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1804 results in Molecular biology, biochemistry, and structural biology

Page 4 of 91

  • 3 - Countercurrrent Separation Processes

  • David Pfister, Lucrèce Nicoud, Massachusetts Institute of Technology, Massimo Morbidelli, Eidgenössische Technische Hochschule Zürich
  • Book:
    Continuous Biopharmaceutical Processes
    Published online:
    22 September 2018
    Print publication:
    11 October 2018, pp 84-109

  • Summary

    This chapter provides basic concepts about multi-column counter-current chromatography. The benefit of counter-current contact in separation processes is demonstrated considering cascades of equilibrium stages. Based on this, the true moving bed (TMB) process and the simulated moving bed (SMB) process are introduced. Then, the design space of the TMB and SMB processes leading to a complete separation is identified, starting with simplistic systems and progressively introducing more complex effects, namely non-linear adsorption isotherms and mass transfer limitations. Finally, two process design approaches for multi-column chromatographic processes are presented: an empirical one, which allows to obtain a first guess of the operating conditions from a single-column experiment, and a model-based one, which allows a more rigorous determination of the process variables.

  • 5 - Countercurrent Chromatography for the Polishing Steps

  • David Pfister, Lucrèce Nicoud, Massachusetts Institute of Technology, Massimo Morbidelli, Eidgenössische Technische Hochschule Zürich
  • Book:
    Continuous Biopharmaceutical Processes
    Published online:
    22 September 2018
    Print publication:
    11 October 2018, pp 153-202

  • Summary

    This chapter focusses on the polishing steps encountered during protein purification by chromatography. The first part provides a detailed description of the mechanistic phenomena at stake, whose complexity may greatly vary from one case to the other depending on the number and type of impurities, the selectivity for the target protein as well as non-linear and competitive effects. Several multi-column counter-current processes allowing the implementation of the polishing steps in a continuous manner are presented in the second part of the chapter. Modifications of the classical SMB process introduced to better cope with the specific needs of the biopharmaceutical industry are discussed. These modifications primarily aim at recovering more than two fractions and at implementing modifier concentration gradients. Two process design approaches for multi-column processes are presented: an empirical one, which provides a first guess of the operating conditions from single-column experiments, and a model-based one, which allows a more rigorous determination of the process variables. The model-based approach is used to compare the performance of multi-column and single-column processes.

  • 6 - Protein Conjugation

  • David Pfister, Lucrèce Nicoud, Massachusetts Institute of Technology, Massimo Morbidelli, Eidgenössische Technische Hochschule Zürich
  • Book:
    Continuous Biopharmaceutical Processes
    Published online:
    22 September 2018
    Print publication:
    11 October 2018, pp 203-246

  • Summary

    This chapter deals with protein bioconjugation, which has emerged as an attractive technique to enhance therapeutic properties of protein-based drugs (e.g. in-vivo circulation, potency). The first part of this chapter aims at answering questions regarding the coupling chemistry and the modelling of the kinetics of bioconjugation. Then, the purification of conjugated proteins is addressed. In particular, chromatography is presented as a technique to purify conjugated proteins according to their extent of conjugation on the one hand and to the position of the conjugated groups on the other hand. Moreover, continuous technologies for both the conjugation reaction and the purification by chromatography are discussed. Finally, the potential benefits from the integration between the reaction and the purification steps in bioconjugation processes are examined. This chapter combines theoretical aspects, model simulations and experimental results from practical examples, for instance dealing with PEGylated proteins and antibody-drug conjugates.

Continuous Biopharmaceutical Processes
  • Chromatography, Bioconjugation, and Protein Stability
  • David Pfister, Lucrèce Nicoud, Massimo Morbidelli
  • Published online:
    22 September 2018
    Print publication:
    11 October 2018
  • This innovative reference provides a coherent and critical view on the potential benefits of a transition from batch to continuous processes in the biopharmaceutical industry, with the main focus on chromatography. It also covers the key topics of protein stability and protein conjugation, addressing the chemical reaction and purification aspects together with their integration. This book offers a fine balance between theoretical modelling and illustrative case studies, between fundamental concepts and applied examples from the academic and industrial literature. Scientists interested in the design of biopharmaceutical processes will find useful practical methodologies, in particular for single-column and multi-column chromatographic processes.

