optim15v1.0 an introduction to proteins, their folding and · pdf file ·...
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Optim® 1000: An introduction to proteins, their folding
and aggregation Technical note
Introduction In recent years there has been a rapid growth in the number of protein-based biopharmaceutical molecules in development. Although there are many advantages to using proteins as therapeutic molecules, their behaviour in solution is often complex. For the vast majority of the time in vivo and in simple dilute solutions in vitro, protein molecules are well behaved, retaining their correct native structure and not forming aggregates. However, the processes typical to the development of a biopharmaceutical are rarely ideally suited to maintaining a protein in its native state. Many candidate molecules are studied before a suitable candidate is found, and even then, devising a formulation which has a stable native conformation when subjected to a wide variety of stresses during scaling up production, storage and delivery is highly non-trivial. Understanding the science behind protein-folding and aggregation has never been more important and this technical note aims to provide an introduction to some basic principles.
Protein structure
Proteins and polypeptides are linear polymers of amino acids (Figure 1) and the distinction in naming is really only dependent on the length of the polymer. Typically, polypeptides are less than 20 amino acid residues in length, whereas proteins are longer. There are twenty different naturally-occurring amino acids that can be incorporated into a protein chain, each with a unique functional group (or side chain). For example, some side chains are acidic whilst others are basic, some are polar whilst others are non-polar and some are aromatic whilst others are aliphatic. It is the particular order of the amino acids in a protein that dictates its final structure and function.
Figure 1. Representation of an amino acid containing a side-chain, R, and a chiral centre Cα.
Protein structure can be thought of in four different levels of organisation, each with increasing complexity (Figure 2).
The primary structure of a protein is simply the sequence of amino acids contained within the molecule. Each amino acid contains a chiral centre, which is an asymmetric carbon molecule (Cα) that is covalently bonded to an amine and a carboxyl group, along with a hydrogen atom and one of a number of possible side-chains. It is possible to have two stereo-isomers in amino acids, L and D, due to the chiral nature of the Cα atom – in natural proteins all amino acids are L.
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There are no restrictions on the shape, size, charge, hydrophobicity or reactivity of the side chain.
(a)
(b)
(c)
(d)
Figure 2. Different levels of protein structure. (a) Primary structure which is the sequence of amino acids. This
contains bonds which have different angles of rotation. These are constrained sterically which leads to (b)
secondary structure. The three dimensional arrangement of secondary structure elements is called (c)
tertiary structure, and the ordered of multimers of proteins containing folded tertiary structures are referred
to as (d) quaternary structure.
In a protein, the amino acids are joined together through a covalent bond between the carboxyl group of one amino acid and the amine group of the next amino acid. This is referred to as the peptide bond and it produces an asymmetric chain of amino acids with a free NH
2
group at the N-terminus and a free COOH group at the C-terminus. The partial double-bond character of the N – C bond, as shown in figure 1, leads to the NH-to-CO region being planar. Other than this constraint there are only steric considerations which determine the angles that the amino acid side chains can orient themselves and it is this sequence of torsion angles which determines the main chain conformation.
This leads to the next level of complexity, secondary structure, which is the conformation adopted by this backbone. Two common types of regular secondary structure exist: the α-helix and the β-strand.
The α-helix is a tightly wound regular spiral of amino acids where the carboxyl group of one amino acid forms a hydrogen bond with the amine group four residues further along the chain. All of the side chains point away from the centre of the helix. The β-strand is fundamentally different to an α-helix as stabilising hydrogen bonds form between β-strands, forming a β-sheet.
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Although also regular, the β-sheet is a much more extended structure compared with an α-helix, where the β-strands may be either parallel or anti-parallel to one another. Both α-helices and β-strands are linked by loop regions. Loop regions do not have regular repeating structure but they are not altogether random. Loops can contain very short secondary structural elements called β-turns, which are often found joining β-strands and abruptly change the direction of the protein backbone.
The next level of complexity is tertiary structure, which dictates how the secondary structures fit together to give the overall three-dimensional structure of an individual protein molecule. They are held together by a combination of hydrogen bonds and a number of hydrophobic forces, sulphide cross-links and salt bridges. Hydrophobic side chains have a tendency to cluster together within the core of the protein whilst in aqueous solution. Polar and charged amino acids form hydrogen and ionic bonds, or cluster on the outside of the protein where they can interact with water. For monomeric proteins, a protein is said to be ‘native’ when it has attained the correct, functional, tertiary structure.
When a protein forms ordered multimers, it is said to have quaternary structure, which is the spatial arrangement of monomers within a multimer.
Denaturation The structure of a protein is held together by a combination of hydrogen bonds, hydrophobic and Van der Waals interactions, covalent disulphide bonds and ionic salt bridges. All of these interactions mean that the native structure of a protein is more stable than its denatured (or unfolded) structure. However, most proteins are only marginally stable because all of these interactions only just out-weigh the large entropic cost of reaching the native state (Figure 3).
Figure 3. Balancing enthalpic and entropic contributions to fold a protein.
In many small, single-domain proteins, the loss of a small number of hydrogen bonds is enough for the unfolded state to be the most favourable, leading to complete unfolding of the tertiary and secondary structure into an unfolded random coil. There are many ways that a protein can be denatured, a number of which can be encountered during the biopharmaceutical development and production process. These include heat or cold stress, mechanical or shear stress, solvent composition (pH, salt concentration, chaotrope concentration, polarity, etc.), surface interactions, incorrect post-translational modification or truncation from partial protease
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degradation. Many small, single-domain proteins can recover their correct, functional native tertiary structure after being denatured. However, larger, more complex multi-domain proteins, such as antibodies, become mis-folded, irreversibly losing their correct structure and function. Many proteins can also form rare partially unfolded states, in which only small regions of structure become unfolded.
Aggregation In native proteins, the water-hating hydrophobic amino acid side chains tend to be packed in the core of the protein, where they are able to interact with other hydrophobic side chains. This leaves the surface of the native protein relatively hydrophilic and able to easily interact with aqueous solvent. Conversely, unfolded, partially unfolded and mis-folded proteins do not bury as many of their normally exposed hydrophobic groups from the surroundings. As this is an energetically unfavourable situation, the hydrophobic groups of neighbouring proteins tend to come together, irreversibly forming aggregates (Figure 4).
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Figure 4. Unfavourable interactions between water (blue) and hydrophobic groups of protein (black) leads
to aggregation in order to minimise number of interactions.
Aggregation is not only driven by hydrophobicity. For example in a solution at a pH close to the pI of the solute protein, electrostatic interactions can lead to aggregation of native protein. Other mechanisms potentially leading to aggregation include non-native disulphide bond formation between protein molecules, protein oxidation and the Maillard reaction. Aggregated protein may initially be soluble, but as the size of the aggregate increases it tends to become insoluble, eventually precipitating out of solution. Aggregates can be disordered, where the protein chains coalesce at random, or they can be ordered, where the proteins arrange themselves in a very specific manner. It is even possible for some native states to form aggregates which may be functional.
Protein aggregation is undesirable in the case of a biopharmaceutical product. Formation of aggregates during the production phase can lead to markedly decreased yields of the correct product, although aggregates can be removed during the purification process. Aggregates formed during the storage of a biopharmaceutical often cannot be removed before administration which can potentially lead to a decreased efficacy of the drugs and unwanted side effects in patients.
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