Development and workflow of a continuous protein crystallization process: A case of lysozyme

In the present work, a workflow on the development of a continuous protein crystallization is introduced, with lysozyme as a model protein, from microliter screening experiments, to small scale batch crystallization experiments in a shaking crystallization platform, and to batch and continuous crystallization experiments in an oscillatory flow platform. The lysozyme crystallizations investigated were for a concentration range from 30 to 100 mg/mL, shaking conditions from 100 to 200 rpm in the batch shaking crystallization platform, and oscillatory conditions with amplitude (x 0 ) from 5 to 30 mm and frequency (f) from 0.1 to 1.0 Hz in the batch oscillatory flow crystallization platform. We propose the use of the Reynold's number (R e ) for scaling up of the process from the shaking batch to the continuous oscillatory flow platform. Additionally, it is shown that the nucleation rate increased with increase in concentration of initial lysozyme solution, or increase in shear rate, inducing smaller size of lysozyme crystals. The properties and qualities of the crystal products indicate that continuous crystallization platforms may offer advantages to the downstream bioprocessing of proteins

Selective crystallisation of carbamazepine polymorphs on surfaces with differing properties

Surface-induced nucleation of carbamazepine (CBZ) in ethanol was investigated with different surface materials: glass, polytetrafluoroethylene (PTFE) and tin. The introduction of foreign surfaces with different areas and surface chemistries into the solution has an impact on the crystal morphology and polymorphic form (Form II or III). With an increase in supersaturation, a higher possibility of crystallisation of CBZ metastable Form II was observed, as expected. Increasing the number of inserts resulted in a direct increase in the surface area available for heterogeneous nucleation. The increase in surface area resulted in the greater possibility of obtaining the metastable Form II of CBZ. The stable Form III preferred to nucleate on tin rather than on glass and PTFE. The results indicate that the two different polymorphs of CBZ can be selectively crystallised out from solution with the aid of a foreign surface. The kinetic mechanism of heterogeneous nucleation of the different polymorphs induced by foreign surfaces was discussed. The potential applications will be used to control and design the crystallisation process

Optimization of vapor diffusion conditions for anti-CD20 crystallization and scale-up to meso batch

© 2019, MDPI AG. All rights reserved. The crystal form is one of the preferred formulations for biotherapeutics, especially thanks to its ability to ensure high stability of the active ingredient. In addition, crystallization allows the recovery of a very pure drug, thus facilitating the manufacturing process. However, in many cases, crystallization is not trivial, and other formulations, such as the concentrate solution, represent the only choice. This is the case of anti-cluster of differentiation 20 (anti-CD20), which is one of the most sold antibodies for therapeutic uses. Here, we propose a set of optimized crystallization conditions for producing anti-CD20 needle-shaped crystals within 24 h in a very reproducible manner with high yield. High crystallization yield was obtained with high reproducibility using both hanging drop vapor diffusion and meso batch, which is a major step forward toward further scaling up the crystallization of anti-CD20. The influence of anti-CD20 storage conditions and the effect of different ions on the crystallization processes were also assessed. The crystal quality and the high yield allowed the first crystallographic investigation on anti-CD20, which positively confirmed the presence of the antibody in the crystals

Optimization of vapor diffusion conditions for anti-CD20 crystallization and scale-up to meso batch

© 2019, MDPI AG. All rights reserved. The crystal form is one of the preferred formulations for biotherapeutics, especially thanks to its ability to ensure high stability of the active ingredient. In addition, crystallization allows the recovery of a very pure drug, thus facilitating the manufacturing process. However, in many cases, crystallization is not trivial, and other formulations, such as the concentrate solution, represent the only choice. This is the case of anti-cluster of differentiation 20 (anti-CD20), which is one of the most sold antibodies for therapeutic uses. Here, we propose a set of optimized crystallization conditions for producing anti-CD20 needle-shaped crystals within 24 h in a very reproducible manner with high yield. High crystallization yield was obtained with high reproducibility using both hanging drop vapor diffusion and meso batch, which is a major step forward toward further scaling up the crystallization of anti-CD20. The influence of anti-CD20 storage conditions and the effect of different ions on the crystallization processes were also assessed. The crystal quality and the high yield allowed the first crystallographic investigation on anti-CD20, which positively confirmed the presence of the antibody in the crystals

General guide to the exhibition halls of the American Museum of Natural History, 1930.

The synthesis and characterization of three barium coordination polymers with one-, two-, and three-dimensional (1-D, 2-D, 3-D) inorganic connectivity based on biphenyl carboxylic acid ligands are described. Employing biphenyl-3,3′,5,5′-tetracarboxylic acid (H₄BTTC) as a ligand, [Ba₂(BTTC)­(H₂O)₂]_<i>n</i> (<b>1</b>, space group = <i>Pn</i>2₁<i>a</i>, <i>a</i> = 7.059(1) Å, <i>b</i> = 12.432(2) Å, <i>c</i> = 19.090(3) Å), a coordination polymer with 1-D inorganic connectivity (I¹O²), can be synthesized. The coordinated water is strongly coordinated and removed at 270 °C. By using 4,4′-biphenyldicarboxylic acid (H₂BPDC), [Ba­(BPDC)]_<i>n</i> (<b>2</b>, space group = <i>C</i>2/<i>m</i>, <i>a</i> = 6.955(2) Å, <i>b</i> = 5.947(1) Å, <i>c</i> = 13.852 (4) Å, β = 92.399(4)°) a coordination polymer with 2-D inorganic connectivity (I²O¹) is obtained. The connection of the Ba–O bonds in each layer is topologically similar to CaF₂. Using biphenyl-3,5,5′-tricarboxylic acid (H₃BPTC) as a ligand, [Ba₃(BPTC)₂(NMF)₅⊃2NMF]_<i>n</i> (<b>3</b>, space group = <i>I</i>4̅2<i>d</i>, <i>a</i> = 25.984(3) Å, <i>c</i> = 13.999(2) Å) (NMF = <i>N</i>-methyl formamide), a structurally porous coordination polymer with rare 3-D inorganic connectivity (I³O⁰) can be synthesized. Hence, barium as a metal is extremely malleable with respect to construction of coordination polymers of different inorganic dimensionalities. <b>2</b> with I²O¹ connectivity demonstrates extraordinary thermal stability and maintains its crystallinity until decomposition at 590 °C. The luminescence behavior of <b>1</b>, <b>2</b>, and <b>3</b> at room temperature has been investigated and is predominantly intraligand based

