Don't shake it! Mechanical stress testing of mRNA-lipid nanoparticles.

Shaking stress studies are typically performed during formulation development to test the liability of a drug product towards interfacial stress occurring during transport, especially if a liquid formulation is desired. We evaluated various shaking procedures using a polyA-surrogate solution and verified our findings by eGFP-LNP cell-expression experiments. Shaking on an orbital shaker in vertical and horizontal orientations at increasing speeds from 300 to 600 rpm resulted in decreasing levels of encapsulated nucleic acid content, larger LNP sizes, and decreasing PDI. We report that vertical and horizontal shaking of both polyA- and eGFP-LNPs led to white deposits on the inner glass vial surface, depending on time, rpm, and temperature. Increasing the fill volume/smaller headspace (0.3 versus 0.9 mL fill) did not mitigate this phenomenon in the studied configuration, and the use of hydrophobic primary packaging even accelerated the formation of white deposits. In contrast, we demonstrated that a lyophilized polyA-LNP dosage form was less susceptible to shaking and maintained cake integrity and product properties. Multiple vortexing steps resulted in an increase in LNP size, PDI, and a decrease in encapsulated polyA content. We conclude that shaking experiments of nucleic acid-loaded LNPs in their final configuration at intended transport conditions need to be considered during technical development.

Intraocular pressure and injection forces during intravitreal injection into enucleated porcine eyes.

Injection of biological molecules into the intravitreous humor is of increasing interest for the treatment of posterior segment eye diseases such as age-related degenerative macular degeneration. The injection volume is limited by an increase in intraocular pressure (IOP) and 50-100 µL are typically used for most intravitreally (IVT) applied commercial products. Direct measurement of IOP is difficult and has not been studied dependent on solution properties and injection rates. We used an instrumental set-up to study IOP ex vivo using healthy enucleated porcine eyes. IOP was determined as a function of injection volume for viscosities between 1 and 100 mPas, injection rates of 0.1, 1, and 1.5 mL/min, and needle length and diameter (27/30G and 0.5/0.75″) using Dextran solutions. IOP increased exponentially for injection volumes larger than 100 µL. We did not observe differences in IOP dependent on viscosity, injection rate, and needle diameter. However, variability increased significantly for injection volumes larger than 100 µL and, unexpectedly, declined with higher viscosities. We demonstrate that the exponential increase in IOP is not reflected by injection force measurements for typical configurations that are used for IVT application. The present findings may guide injection volumes for intravitreal injection and inform injection force considerations during technical drug product development.

Opportunities in an Evolving Pharmaceutical Development Landscape: Product Differentiation of Biopharmaceutical Drug Products.

The current perspective reviews the biopharmaceutical market until end of 2020 and highlights the transforming biopharmaceutical landscape during the recent decade. In particular, the rise of biosimilars and the development of new therapeutic modalities through recent advancement in molecular biology research sustainably change the product scenery. The present manuscript describes opportunities for pharmaceutical technical development, highlighting concepts such as product differentiation to succeed in a competitive product landscape. Product differentiation offers the opportunity for numerous life-cycle options and market exclusivity through incremental improvements in standard of care treatment. In particular, different formulation options and formulation-device combinations are described, focusing on systemic delivery of monoclonal antibody products and patient-centered development. The concept of product differentiation is exemplified in a case study about HER2+ breast cancer therapy, underlining pharmaceutical technical solutions and major improvements for the patient.

Tissue Resistance during Large-Volume Injections in Subcutaneous Tissue of Minipigs.

PURPOSE: Injection devices for administration of biopharmaceuticals enable subcutaneous self-administration by patients. To meet patient specific capabilities, injection forces need to be characterized. We address the open question of whether tissue resistance significantly contributes to overall injection forces, especially for large injection volumes. METHODS: Subcutaneous tissue resistance was systematically quantified for injection volumes up to 11 mL depending on viscosity (1-20 mPa·s) and injection rates (0.025-0.2 mL/s) using Göttingen Minipigs as the animal model. The contribution of an artificially applied external force at the injection site simulating autoinjector needle cover depression was tested between 2.5-7.5 N. RESULTS: Tissue resistance reached average values of ~120 mbar for injection volumes up to 11 mL independent of viscosity and injection rate, and maximum values of 300 mbar were determined. Artificially applied external forces led to higher values, independent of the absolute applied force - maximum values of 1 bar were obtained when injecting 4.5 mL of the 20 mPa·s solution at an injection rate of 0.1 mL/s with the application of an artificial 5 N force, corresponding to ~450 mbar. All conditions yield defined injection sites suggesting tissue resistance is defined by mechanical properties of the subcutaneous tissue. CONCLUSIONS: We set our results in relation to overall injection forces, concluding that maximum values in tissue resistance may cause challenges during subcutaneous injection when using injection devices. Graphical abstract.

Excipients for Room Temperature Stable Freeze-Dried Monoclonal Antibody Formulations.

