Raman spectroscopy as a process analytical technology to investigate biopharmaceutical freeze concentration processes.

Freezing processes are a well-established unit operation in the biopharmaceutical industry to increase the shelf-life of protein-based drugs. While freezing reduces degradation reaction rates, it may also exert stresses such as freeze concentration. Macroscopic freeze concentration in large-scale freezing processes has been described thoroughly by examination of frozen bulk material, but the transient process leading to such freeze concentration profiles has not been monitored yet for biopharmaceutical solutions. In this study, Raman spectroscopy as a process analytical technology is demonstrated for model formulations containing monoclonal antibodies (mAbs) or bovine serum albumin (BSA) in varying concentrations of sucrose and buffer salts. Therefore, a Raman probe was immersed into a bulk volume at different heights, monitoring the freeze concentration in the liquid phase during the freezing processes. Partial least square regression models were used to quantitatively discriminate between the protein and excipients simultaneously. The freeze concentration profiles were dependend on freezing temperature and formulation with freeze concentrations up to 2.4-fold. Convection currents at the bottom of the freezing container were observed with a maximum height of 1 mm. Furthermore, freeze concentration was correlated with the sucrose concentration in a formulation. Analysis of the freeze concentration slope indicated diffusion from the bottom to the top of the container. In summary, Raman spectroscopy is a valuable tool for process validation of freeze concentration simulations and to overcome scale-dependent challenges.

Rituximab versus mitoxantrone: comparing effectiveness and safety in advanced relapsing multiple sclerosis.

BACKGROUND: Rituximab (RTX), a CD20 depleting agent, is a frequently used off-label treatment for multiple sclerosis (MS), while mitoxantrone (MTX) is approved, albeit rarely used for active relapsing MS (RMS). However, observational data comparing RTX and MTX effectiveness and safety are scarce. OBJECTIVE: We aimed to compare effectiveness and safety of MTX and RTX in patients with active RMS. METHODS: From combined retrospective clinical data of three MS centers, we selected patients who had received at least one infusion of RTX or MTX and had at least a 6-month clinical follow-up available. Treatment groups were compared by propensity score (PS)-adjusted regression and inverse PS-weighted generalized estimated equation models regarding disability progression, relapse activity, and adverse events (AEs). RESULTS: We included 292 RMS patients (mean age 41.8 years, 71.6% female) who received RTX (119 patients, mean age 36.8 years, 74.8% female) or MTX (173 patients mean age 45.3 years, 69.4% female). Using both PS methods, we did not find a significant effect favoring RTX or MTX treatment regarding the probability of disability worsening or relapse occurrence. However, RTX treatment was associated with a significantly lower probability of severe AEs and AEs. CONCLUSIONS: RTX shows comparable effectiveness but a favorable safety profile compared with MTX in active RMS.

Impact of freeze-thaw processes on monoclonal antibody platform process development.

Freezing of cell culture supernatant (CCS) is a standard procedure in process development of monoclonal antibody (mAb) platform processes as up- and downstream development are usually separated. In the manufacturing process of mAb, however, freezing is avoided, which poses the question of comparability and transferability from process development to manufacturing. In this case study, mAb CCS from Chinese hamster ovary (CHO) cells is frozen and thawed in a novel active freezing device and subsequently captured by protein A chromatography. Critical quality attributes such as host cell protein (HCP) concentration and soluble mAb dimer shares have been monitored throughout the case study. Furthermore, cryo-concentration of individual proteins was investigated. The main factors that drive cryo-concentration are diffusion and natural convection. Natural convection in freezing processes was found to increase at warmer freezing temperatures and thus slower freezing, leading to higher concentration gradients from top to bottom of a freezing chamber. The freeze concentration was dependent on protein size and correlated to diffusivity, where smaller proteins are exposed to higher cryo-concentration. Our results suggest that as a result of freezing processes, large particles based on mAb and specific host cell proteins (HCPs) expressing a certain affinity to mAbs are formed that have to be removed before purification. This leads to a significant improvement in HCP reduction by the protein A step, when compared with reference samples, where twice as much HCP remained in the eluate. Furthermore, HCP and mAb dimer concentrations in protein A eluate were dependent on the freezing temperature. As a conclusion, CCS should be frozen as rapidly as possible during process development to minimize issues of transferability from process development to manufacturing.

