Bulk Protein Crystallization

A special issue of Crystals (ISSN 2073-4352). This special issue belongs to the section "Biomolecular Crystals".

Deadline for manuscript submissions: closed (21 May 2021) | Viewed by 9541

Special Issue Editors

Faculty of Engineering, University of Porto, Porto, Portugal
Interests: crystallization/precipitation processes; protein crystallization; calcium phosphates for biomedical applications; multiphase reactors; materials characterization
Karlsruhe Institute of Technology, Karlsruhe, Germany
Interests: suspension crystallization; industrial crystallization; preparative protein crystallization; pharmaceutical crystallization; precipitation; product design

Special Issue Information

Dear Colleagues,

In the last decade, protein-based therapy has emerged as a safe and powerful tool for the administration of biopharmaceuticals. However, its manufacturing process, in particular the purification stage, remains costly. Unlike conventional protein purification techniques (i.e., chromatography), protein crystallization requires no costly equipment or consumables (e.g., resins) and can yield highly concentrated slurries of pure protein ready for further formulation in a single step.

Although its viability has been demonstrated, protein crystallization remains a largely empirical process due to the complexity of the underlying mechanisms and the unavailability of generalized crystallization strategies and scale-up criteria.

This is the motivation for this Special Issue, "Bulk Protein Crystallization ", which will gather the latest achievements in the search for better control and more rational design of protein crystallization processes. To this is added the recent advances in in situ monitoring and modelling. Studies on the influence of fluid dynamics on the process and crystal quality as well as on the specific crystallization of a given protein from a more realistic matrix (e.g., protein mixtures, clarified concentrated fermentation broths) are also crucial. 

With the contribution of experts from chemical engineering, biotechnology and biochemistry, mathematics, and physics, this Special Issue is intended to contribute with robust and scalable protein crystallization strategies to boost its implementation in the pharmaceutical and biotechnological industry.

Reports on the following topics are welcome:

- Strategies to enhance the crystallization efficiency and decrease the process time;
- Strategies to enable the rapid establishment of phase diagrams;
- Advanced imaging, spectroscopy, and light scattering techniques;
- Modelling of protein crystallization processes (population balance modelling, etc.);
- Crystallization from “impure” protein solutions;
- Influence of hydrodynamics (mixing, shear, etc.) on protein crystallization.

Dr. Filipa Castro
Prof. Dr. Matthias Kind
Guest Editors

Manuscript Submission Information

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Keywords

  • Protein crystallization
  • Bioseparation/downstream processing
  • Systematic crystallization strategies
  • In situ monitoring
  • Hydrodynamics
  • Modelling

Published Papers (3 papers)

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Research

14 pages, 4924 KiB  
Article
Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases
Crystals 2021, 11(8), 975; https://doi.org/10.3390/cryst11080975 - 17 Aug 2021
Cited by 4 | Viewed by 2929
Abstract
Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected [...] Read more.
Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of Lactobacillus brevis (LbADH) to Lactobacillus kefir (LkADH). Proteins were expressed in E. coli and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of LkADH wild type compared to LbADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of LkADH mutants with the wild type, the mean protein crystal size of LkADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the LkADH variants studied results in an order of crystallizability T102E > Q126K > LkADH wild type, which corresponds to the results with LbADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies. Full article
(This article belongs to the Special Issue Bulk Protein Crystallization)
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13 pages, 10394 KiB  
Article
Quasi-Continuous Production and Separation of Lysozyme Crystals on an Integrated Laboratory Plant
Crystals 2021, 11(6), 713; https://doi.org/10.3390/cryst11060713 - 21 Jun 2021
Cited by 6 | Viewed by 1882
Abstract
Vacuum crystallization with subsequent solid–liquid separation is a suitable method to produce and separate the temperature-sensitive protein lysozyme. The conventional process is performed batch-wise and on different devices, which in turn leads to disadvantages in terms of energy efficiency, contamination risk and process [...] Read more.
Vacuum crystallization with subsequent solid–liquid separation is a suitable method to produce and separate the temperature-sensitive protein lysozyme. The conventional process is performed batch-wise and on different devices, which in turn leads to disadvantages in terms of energy efficiency, contamination risk and process control. This publication therefore focuses on the application of the previously multistage process to a quasi-continuous, integrated single plant. The transfer occurs successively and starts with the substitution of the batch vessel by a process chamber. Afterwards, the filtration scale is increased and the formerly deployed membrane is replaced by an industrial filter cloth. Based on the results of these experiments, the complete process chain is successfully transferred to an integrated laboratory plant. Full article
(This article belongs to the Special Issue Bulk Protein Crystallization)
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18 pages, 26874 KiB  
Article
Controlling Protein Crystallization by Free Energy Guided Design of Interactions at Crystal Contacts
Crystals 2021, 11(6), 588; https://doi.org/10.3390/cryst11060588 - 24 May 2021
Cited by 4 | Viewed by 3715
Abstract
Protein crystallization can function as an effective method for protein purification or formulation. Such an application requires a comprehensive understanding of the intermolecular protein–protein interactions that drive and stabilize protein crystal formation to ensure a reproducible process. Using alcohol dehydrogenase from Lactobacillus brevis [...] Read more.
Protein crystallization can function as an effective method for protein purification or formulation. Such an application requires a comprehensive understanding of the intermolecular protein–protein interactions that drive and stabilize protein crystal formation to ensure a reproducible process. Using alcohol dehydrogenase from Lactobacillus brevis (LbADH) as a model system, we probed in our combined experimental and computational study the effect of residue substitutions at the protein crystal contacts on the crystallizability and the contact stability. Increased or decreased contact stability was calculated using molecular dynamics (MD) free energy simulations and showed excellent qualitative correlation with experimentally determined increased or decreased crystallizability. The MD simulations allowed us to trace back the changes to their physical origins at the atomic level. Engineered charge–charge interactions as well as engineered hydrophobic effects could be characterized and were found to improve crystallizability. For example, the simulations revealed a redesigning of a water mediated electrostatic interaction (“wet contact”) into a water depleted hydrophobic effect (“dry contact”) and the optimization of a weak hydrogen bonding contact towards a strong one. These findings explained the experimentally found improved crystallizability. Our study emphasizes that it is difficult to derive simple rules for engineering crystallizability but that free energy simulations could be a very useful tool for understanding the contribution of crystal contacts for stability and furthermore could help guide protein engineering strategies to enhance crystallization for technical purposes. Full article
(This article belongs to the Special Issue Bulk Protein Crystallization)
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