Protein crystal occurrence domains in selective protein crystallisation for bio-separation

Bio-separation is a key bottleneck in the manufacture of biopharmaceuticals. In this work, we report experimental evidence of direct selective protein crystallisation from a binary protein mixture solution. Lysozyme–thaumatin mixtures with a wide protein composition range (0–100 mg mL−1, respectively) were tested under the same crystallisation cocktail conditions using the hanging-drop vapour-diffusion (HDVD) crystallisation method. This work demonstrates the selectivity of crystallisation from a model binary protein mixture and four crystal occurrence domains were determined as the operation windows of selective crystallisation of the target protein: 1) an unsaturated region with no crystal formation, 2 & 3) target regions with only a single type of protein crystals (lysozyme crystals only or thaumatin crystals only) and 4) a mixture region which have a mixture of both types of protein. This study demonstrates that protein crystallisation is not only applicable to high-purity protein solutions and emphasizes the vital impacts of the presence of protein impurities in the process of target protein crystallisation. The study concludes that protein crystallisation is a feasible approach to separate a target protein from a complex mixture environment which can be achieved by manipulating the crystallisation operation conditions such as mixture composition, precipitant concentration, and operation time.</p

Novel Globular Polymeric Supports for Membrane-Enhanced Peptide Synthesis

Membrane-enhanced peptide synthesis (MEPS), a technique that combines liquid-phase peptide synthesis (LPPS) with organic solvent nanofiltration (OSN), has emerged as a new methodology to tackle current challenges in solid-phase peptide synthesis (SPPS), the current strategy of choice for the preparation of peptides. This new technology platform is scalable beyond kilogram scale, automatable, and compatible with established Fmoc chemistry, as it combines chemistry in solution with expedient membrane purification. Here we screened novel highly rejected soluble polymeric supports and studied their application for the preparation of a model peptide. Our findings make a significant contribution to the development of MEPS

Metal-Mediated Controllable Creation of Secondary, Tertiary, and Quaternary Carbon Centers: A Powerful Strategy for the Synthesis of Iron, Cobalt, and Copper Complexes with in Situ Generated Substituted 1‑Pyridineimidazo[1,5‑<i>a</i>]pyridine Ligands

An efficient strategy for the synthesis of a wide variety of coordination complexes has been developed. The synthetic protocol involves a solvothermal in situ metal–ligand reaction of picolinaldehyde, ammonium acetate, and transition-metal ions, leading to the generation of 12 coordination complexes supported by a novel class of substituted 1-pyridineimidazo­[1,5-<i>a</i>]­pyridine ligands (<b>L1</b>–<b>L5</b>). The ligands <b>L1</b>–<b>L5</b> were afforded by metal-mediated controllable conversion of the aldehyde group of picolialdehyde into a ketone and secondary, tertiary, and quaternary carbon centers, respectively. Complexes of various nuclearities were obtained: from mono-, di-, and tetranuclear to 1D chain polymers. The structures of the in situ formed complexes could be controlled rationally via the choice of appropriate starting materials and tuning of the ratio of the starting materials. The plausible mechanisms for the formation of the ligands <b>L1</b>–<b>L5</b> were proposed

Metal-Mediated Controllable Creation of Secondary, Tertiary, and Quaternary Carbon Centers: A Powerful Strategy for the Synthesis of Iron, Cobalt, and Copper Complexes with in Situ Generated Substituted 1‑Pyridineimidazo[1,5‑<i>a</i>]pyridine Ligands

An efficient strategy for the synthesis of a wide variety of coordination complexes has been developed. The synthetic protocol involves a solvothermal in situ metal–ligand reaction of picolinaldehyde, ammonium acetate, and transition-metal ions, leading to the generation of 12 coordination complexes supported by a novel class of substituted 1-pyridineimidazo­[1,5-<i>a</i>]­pyridine ligands (<b>L1</b>–<b>L5</b>). The ligands <b>L1</b>–<b>L5</b> were afforded by metal-mediated controllable conversion of the aldehyde group of picolialdehyde into a ketone and secondary, tertiary, and quaternary carbon centers, respectively. Complexes of various nuclearities were obtained: from mono-, di-, and tetranuclear to 1D chain polymers. The structures of the in situ formed complexes could be controlled rationally via the choice of appropriate starting materials and tuning of the ratio of the starting materials. The plausible mechanisms for the formation of the ligands <b>L1</b>–<b>L5</b> were proposed