Temperature Based Process Characterization of Pharmaceutical Freeze-Thaw Operations.

In biopharmaceutical production processes, freeze-thaw operations are used to ensure product integrity during long hold times, but they also introduce additional stresses such as freeze concentration gradients that might lead to a loss of protein activity. Process characterization of freeze-thaw operations at different scales should be conducted with attention to freezing time and boundary effects to ensure the product stability throughout the process and process development. Currently, process characterization often relies on one or very few temperature probes that detect freezing times based on raw temperature, which is largely influenced by freezing-point depression in case of concentrated solutions. A method to detect freezing based on the second derivative of temperature measurements from Fiber-Bragg-Grating sensors is presented to overcome this issue. The applicability of the method is demonstrated by process characterization of a novel small-scale freeze-thaw device with minimized boundary effects using freezing times of purified water and concentrated formulations. Freezing times varied from 35 to 81 min for temperatures between -60 and -20°C and impacted freeze concentration profiles. Furthermore, freezing time estimations based on the Plank equation revealed model limitations due to start-up temperature gradients, that can be corrected by an empirically extended Plank model. As a hypothesis, we conclude that freezing temperature, from a freeze concentration view, is less important in containers with small characteristic freezing distances such as freeze bags. Using a 2D-resolved temperature profile, a shift of the last point to freeze position from top to bottom of a container was observed when freezing above -30°C.

Soluble Metal Oxo Alkoxide Inks with Advanced Rheological Properties for Inkjet-Printed Thin-Film Transistors.

Semiconductor inks containing an indium-based oxo alkoxide precursor material were optimized regarding rheology requirements for a commercial 10 pL inkjet printhead. The rheological stability is evaluated by measuring the dynamic viscosity of the formulations for 12 h with a constant shear rate stress under ambient conditions. It is believed that the observed superior stability of the inks is the result of effectively suppressing the hydrolysis and condensation reaction between the metal oxo alkoxide precursor complex and atmospheric water. This can be attributed to a strong precursor coordination and the resulting reduction in ligand exchange dynamics of the solvent tetrahydrofurfuryl alcohol which is used as the main solvent in the formulations. It is also shown that with a proper selection of cosolvents, having high polar Hansen solubility parameter values, the inks drop formation properties and wettability can be fine-tuned by maintaining the inks rheological stability. Good drop jetting performance without satellite formation and high drop velocities of 8.25 m/s were found with the support of dimensionless numbers and printability windows. By printing single 10 pL ink dots onto short channel indium-tin-oxide electrodes, In2O3 calcination at 350 °C and a solution-processed back-channel protection, high average saturation mobility of approximately 10 cm2/(V s) are demonstrated in a bottom-contact coplanar thin-film transistor device structure.

Size distribution analysis of influenza virus particles using size exclusion chromatography.

Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity.

Mono- and polyprotic buffer systems in anion exchange chromatography of influenza virus particles.

Different ions typically used in downstream processing of biologicals are evaluated for their potential in anion exchange chromatography of an industrially produced, pandemic influenza H1N1 virus. Capacity, selectivity and recovery are investigated based on single step elution parallel chromatography experiments. The inactivated H1N1 feedstream is produced in Madin-Darby Bovine Kidney cells. Interesting effects are found for sodium phosphate and sodium citrate. Both anions are triprotic kosmotropes. Anion exchange chromatography generally offers high scalability to satisfy sudden demands for vaccines, which may occur in case of an emerging influenza outbreak. Appropriate pH conditions for H1N1 adsorption are determined by Zeta potential measurements. The dynamic binding capacity of a salt tolerant polyamine-type resin is up to 6.4 times greater than the capacity of a grafted Q-type resin. Pseudo-affinity interactions of polyamines with the M2 protein of influenza may contribute to the obtained capacity increase. Both resins achieve greater capacity in sodium phosphate buffer compared to Tris/HCl. A recovery of 67% and DNA clearance close to 100% without DNAse treatment are achieved for the Q-type resin. Recovery of the virus from the salt tolerant resin requires the use of polyprotic acids in the elution buffer. 85% of the DNA and 60% of the proteins can be removed by the salt tolerant resin. The presence of sodium phosphate during anion exchange chromatography seems to support stability of the H1N1 particles in presence of hydrophobic cations.

