Fully integrated downstream process to enable next-generation manufacturing.

Next-generation manufacturing (NGM) has evolved over the past decade to a point where large biopharmaceutical organizations are making large investments in the technology and considering implementation in clinical and commercial processes. There are many well-considered reasons to implement NGM. For the most part, organizations will not fund NGM unless the implementation benefits the funding organization by providing reduced costs, reduced time, or additional needed capabilities. Productivity improvements gained from continuous purification are shown in this work, which used a new system that fully integrates and automates several downstream unit operations of a biopharmaceutical process to provide flexibility and easy implementation of NGM. The equipment and automation needed to support NGM can be complicated and expensive. Biopharmaceutical Process Development considered two options as follows: (1) design its own NGM system or (2) buy a prebuilt system. PAK BioSolutions offers a turn-key automated and integrated system that can operate up to four continuous purification stages simultaneously, while maintaining a small footprint in the manufacturing plant. The system provides significant cost benefits (~10× lower) compared with the alternative-integration of many different pieces of equipment through a Distributed Control System that would require significant engineering time for design, automation, and integration. Integrated and Continuous Biomanufacturing can lead to significant reductions in facility size, reduced manufacturing costs, and enhanced product quality when compared with the traditional batch mode of operation. The system uses new automation strategies that robustly link unit operations. We present the optimized process fit, sterility and bioburden control strategy, and automation features (such as pH feedback control and in-line detergent addition), which enabled continuous operation of a 14-day end-to-end monoclonal antibody purification process at the clinical manufacturing scale.

End-to-end collaboration to transform biopharmaceutical development and manufacturing.

An ambitious 10-year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end-to-end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program.

Integration of rapid bioburden testing into production quality management systems and process control.

The move to integrated continuous bioprocessing (ICB), while providing a means for process intensification, can put added strain on process analytics when conventional methods are used. For instance, traditional microbial methods provide minimal value to ICB processes given that the time required for data to become available is much longer than a typical full cycle of the manufacturing process. Although rapid microbial detection has been in discussion for over 30 years, it is still not routinely deployed in commercial biopharmaceutical manufacturing. One contributing factor is the ability to integrate this technology into a process control strategy and existing quality systems. An understanding of the capability of microbial detection technology available today can be leveraged to implement a control strategy for bioburden monitoring in real time for process intermediates. One key tenet of this proposed control strategy is the use of a "two-tiered approach" wherein a fast (but possibly less sensitive) test is used to monitor the process and trigger further action for a second, longer duration test which is used to confirm and quantify the presence of bioburden and identify the organism. This approach, presented here alongside several case studies for microbial monitoring, can have broader application for other process analytical technologies where fit for purpose methods could be employed to establish process control alongside real time continuous processes.

Kinetic study of beta-amyloid residue accessibility using reductive alkylation and mass spectrometry.

Beta-amyloid peptide (Abeta) is the major protein constituent found in senile plaques in Alzheimer's disease (AD). It is believed that Abeta plays a role in neurodegeneration associated with AD and that its toxicity is related to its structure or aggregation state. In this study, an approach based on chemical modification of primary amines and mass spectrometric (MS) detection was used to identify residues on Abeta peptide that were exposed or buried upon changes in peptide structure associated with aggregation. Results indicate that the N terminus was the most accessible primary amine in the fibril, followed by lysine 28, then lysine 16. A kinetic analysis of the data was then performed to quantify differences in accessibility between these modification sites. We estimated apparent equilibrium unfolding constants for each modified site of the peptide, and determined that the unfolding constant for the N terminus was approximately 100 times greater than that for K28, which was about six times greater than that for K16. Understanding Abeta peptide structure at the residue level is a first step in designing novel therapies for prevention of Abeta structural transitions and/or cell interactions associated with neurotoxicity in Alzheimer's disease.

The effect of new compounds in stabilizing downstream monoclonal antibody (mAb) process intermediates.

