Visible and Sub-visible Particle Formation for a Model Bioconjugate.

The time-course and extent of visible particle (VP) and sub-visible particle (SVP) formation was monitored as a function of interfacial area (IA) for a model bioconjugate. To facilitate particle formation, the bioconjugate was agitated in a glass vial and exposed to IAs up to 478 mm2. Since vials had equal fill and headspace volumes, the area of the air-water interface was varied by placing vials on angled blocks at 0°, 30°, 60°, or 90° from the horizontal. A significant increase in visible and sub-visible particle formation was observed with increasing air-water IA. Exposure to IAs below ∼305 mm2 resulted in the formation of very few particles, while IAs > ∼305 mm2 resulted in substantial particle formation. Visible and sub-visible particle morphology varied with interfacial area and time. The sub-visible particles initially increased with time but did not reach steady state; instead the initial increase was followed by complete depletion. These phenomena indicate that visible particle formation likely increased at the expense of the sub-visible particle population and demonstrate a potential link between the two particle populations for this model bioconjugate. Initiation of particle formation did not result in corresponding decreases in protein concentration or increases in soluble aggregates. However, extended agitation time resulted in a significant decrease in protein concentration.

The Race to Develop the Pfizer-BioNTech COVID-19 Vaccine: From the Pharmaceutical Scientists' Perspective.

At the outset of the coronavirus disease 2019 (COVID-19) pandemic, it was clear that a vaccine would be crucial for global health efforts. The Pfizer and BioNTech teams came together in a race against the virus, working to design, test, manufacture, and distribute a safe and efficacious vaccine in record time for people around the world. Here, we provide backstory commentary from the pharmaceutical scientist perspective on the challenges and solutions encountered in the development of the Pfizer-BioNTech mRNA COVID-19 vaccine (BNT162b2; b2; Comirnaty®; tozinameran). We discuss the foundational science that led to the decision to use an mRNA-based approach. We also describe key challenges in the identification of an optimal vaccine candidate and testing in clinical trials, the continuous efforts to improve the vaccine formulation in response to changing global health priorities and facilitate vaccine accessibility, and how vast quantities of vaccine doses were manufactured and safely delivered to every corner of the globe, all without compromising quality, science, and safety. The key to successfully delivering a safe and efficacious vaccine within nine months was a result of extraordinary, real-time, parallel effort and across-the-board collaboration between stakeholders on a global scale.

Characterizing the freeze-drying behavior of model protein formulations.

The freeze-drying behavior of three model proteins, namely, lysozyme, BSA, and IgG, has been studied using a variety of techniques under two different primary drying conditions (shelf temperatures of -25°C and +25°C, respectively) in an amorphous formulation. Manometric temperature measurements were used to characterize product temperature (T (pr)), sublimation rates, and product resistance (R (p)) during primary drying. Biophysical techniques such as circular dichroism, fluorescence, and Fourier transform infrared spectroscopy were used to study protein conformation. Size exclusion chromatography was used to monitor the formation of high-molecular-weight species (HMWS) over time on storage, and cake morphology was studied using scanning electron microscopy. The differences in the freeze-drying behavior of the three proteins were more evident at higher protein concentrations, where the protein significantly influences the behavior of the formulation matrix. However, these differences were minimized in the aggressive mode and were insignificant at lower protein concentrations where excipients dominated the freeze-drying behavior. Differences in cake morphology were observed between the two drying conditions employed as well as between the three proteins studied. The stability and the protein structure, however, were equivalent for the protein cakes generated using the two different primary drying conditions.

Ex Situ and In Situ Characterization of Vaccine Suspensions in Pre-Filled Syringes.

Ex situ and in situ techniques were used to characterize the suspended phase over time for a vaccine drug product supplied in syringes. Micro-computed tomography was used to characterize the suspended sediment in situ in the syringe, while traditional techniques such as particle size distribution, charge (zeta potential), settling rate, and front-faced fluorescence were used to characterize the suspension ex situ. In addition, analytical chemical measurements were conducted in parallel during the course of the study. The ex situ and in situ techniques together with the chemical analyses provided different sets of data, but all leading to the same conclusion that the older, hard to re-disperse vaccine product syringes were similar in product quality attributes (both physical and chemical) to the freshly made, easy to re-disperse syringes. Longer re-dispersion time with age was not a result of any altered physical or chemical attributes of the product but simply because of the distance travelled by the sediment from the neck of the syringe barrel deeper into the bore of the syringe over time under the influence of gravity in the tip down orientation, making it harder for the continuous external phase to access the sediment in the bore and enable easy re-dispersion.

The Effect of Shipping Stresses on Vaccine Re-dispersion Time.

A case study is presented for a vaccine drug product (DP) that showed variable re-dispersion times between syringes within a given DP lot and between different DP lots when shipped from the manufacturing site to the receiving site. A simulated shipping study was designed to understand the effect of individual shipping stresses on re-dispersion time and product quality. Shipping stresses simulating shock/drop, aircraft, and truck vibrations were applied separately to 3 syringe orientations, namely tip up, tip down, and tip horizontal (TH). Results from the simulated shipping study showed that shock/drop reduced re-dispersion time while truck and aircraft vibrations increased re-dispersion time in the tip down orientation. The dissimilar effects of different shipping stresses on re-dispersion resulted in the observed intra and inter DP lot variability in re-dispersion time. Shipping stresses did not impact re-dispersion in the TH or tip up orientation. No vaccine product quality attributes or physical properties were affected by shipping stresses. Actual shipping results correlated well with simulated shipping data. Because re-dispersion time was influenced mainly by shipping stress and syringe orientation, the mitigation measure to reduce end-user re-dispersion time was to implement the TH orientation for DP syringes during shipment and storage.