Insights into Interprotein Electron Transfer of Human Cytochrome c Variants Arranged in Multilayer Architectures by Means of an Artificial Silica Nanoparticle Matrix.

The redox behavior of proteins plays a crucial part in the design of bioelectronic systems. We have demonstrated several functional systems exploiting the electron exchange properties of the redox protein cytochrome c (cyt c) in combination with enzymes and photoactive proteins. The operation is based on an effective reaction at modified electrodes but also to a large extent on the capability of self-exchange between cyt c molecules in a surface-fixed state. In this context, different variants of human cyt c have been examined here with respect to an altered heterogeneous electron transfer (ET) rate in a monolayer on electrodes as well as an enhanced self-exchange rate while being incorporated in multilayer architectures. For this purpose, mutants of the wild-type (WT) protein have been prepared to change the chemical nature of the surface contact area near the heme edge. The structural integrity of the variants has been verified by NMR and UV-vis measurements. It is shown that the single-point mutations can significantly influence the heterogeneous ET rate at thiol-modified gold electrodes and that electroactive protein/silica nanoparticle multilayers can be constructed with all forms of human cyt c prepared. The kinetic behavior of electron exchange for the mutant proteins in comparison with that of the WT has been found altered in some multilayer arrangements. Higher self-exchange rates have been found for K79A. The results demonstrate that the position of the introduced change in the charge situation of cyt c has a profound influence on the exchange behavior. In addition, the behavior of the cyt c variants in assembled multilayers is found to be rather similar to the situation of cyt c self-exchange in solution verified by NMR.

An unusual recovery from traumatic brain injury in a young man.

CONTEXT: Traumatic brain injury (TBI), a complex neurological traumatic incident where brain function is disrupted due to physical trauma, can be categorized in multiple ways and is commonly scored using the Glasgow Coma Scale. Severe closed head injury is a form of TBI with a Glasgow Coma Scale less than 8. The outcomes and prognosis are not uniform in the population but mortality is estimated at 30-50 percent. In this case of severe closed head injury, the patient was able to make a near full recovery after several neurosurgery and medical treatments and intercessory prayer to Saint Luigi Guanella. FINDINGS: A 21-year-old male patient received a severe closed head TBI and bilateral hemotympanum while rollerblading without a helmet. After imaging, a left frontal craniotomy and evacuation of epidural and subdural hematomas and resection of a left frontal contusion were performed. Intracranial pressure increased and the patient experienced a transtentorial herniation. He underwent a right frontotemporal and subtemporal craniectomy and evacuation of a frontotemporal subdural hematoma. The patient had intraventricular hemorrhage to which a ventriculostomy was performed and later converted to a ventriculo-peritoneal shunt for recurrent hydrocephalus. The patient was not expected to regain consciousness, but made a recovery after 24 days in the hospital and 10 days in rehabilitation. The patient followed up 6 months after injury for a cranioplasty and soon after returned to near baseline. CONCLUSIONS/CLINICAL RELEVANCE: In this extraordinary case, the severe closed head injury the patient sustained required intensive neurosurgical and medical treatment and the prognosis for recovery of consciousness was very poor; however, with treatment and rehabilitation and intercessory prayer to Saint Luigi Guanella, this patient was able to recover close to baseline from a Glasgow Coma Scale of 7. LAY SUMMARY: Head injuries vary in severity and traumatic brain injuries can be extremely serious leading to bleeding, loss of consciousness, and can affect verbal responses, muscles movement in motor responses, and responses with eye movement. Traumatic brain injuries require medical care to assess the severity and treat the injury. In this case report, we discuss a patient's very severe closed head injury while rollerblading without a helmet from which he was not expected to make a full recovery, but did so following intensive medical treatment, rehabilitation, and intercessory prayer to Saint Don Guanella to combat the initial injury and subsequent issues.