  • 6 - Electrophoretic Techniques

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 219-252

  • Summary

    GENERAL PRINCIPLES

    The term electrophoresis describes the migration of a charged particle under the influence of an electric field. Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species, either cations (positively charged) or anions (negatively charged). Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.

    The equipment required for electrophoresis consists basically of two items, a power pack and an electrophoresis unit. Electrophoresis units are available for running either vertical or horizontal gel systems. Vertical slab gel units are commercially available and routinely used to separate proteins in acrylamide gels (Section 6.2). The gel is formed between two glass plates that are clamped together, but held apart by plastic spacers. A commonly used equipment in this context is the so-called minigel apparatus (Figure 6.1). Gel dimensions are typically 8.5 cm wide × 5 cm high, with a thickness of 0.5–1 mm. A plastic comb is placed in the gel solution and is removed after polymerisation to provide loading wells for up to 10 samples. When the apparatus is assembled, the lower electrophoresis tank buffer surrounds the gel plates and affords some cooling of the gel plates. A typical horizontal gel system is shown in Figure 6.2. The gel is cast on a glass or plastic sheet and placed on a cooling plate (an insulated surface through which cooling water is passed to conduct away generated heat). Connection between the gel and electrode buffer is made using a thick wad of wetted filter paper (Figure 6.2); note, however, that agarose gels for DNA electrophoresis are run submerged in the buffer

    (Section 6.4.1). The power pack supplies a direct current between the electrodes in the electrophoresis unit. All electrophoresis is carried out in an appropriate buffer, which is essential to maintain a constant state of ionisation of the molecules being separated. Any variation in pH would alter the overall charge and hence the mobilities (rate of migration in the applied field) of the molecules being separated.

  • 14 - Basic Techniques Probing Molecular Structure and Interactions

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 500-534

  • Summary

    INTRODUCTION

    Determining the structure of molecules and the interactions between molecules is critical for understanding biochemical processes. All experimental techniques discussed in the following sections revolve around investigations of the three-dimensional structure of molecules as well as the elucidation of interactions between different groups of molecules. As such, some of these techniques are characterised by a somewhat higher level of complexity in undertaking and are often employed at a later stage of biochemical characterisation.

    Techniques probing the thermodynamics of a system (such as isothermal titration calorimetry) or atomic/molecular structure and interactions without requirements for washing or flow (NMR spectrosocopy, magnetic resonance imaging, X-ray diffraction, light scattering) can be summarised as direct techniques. Alternatively, molecular interactions can be analysed by applying tracers to the molecules themselves, resulting in molecular switch techniques. Last, there are in vitro methods that combine the use of analytical techniques (such as the spectroscopic techniques outlined in Chapter 13) with physical sampling – these can be classified as indirect techniques.

    The analysis of molecular interactions can result in the development of a biosensor: a device that is composed of a biological element and a physico-chemical transduction part that converts signal reception by the biological entity into a physical quantifiable response. Such technology gives rise to the field of biosensing.

    ISOTHERMAL TITRATION CALORIMETRY

    Principles

    Isothermal titration calorimetry (ITC) enables study of the thermodynamics of molecules binding to each other. This is a general method for studying the thermodynamics of any binding (association) process in solution. It detects and quantifies small heat changes associated with the binding and has the advantages of speed, accuracy and no requirement for either of the reacting species to be chemically modified or immobilised. The apparatus consists of a pair of matched cells (sample and reference) of approximately 2 cm 3 volume contained in a microcalorimeter (Figure 14.1a). One of the reactants (say the enzyme preparation) is added to the sample cell and the ligand (substrate, inhibitor or effector) is added via a stepper-motor-driven syringe. The mixture is stirred to ensure homogeneity. The reference cell contains an equal volume of reference liquid. A constant power of less than 1 mW is applied to the reference cell.