Application of process analytical technology for monitoring freeze-drying of an amorphous protein formulation: use of complementary tools for real-time product temperature measurements and endpoint detection.

Process analytical technology (PAT) and quality by design have gained importance in all areas of pharmaceutical development and manufacturing. One important method for monitoring of critical product attributes and process optimization in laboratory scale freeze-drying is manometric temperature measurement (MTM). A drawback of this innovative technology is that problems are encountered when processing high-concentrated amorphous materials, particularly protein formulations. In this study, a model solution of bovine serum albumin and sucrose was lyophilized at both conservative and aggressive primary drying conditions. Different temperature sensors were employed to monitor product temperatures. The residual moisture content at primary drying endpoints as indicated by temperature sensors and batch PAT methods was quantified from extracted sample vials. The data from temperature probes were then used to recalculate critical product parameters, and the results were compared with MTM data. The drying endpoints indicated by the temperature sensors were not suitable for endpoint indication, in contrast to the batch methods endpoints. The accuracy of MTM Pice data was found to be influenced by water reabsorption. Recalculation of Rp and Pice values based on data from temperature sensors and weighed vials was possible. Overall, extensive information about critical product parameters could be obtained using data from complementary PAT tools.

Effect of dye aggregation on triarylmethane-mediated photoinduced damage of hexokinase and DNA.

The observation that enhanced mitochondrial membrane potential is a prevalent cancer cell phenotype has provided the conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemo- and photochemotherapy of neoplastic diseases. Cationic triarylmethane (TAM(+)) dyes represent a series of photosensitizers whose phototoxic effects develop at least in part at the mitochondrial level. In this report we describe how the molecular structure of four representative TAM(+) dyes (Crystal Violet, Ethyl Violet, Victoria blue R, and Victoria pure blue BO) affects their efficiency as mediators of the photoinduced inactivation of two model mitochondrial targets, hexokinase (HK) and DNA. Our results have indicated that TAM(+) dyes efficiently bind to HK and DNA in aqueous media both as dye monomers and aggregates, with the degree of aggregation increasing with increasing the lipophilic character of the photosensitizer. The efficiency with which HK and DNA are damaged upon 532 nm photolysis of biopolymer-TAM(+) complexes was found to decrease upon increasing the degree of dye aggregation over these macromolecular templates. Comparative experiments carried out both in water and in D(2)O, and in air-equilibrated and nitrogen-purged samples have also indicated that, at least when Crystal Violet is used as the photosensitizer, the mechanism of macromolecular damage does not require the involvement of molecular oxygen to operate. This finding makes Crystal Violet a potential candidate for use in photochemotherapy of hypoxic or poorly perfused tumor areas.

Use of manometric temperature measurements (MTM) to characterize the freeze-drying behavior of amorphous protein formulations.

The freeze-drying behavior and cake morphology of a model protein in an amorphous formulation were studied at varying protein concentrations using conservative (-25 degrees C) and aggressive (+25 degrees C) shelf temperatures at constant chamber pressure during primary drying. The two cycles were characterized by manometric temperature measurements (MTM) in a SMART freeze dryer that estimates the sublimation rate (dm/dt), product temperature at the freeze-drying front (T(p-MTM)) and product resistance (R(p)) during a run. The calculated sublimation rates (dm/dt) were 3-4 times faster in the aggressive cycle compared to the conservative cycle. For conservatively dried cakes R(p) increased with both dry layer thickness and protein concentration. For aggressively dried cakes (where freeze-drying occurs at the edge of microcollapse), R(p) also increased with protein concentration but was independent of the dry layer thickness. The sublimation rate was influenced by R(p), dry layer thickness and T(p-MTM) in the conservative cycle, but was governed mainly by T(p-MTM) in the aggressive cycle, where R(p) is independent of the dry layer thickness. The aggressively dried cakes had a more open and porous structure compared to their conservatively dried counterparts.

Comparative evaluation of disodium edetate and diethylenetriaminepentaacetic acid as iron chelators to prevent metal-catalyzed destabilization of a therapeutic monoclonal antibody.

Understanding the effect of metal chelators with respect to their ability to inhibit metal-catalyzed degradation in biologic products is a critical component for solution formulation development. Two metal chelators, disodium edetate (Na(2)EDTA) and diethylenetriaminepentaacetic acid (DTPA), were evaluated for their ability to stabilize IgG2 mAb in solution formulations spiked with various levels of iron. Real-time stability attributes such as oxidation, soluble aggregate formation, deamidation, and fragmentation demonstrated that DTPA was equivalent to Na(2)EDTA with respect to inhibiting iron-induced degradation over the range of iron concentrations studied. When sufficient chelator was present to stoichiometrically complex trace iron contamination, both Na(2)EDTA and DTPA exhibited the capacity to reduce protein degradation. However, substoichiometric ratios of both chelators were unable to inhibit the degradation induced by free iron ions, which were found to bind weakly to the mAb. This bound iron did not measurably alter the secondary or the tertiary structure of the mAb but appeared to decrease its intrinsic thermodynamic stability, probably by causing subtle perturbations in the tertiary structure. These destabilization effects were not observed when the chelators were present at stoichiometric ratios highlighting the feasibility of using DTPA as an alternate trace metal chelator to Na(2)EDTA in biologic protein formulations.