On-demand manufacturing of clinical-quality biopharmaceuticals

Conventional manufacturing of protein biopharmaceuticals in centralized, large-scale, single-product facilities is not well-suited to the agile production of drugs for small patient populations or individuals. Previous solutions for small-scale manufacturing are limited in both process reproducibility and product quality, owing to their complicated means of protein expression and purification. We describe an automated, benchtop, multiproduct manufacturing system, called Integrated Scalable Cyto-Technology (InSCyT), for the end-to-end production of hundreds to thousands of doses of clinical-quality protein biologics in about 3 d. Unlike previous systems, InSCyT includes fully integrated modules for sustained production, efficient purification without the use of affinity tags, and formulation to a final dosage form of recombinant biopharmaceuticals. We demonstrate that InSCyT can accelerate process development from sequence to purified drug in 12 weeks. We used integrated design to produce human growth hormone, interferon α-2b and granulocyte colony-stimulating factor with highly similar processes on this system and show that their purity and potency are comparable to those of marketed reference products

High temperature creep performance and microstructure of SiC-C-SiC composites

The high temperature creep behaviour of a 2D SiC/C/SiC composite is studied at temperatures ranging from 1200 to 1673K, under stresses from 50 to 500MPa. Due to the microstructural evolution of the material, steady state creep is never reached ; creep toughening is observed. Microcrack formation and propagation in the interface and in the matrix were identified as contributing to the deformation

Impurities in silicon carbide ceramics and their role during high temperature creep

The high-temperature compressive creep behaviour of hot-pressed silicon carbide ceramics with different additive packages (boron and carbon or no additive) is investigated as a function of several parameters: the microstructure, the nature of the additives and that of the impurities. Additional carbon is present in all the materials investigated, as graphite precipitates of various size and amount. In materials densified with addition of boron, large precipitates of B$_{25}$C and small amorphous silica pockets are identified. In the case of materials containing impurities, small precipitates of FeSi, Fe or Ti$_5$Si$_3$ are detected. Creep experiments are conducted on materials with no additives and on others containing boron and carbon additives, at temperatures ranging from 1 773 K to 1 973 K and under stresses from 100 to 1 100 MPa. A comparison of the creep behaviour of the various materials points out to the destructive effect of carbon precipitates on the creep rate: the stationary creep rate of the material containing carbon (and boron) additives is by a factor 2.5-5 faster, eventhough its grain size is much larger! The creep of both investigated materials is described by a power law with a stress exponent of 1.5 in a low stress range and 3.5-4 in a high stress range. The corresponding activation energies are 364 kJ/mole and 453 kJ/mole in the low stress range and about 629 kJ/mole in the high stress range. At low stresses the materials deform by grain boundary sliding compensated mainly by diffusion along the grain boundaries and to a lesser extent by limited cavitation, as a result of the barrier role played by grain boundaries for dislocations. At high stresses the grain boundaries are no longer an obstacle to dislocation motion, which becomes the dominant deformation mechanism.La microstructure de deux céramiques, SiC sans ajouts et SiC avec bore et carbone, est étudiée par microscopie électronique à transmission dans le but d'évaluer l'influence des additifs sur les propriétés mécaniques à haute température. Dans tous les matériaux, des précipités de graphite de différentes tailles sont observés. Le carbure de silicium fabriqué avec du bore contient des grands précipités de B$_{25}$C et des petites poches de silice amorphe. A partir de nos observations de la microstructure, une prévision des propriétés mécaniques des matériaux est possible. Ces prévisions sont comparées aux résultats de fluage à haute température. Les matériaux sans ajouts et ceux avec carbone et bore sont déformés entre 1 773 K et 1 973 K, sous des contraintes de 100 à 1 100 MPa. Le comportement des deux matériaux suit une loi de puissance avec un exposant de contrainte de 1,5 pour les faibles contraintes et de 3,5-4 pour les fortes contraintes. Les valeurs d'énergie d'activation des deux types de matériaux sont respectivement 364 et 453 kJ/mole dans le domaine de faibles contraintes et 629 kJ/mole aux fortes contraintes. L'observation de la microstructure des matériaux déformés montre comme mécanisme principal de fluage le glissement aux joints de grains accommodé par la diffusion et accompagné par une faible cavitation due à la non-perméabilité des joints de grains pour les dislocations. Aux plus fortes contraintes, les joints de grains deviennent plus perméables, et la déformation par mécanismes de dislocations devient alors prépondérante

Integrated Bottom-Up and Top-Down Liquid Chromatography–Mass Spectrometry for Characterization of Recombinant Human Growth Hormone Degradation Products

With the advent of biosimilars to the U.S. market, it is important to have better analytical tools to ensure product quality from batch to batch. In addition, the recent popularity of using a continuous process for production of biopharmaceuticals, the traditional bottom-up method, alone for product characterization and quality analysis is no longer sufficient. Bottom-up method requires large amounts of material for analysis and is labor-intensive and time-consuming. Additionally, in this analysis, digestion of the protein with enzymes such as trypsin could induce artifacts and modifications which would increase the complexity of the analysis. On the other hand, a top-down method requires a minimum amount of sample and allows for analysis of the intact protein mass and sequence generated from fragmentation within the instrument. However, fragmentation usually occurs at the N-terminal and C-terminal ends of the protein with less internal fragmentation. Herein, we combine the use of the complementary techniques, a top-down and bottom-up method, for the characterization of human growth hormone degradation products. Notably, our approach required small amounts of sample, which is a requirement due to the sample constraints of small scale manufacturing. Using this approach, we were able to characterize various protein variants, including post-translational modifications such as oxidation and deamidation, residual leader sequence, and proteolytic cleavage. Thus, we were able to highlight the complementarity of top-down and bottom-up approaches, which achieved the characterization of a wide range of product variants in samples of human growth hormone secreted from <i>Pichia pastoris</i>