  • 24 - Drug Discovery and Development

    • By David Camp, School of Environment, Griffith University
  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 864-905

  • Summary

    INTRODUCTION

    An enormous cache of human biology remains to be explored as discoveries and new insights emerge from the ‘-omics’ sciences. The goal for many pharmaceutical companies (and increasingly also for academic organisations) is to find selective small molecules that can modulate these newly identified targets (mainly proteins), so that they can ultimately be developed into therapeutics. In this context, the term small molecules is used for organic compounds, typically with a molecular weight < 1000 dalton (Da) comprised mainly of carbon, hydrogen, nitrogen and oxygen atoms, but frequently also sulfur and fluorine, and less often chlorine, bromine and phosphorus. The modulation of new biological targets may require novel structural frameworks (called scaffolds), which contain various functional groups in strategic positions. The correlation between the chemical structure of compounds and their biological activities constitutes the so-called structure–activity relationships and their analysis is an integral part of drug development. Indeed, the identification of new structural motifs of small molecules is one of the many drivers en route to understanding biological systems and developing innovative, safer therapeutics with novel modes of action.

    The task of finding a selective molecule is truly daunting considering there is an estimated 10 20 –10 200 compounds with a molecular weight below 500 Da comprised of the atoms that make up current small-molecule therapies. To put this into perspective, approximately 2.7 × 10 7 molecules have been reported to date, which equates roughly to the ratio of the mass of the Sun compared to the mass of a proton, if the Bohecek number (6 × 10 62) is used to represent the set of small molecules with drug-like properties (see Section 24.2.2). Moreover, it would be impossible to screen this large number of molecules across multiple assays if we were limited to using the available resources on our planet, given the Earth is estimated to contain ‘only’ 10 51 atoms. The real challenge, which will be the primary focus of this chapter, lies in:

  • 2 - Basic Principles

  • Edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie
  • Book:
    Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
    Published online:
    06 August 2018
    Print publication:
    19 April 2018, pp 8-39

  • Summary

    BIOLOGICALLY IMPORTANT MOLECULES

    Molecules of biological interest can be classified into ions, small molecules and macromolecules. Typical organic small molecules include the ligands of enzymes, substrates such as adenosine triphosphate (ATP) and effector molecules (inhibitors, drugs). Ions such as Ca 2+ play a key role in signalling events. Biological macromolecules are polymers which, by definition, consist of covalently linked monomers, the building blocks. The four types of biologically relevant polymers are summarised in Table 2.1.

    Proteins

    Proteins are formed by a condensation reaction of the α-amino group of one amino acid (or the imino group of proline) with the α-carboxyl group of another. Concomitantly, a water molecule is lost and a peptide bond is formed. The peptide bond possesses partial double-bond character and thus restricts rotation around the C–N bond. The progressive condensation of many amino acids gives rise to an unbranched polypeptide chain. Since biosynthesis of proteins proceeds from the N- to the C-terminal amino acid, the N-terminal amino acid is taken as the beginning of the chain and the C-terminal amino acid as the end. Generally, chains of amino acids containing fewer than 50 residues are referred to as peptides, and those with more than 50 are referred to as proteins. Most proteins contain many hundreds of amino acids; ribonuclease, for example, is considered an extremely small protein with only 103 amino-acid residues. Many biologically active peptides contain 20 or fewer amino acids, such as the mammalian hormone oxytocin (nine amino-acid residues) which is clinically used to induce labour since it causes contraction of the uterus, and the neurotoxin apamin (18 amino-acid residues) found in bee venom.

    THE IMPORTANCE OF STRUCTURE

    Three main factors determine the three-dimensional structure of a macromolecule:

  • • allowable backbone angles

  • • i nteractions between the monomeric building blocks

  • • interactions between solvent and macromolecule.

    • The solvent interactions can be categorised into two types: binding of solvent molecules (solvation) and hydrophobic interactions. The latter arise from the inability or reluctance of parts of the macromolecule to interact with solvent molecules (hydrophobic effect), which, as a consequence, leads to exclusive solvent–solvent interactions. Phenomenologically, a collection of molecules that cannot be solvated will stick close to one another and minimise solvent contact.

Page 4 of 91