interface and in the middle of the bilayer of the model lipid membranes. Different water-soluble carrier systems were tested. These data show that different systems can facilitate penetration of CoQ10 in the lipid membranes, where an increase in the lipid order parameter was observed. In addition, water soluble CoQ10 formulations better protect lipids from oxidation in liposome solution. With the exception of the carriers in an emulsified formulation of CoQ10, those in the other samples did not have any significant effects on membrane fluidity.