Sucrose is a common cryoprotectant and lyoprotectant to stabilize labile biopharmaceuticals during freeze-drying and storage. Sucrose-based formulations require low primary drying temperatures to avoid collapse and monoclonal antibody (mAb) containing products need to be stored refrigerated. The objective of this study is to investigate different excipients enabling storage at room temperature and aggressive, shorter lyophilization cycles. We studied combinations of 2-hydroxypropyl-beta-cyclodextrin (CD), recombinant human albumin, polyvinylpyrroldione (PVP), dextran 40 kDa (Dex), and sucrose (Suc) using 2 mAbs. Samples were characterized for collapse temperature (Tc), glass transition temperature of the liquid (Tg') and freeze-dried formulation (Tg), cake appearance, residual moisture, and reconstitution time. Freeze-dried formulations were stored at 5°C, 25°C, and 40°C for up to 9 months and mAb stability was analyzed for color, turbidity, visible and sub-visible particles, and monomer content. Formulations with CD/Suc or CD/PVP/Suc were superior to pure Suc formulations for long-term storage at 40°C. When using aggressive freeze-drying cycles, these formulations were characterized by pharmaceutically elegant cakes, short reconstitution times, higher Tg', Tc, and Tg. We conclude that the addition of CD allows for shorter freeze-drying cycles with improved cake appearance and enables storage at room temperature, which might reduce costs of goods substantially.

Assessment of dual life stage antiplasmodial activity of british seaweeds.

Terrestrial plants have proven to be a prolific producer of clinically effective antimalarial drugs, but the antimalarial potential of seaweeds has been little explored. The main aim of this study was to assess the in vitro chemotherapeutical and prophylactic potential of the extracts of twenty-three seaweeds collected from the south coast of England against blood stage (BS) and liver stage (LS) Plasmodium parasites. The majority (14) of the extracts were active against BS of P. falciparum, with brown seaweeds Cystoseira tamariscifolia, C. baccata and the green seaweed Ulva lactuca being the most active (IC(50)s around 3 µg/mL). The extracts generally had high selectivity indices (>10). Eight seaweed extracts inhibited the growth of LS parasites of P. berghei without any obvious effect on the viability of the human hepatoma (Huh7) cells, and the highest potential was exerted by U. lactuca and red seaweeds Ceramium virgatum and Halopitys incurvus (IC50 values 14.9 to 28.8 µg/mL). The LS-active extracts inhibited one or more key enzymes of the malarial type-II fatty acid biosynthesis (FAS-II) pathway, a drug target specific for LS. Except for the red seaweed Halopitys incurvus, all LS-active extracts showed dual activity versus both malarial intracellular stage parasites. This is the first report of LS antiplasmodial activity and dual stage inhibitory potential of seaweeds.

Antiprotozoal, antitubercular and cytotoxic potential of cyanobacterial (blue-green algal) extracts from Ireland.

Cyanobacteria (= blue-green algae) are prolific producers of structurally distinct and biologically active metabolites. In the continuation of our search for new sources of anti-infective natural products, we have assessed the in vitro antiprotozoal (Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani) and antitubercular (Mycobacterium tuberculosis) potential of samples of two terrestrial cyanobacteria, Nostoc commune (collected when desiccated and wet) and Rivularia biasolettiana. The cytotoxic potential of the extracts was also evaluated against primary L6 cells. Except for T. cruzi and M. tuberculosis, the crude extracts were active against all the organisms tested and showed no toxicity. The crude extracts were then partitioned between n-hexane, chloroform and aqueous methanol and retested against the same panel of pathogens. The chloroform sub-extracts of both N. commune samples showed significant activity against T. b. rhodesiense (IC50 values 2.0 and 3.5 microg/mL) and P. falciparum (IC50s 7.4 and 5.8 microg/mL), with low toxicity. This trend was also true for R. biasolettiana extracts, and its chloroform sub-extract showed notable activity against all parasitic protozoa. There were differences in the biological activity profiles of extracts derived from desiccated and hydrated forms of N. commune. To our knowledge, this is the first study assessing the anti-infective activity of desiccated and hydrated forms of N. commune, as well as R. biasolettiana. Furthermore, the present work reports such biological activity in terrestrial cyanobacteria from Ireland for the first time. These results warrant the further study of Irish terrestrial cyanobacteria as a valuable source of new natural product leads for the treatment of parasitic protozoal infections.

Antimycobacterial, antiprotozoal and cytotoxic potential of twenty-one brown algae (Phaeophyceae) from British and Irish waters.

In the continuation of our research on seaweeds, crude extracts of 21 brown algae collected from the south coast of England and the west coast of Ireland were screened for in vitro trypanocidal, leishmanicidal and antimycobacterial activities. Mammalian stages of a small set of parasitic protozoa; i.e. Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, and the tubercle bacillus Mycobacterium tuberculosis were used as test organisms. The extracts were also evaluated for selectivity by testing on a mammalian cell line (L6 cells). Only four extracts were moderately active against T. cruzi, whereas all algal extracts showed significant activity against T. brucei rhodesiense, with Halidrys siliquosa and Bifurcaria bifurcata (Sargassaceae) being the most potent (IC50 values 1.2 and 1.9 µg/mL). All algal extracts also displayed leishmanicidal activity, with H. siliquosa and B. bifurcata again being the most active (IC50s 6.4 and 8.6 µg/mL). When tested against M. tuberculosis, only the B. bifurcata extract was found to have some antitubercular potential (MIC value 64.0 µg/mL). Only three seaweed extracts, i.e. H. siliquosa, B. bifurcata and Cystoseira tamariscifolia showed some cytotoxicity. To our knowledge, this is the first study on the antiprotozoal and antimycobacterial activity of brown algae from British and Irish waters.