Real-time monitoring of biopharmaceutical crystallization: chord length distribution to crystal size distribution for lysozyme, rHu insulin and vitamin B12 (original) (raw)

High Resolution Imaging as a Characterization Tool for Biological Crystals

Annals of the New York Academy of Sciences, 2004

A BSTRACT : Biomolecular crystals consist of large unit cells that form a rather flexible medium that is able to accommodate a certain degree of lattice distortion, leading to several interesting issues ranging from structural to physical properties. Several techniques, from X-ray diffraction to microscopy, have been adapted to study the structural and physical properties of biomolecular crystals systematically. The use of synchrotron-based monochromatic X-ray diffraction topography, with triple axis diffractometry and rocking curve measurements, to characterize biomolecular crystals is reviewed. Recent X-ray diffraction images from gel and solution grown lysozyme crystals are presented. Defect structures in these crystals are discussed, together with reciprocal space mapping, and compared with results obtained from crystals grown in a low gravity environment.

In situ monitoring of pharmaceutical crystallisation

2012

Using confocal Raman spectroscopy/microscopy, we have monitored pharmaceutical crystallisation 'in situ' in three model (well characterised polymorphic systems) Active Pharmaceutical Ingredients (APIs) and one previously unstudied system where polymorphism had not being reported prior to this study: flufenamic acid, a Non Steroidal Non-Inflammatory Drug (NSAID); nifedipine, an antihypertensive; tolbutamide, used in the treatment of type II diabetes; and imipramine hydrochloride, an antidepressant respectively. Constrained crystallisation from the solid amorphous state was utilised to kinetically trap polymorphs via the Ostwald's rule of stages. Particular emphasis was placed on the phonon-mode/low wavenumber region (4-400cm-1) of the Raman spectral window (this region provides useful information about lattice environment). In all cases our results from the Raman experiments were complemented with similar experiments using Differential Scanning Calorimetry (DSC) and Varia...

Protein crystal quality studies using rod-shaped crystals

Journal of Crystal Growth, 1996

Lysozyme single crystals were grown into X-ray capillaries to a size larger than the capillary diameter thus filling it. The two ends of the same crystal grow at different rates, the difference being at least one order of magnitude. These rod-shaped crystals allow ideal diffraction experiments to test crystal quality as a function of the growth rate. In situ X-ray diffraction experiments were carded out using the capillary where the crystal grew. Oscillation pictures yield different values of the maximum resolution level, ranging from 2.5 to 1.2 A for the opposite ends of the crystal suggesting a large influence of growth rate on protein crystal quality. * Corresponding

Fully automatic characterization and data collection from crystals of biological macromolecules

Acta crystallographica. Section D, Biological crystallography, 2015

Considerable effort is dedicated to evaluating macromolecular crystals at synchrotron sources, even for well established and robust systems. Much of this work is repetitive, and the time spent could be better invested in the interpretation of the results. In order to decrease the need for manual intervention in the most repetitive steps of structural biology projects, initial screening and data collection, a fully automatic system has been developed to mount, locate, centre to the optimal diffraction volume, characterize and, if possible, collect data from multiple cryocooled crystals. Using the capabilities of pixel-array detectors, the system is as fast as a human operator, taking an average of 6 min per sample depending on the sample size and the level of characterization required. Using a fast X-ray-based routine, samples are located and centred systematically at the position of highest diffraction signal and important parameters for sample characterization, such as flux, beam s...

CLSM as Quantitative Method to Determine the Size of Drug Crystals in a Solid Dispersion

Pharmaceutical Research, 2011

Purpose To test whether confocal laser scanning microscopy (CLSM) can be used as an analytical tool to determine the drug crystal size in a powder mixture or a crystalline solid dispersion. Methods Crystals of the autofluorescent drug dipyridamole were incorporated in a matrix of crystalline mannitol by physical mixing or freeze-drying. Laser diffraction analysis and dissolution testing were used to validate the particle size that was found by CLSM. Results The particle size of the pure drug as determined by laser diffraction and CLSM were similar (D 50 of approximately 22 μm). CLSM showed that the dipyridamole crystals in the crystalline dispersion obtained by freeze-drying of less concentrated solutions were of sub-micron size (0.7 μm), whereas the crystals obtained by freeze-drying of more concentrated solutions were larger (1.3 μm). This trend in drug crystal size was in agreement with the dissolution behavior of the tablets prepared from these products. Conclusion CLSM is a useful technique to determine the particle size in a powder mixture. Furthermore, CLSM can be used to determine the drug crystal size over a broad size distribution. A limitation of the method is that the drug should be autofluorescent.

Measuring Size Distribution of Organic Crystals of Different Shapes Using Different Technologies

Particle & Particle Systems Characterization, 2006

The ultrasonic attenuation (UA) technique was developed to measure not only particle size distribution (PSD) but also concentration for on-line and off-line analysis of high concentration dispersions. Another significant characteristic along with particle size is particle shape. Particle shape influences the apparent particle size as measured by various instruments. Shape factors have been calculated from microscopic images using conventional image analysis (IA) techniques. Particle sizing data measured by UA is more representative than by IA because much larger samples are used for analysis. In this study UA, laser diffraction (LD) and IA size measurements are compared and the influence of particle shape is investigated. Data obtained from UA has been combined with information on shape factor from imaging to obtain equivalent particle size distributions. In a previous study , the influence of shape on size measurement has been investigated using different sizing techniques for non-fragile materials. These particles showed significant variation in measured PSD with their different shapes. In this paper, the same phenomenon was observed with evolving polymorphs of L-glutamic acid crystals (a and b form), oxalic acid, sucrose and monosodium glutamate.

Adaptation of in-situ microscopy for crystallization processes

Journal of Crystal Growth, 2009

In biotechnological and pharmaceutical engineering, the study of crystallization processes gains importance. An efficient analytical inline sensor could help to improve the knowledge about these processes in order to increase efficiency and yields. The in-situ microscope (ISM) is an optical sensor developed for the monitoring of bioprocesses. A new application for this sensor is the monitoring in downstream processes, e.g. the crystallization of proteins and other organic compounds. This contribution shows new aspects of using in-situ microscopy to monitor crystallization processes. Crystals of different chemical compounds were precipitated from supersaturated solutions and the crystal growth was monitored. Exemplified morphological properties and different forms of crystals could be distinguished on the basis of offline experiments. For inline monitoring of crystallization processes, a special 0.5 L stirred tank reactor was developed and equipped with the insitu microscope. This reactor was utilized to carry out batch experiments for crystallizations of Oacetylsalicyclic acid (ASS) and hen egg white lysozyme (HEWL). During the whole crystallization process, the in-situ microscope system acquired images directly from the crystallization broth. For the data evaluation, an image analysis algorithm was developed and implemented in the microscope analysis software.

Optimization of crystallization conditions for biological macromolecules

Acta crystallographica. Section F, Structural biology communications, 2014

For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection. Even when the initial samples are suitable, often marginally, refinement of conditions is recommended in order to obtain the highest quality crystals that can be grown. The quality of an X-ray structure determination is directly correlated with the size and the perfection of the crystalline samples; thus, refinement of conditions should always be a primary component of crystal growth. The improvement process is referred to as optimization, and it entails sequential, incremental changes in the chemical parameters tha...