Determining the stability of downstream process (DSP) intermediates is an extremely important parameter used to maintain product quality attributes within their acceptance ranges. The IgG4 monoclonal antibody studied (mAb1) showed aggregation under acidic conditions, inhibiting the use of low pH treatment to inactivate endogenous retroviruses, and poor virus filtration performance. Both manufacturing steps are included in mAb DSP for viral clearance. The impact of several new compounds on the aggregation and stabilization of mAb1 in process intermediate pools encountered during these critical DSP steps was investigated. Results showed that, in the presence of a protein stabilizer at pH 3.2, 27% less aggregation was observed compared to controls, during the low pH treatment for viral inactivation. The impact of a novel protein stabilizer on virus filter throughput during mAb1 filtration was compared to L-arginine using an innovative high-throughput automation technique. Compared to control experiments without additives, conditions were found where a 70% increase in filter volumetric throughput was achieved in the presence of the novel stabilizer, and a 56% decrease in volumetric throughput observed with L-arginine. These findings present the possibility of using these novel compounds to stabilize proteins during DSP and permitting the use of platform DSP elements such as low pH treatment and high-throughput virus filtration to challenging and unstable proteins.

A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins.

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.

Hematemesis With Gastric Laceration After Tattooing a Polyp With Purified Carbon: A Review of the Literature.

Endoscopic tattooing is a simple and effective technique for marking small lesions, so they can be localized during surgery or in later endoscopies. Various agents can be used such as India ink or a solution of purified carbon particles. The number of complications from tattooing is relatively small, but not rare. The majority of the literature on the subject refers to complications in the colon. We present a case of gastric bleeding secondary to a laceration following tattooing with purified carbon, and a literature review.

Efecto antiulcerso de formulas que contienen un extracto de Aloe vera L. (Sabila) / Antiulcerous effect of formulas containing extract of Aloe vera L. (sabila)

Se estudio el efecto de formulas que contenian un extracto acuoso de Aloe vera L. sobre las lesiones de la mucosa gastrica de ratas, producidas por los modelos experimentales de estres, etanol e indometacina. Se usaron tres formulas que contenian un extracto de la planta en concentraciones de 12,5; 25 y 50 porciento, respectivamente, en un vehiculo en forma de gel. Se usaron cinco grupos de tratamiento que recibieron, por via oral, cada una de las formulas en dosis que correspondienron a 3,6; 7,4 y 14,6 mg del material vegetal/kg de peso, respectivamente, durante cinco dias; un grupo control que recibio el vehiculo solamente y un grupo que recibio agua comun. Se determino tambien el efecto de la formula que contenia el extracto al 50 porciento sobre la secrecion acida basal y sobre la generacion de prostaglandinas (PGE2 y 6-keto-PGF1) en la mucosa gastrica. De las formulas probadas, solo la que contenia el extracto al 50 porciento disminuyeron significativamente el numero y la severidad de las lesiones gastricas inducidas por los tres agentes ulcerogenos, sin afectar la secrecion acida. Esta formula tampoco afecto la generacion mucosal de prostaglandinas. Se concluye que la formula con extracto de Aloe vera al 50 porciento podria constituir una laternativa terapeutica en el tratamiento de la ulcera gastroduodenal y que su accion gastroprotectora parece ser independiente de la secrecion de acido y de la generacion de prostaglandinas en la mucosa gastrica

Bioequivalencia y farmacodinamia de 2 formulaciones de quinidina de liberación prolongada en perros / Bioequivalence and pharmacodynamics of 2 sustained-release quinidine formulations in dogs

Se comprobó la bioequivalencia y los efectos farmacodinámicos de 2 formulaciones de quinidina, tabletas de liberación prolongada desarrollada en el CIDEM (IMEFA, Cuba). Ambas contenían 166 mg de la sustancia biológicamente activa en forma de base. En el estudio se utilizaron perros beagles (n=6), siguiendo un diseño aleatorio cruzado. No se detectaron diferencias significativas entre las concentraciones máximas, el tiempo al que se alcanza la concentración máxima y las áreas bajo la curva, lo cual permite plantear que ambas formulaciones desde el punto de vista farmacocinético son bioequivalentes

Comparación farmacocinética de 2 formulaciones de quinidina en voluntarios sanos / Comparison of pharmacokinetics of 2 quinidine formulations in healthy volunteers

Se realizó un estudio comparativo del curso temporal de las concentraciones plasmáticas de quinidina en voluntarios sanos, y se determinaron los parámetros farmacocinéticos fundamentales en tabletas convencionales de quinidina sulfato y tabletas de liberación prolongada elaboradas a partir de un complejo polimetacrilato-quinidina. Se concluyó que resultó adecuado al régimen de dosificación de 2 tabletas de quinidina de liberación prolongada cada 12 horas para lograr un perfil de concentración plasmática vs. tiempo, característico de este tipo de formulación, con el valor de tiempo de vida media de eliminación de 12,8 horas en comparación con 4,5 horas que presentó la tableta convencional de sulfato de quinidina