PowerPoint-presentation

[Virtual Presenter] Welcome, to this video lecture from RealHope that describes the different levels of protein structures and how these affects the function of protein pharmaceuticals.

[Audio] There are four levels of protein structures that we normally uses, from the primary structure that gives the sequence of amino acids to quaternary structure that describes how different monomers of a protein or groups of proteins interact with each other. We will in this lecture go through all four and discuss what we need to know from these to understand the formulation and safety of drug products..

[Audio] The primary structure of a protein is the amino acid sequence and this needs to be characterized for our product. According to guidelines the amino acid sequence of the desired product should be determined to the extent possible and compared to the gene-sequence. There are a few recommended analysis's that could be performed. These are Amino Acid Composition: Here the overall composition of amino acids are determined which provides valuable insights. For example, the relative abundance of different amino acids present,. This can be give indications of specific structural or functional characteristics of the protein. Terminal Amino Acid Sequence: Here the amino acids at the ends of the protein chain is determined. By determining the terminal amino acid sequence, we can gain insights into post-translational modifications or processing events that might have occurred. Peptide Map: This involves cleaving the protein into smaller fragments using proteolytic enzymes. The resulting fragments, or peptides, can be separated and analysed to create a map of their positions within the protein sequence. This information helps in identifying potential domains or regions of interest within the protein. Sulfhydryl Groups and Disulphide Bridges: Proteins often contain cysteine residues that can form intramolecular or intermolecular disulphide bridges. These bridges play a crucial role in stabilizing the protein structure. Analysing the presence and location of sulfhydryl groups and disulphide bridges helps in understanding the protein's folding pattern and potential functional properties.

[Audio] the secondary structures, is a part of their overall three-dimensional shape and function of the protein. they are segment of well defined structures primarally stabilsed by hydrogen bonds. Three common types of secondary structures are: The alpha-helix which is a tightly coiled, right-handed helical structure. That results in a stable and compact helix-like arrangement. Beta-sheets which are formed when segments of the polypeptide chain lie parallel or antiparallel to each other, and hydrogen bonds form between adjacent strands. Beta-sheets can be either parallel or antiparallel, and they often contribute to the stability of protein structures. Beta-barrels are a type of secondary structure commonly found in membrane proteins and certain globular proteins. They consist of multiple beta-strands arranged in a barrel-like shape. To study the secondary structure of proteins, researchers use various experimental techniques, and one of the common methods is Circular Dichroism (CD). Circular Dichroism measures the differential absorption circularly polarized light by chiral molecules, such as proteins. It is particularly useful in providing information about the protein's secondary structure, as different secondary structures exhibit characteristic CD spectra, as shown in the picture. It is important to note that even misfolded or partially denatured proteins can retain elements of secondary structure. Therefore, understanding the secondary structure of proteins, including misfolded variants, is essential for the understanding of the quality of a protein drug..

[Audio] The tertiary structure of a protein refers to its three-dimensional arrangement. It involves the folding of the secondary structural elements into a specific overall 3D conformation. The correct tertiary structure is essential for the protein to carry out its biological function effectively. Any alterations or misfolding in the tertiary structure can lead to loss of function, and can also lead to aggregation. Therefore, it is essential to ensure that a protein has the right tertiary structure for the drug product to perform as it should. According to guidelines this can be done by adding assays that measure the biological function of the protein. Its potency. Two commonly used assays are: Binding Assays: Here one measures the ability of the protein to bind to a specific targets. For instance, antibodies binding antigens or receptors binding to ligands. Enzymatic Assays: Enzymatic assays monitor the enzyme's activity by measuring its ability to catalyses a specific reaction Proteins' three-dimensional structure can be significantly effected by the surrounding solution conditions. Three key factors include: pH: As the acidity or alkalinity of the environment can alter the protein's charge distribution, affecting its folding and stability. Ionic Strength: This as the concentration of ions in the solution can influence electrostatic interactions within the protein. What ions that are used is also important as the protein can interact for example with divalent ions. The Hofmeister series can be used to understand how ions effects, protein structure. More on this in the lecture on how to formulate protein drugs and choice of excipients. Interaction with Excipients: Excipients, which are additives often used in pharmaceutical formulations, can interact with proteins and affect their folding behaviour. The structure of proteins can be disrupted due to heating, denaturation by chemical compounds such as Urea and Guanidinium chloride or Surfactants such as SDS and finally though adsorption to interfaces Conformational changes can often lead to aggregation of the protein and this will lead to both a loss of active substance and a risk for adverse events such as immune reactions and blood clotting..

[Audio] While the guidelines strongly recommends a biological assay for the specification of a biologics. It does not demand that the structure is determined. However, in development of a product we still often wants to determine the structure. Today the most common way is to use X-ray crystallography. However, this gives the structure in a crystal and not in solution. Several proteins are also difficult to crystallize. An alternative or complement to X-ray crystallography is 2-D NMR. The advantage is that this can be done in solution and thus formulation effects on structure can be investigated. However, until just recently the complexity of the date obtained has made it difficult to determine the full structure using only NMR. Finally computational modelling has increased substantially during the last years and made it possible to get a good prediction of the 3-D structure from the primary structure. The state of the art program for this is currently Alphafold..

[Audio] The quaternary structure of a protein refers to the arrangement and interaction of multiple protein subunits to form a functional, multi-subunit protein complex. The figure for example shows a hexamer of insulin It involves the assembly of two or more protein subunits, each contributing to the overall functionality of the protein or its beahvior in the formulation. For example quartenary structure can affect uptake or stability of the protein. It is a equlibrium structure and can be affected by solution conditions. Thus changing things like pH, presence of specific ions or concentration of the protein solution might affect the quatenary structure of the protein..

[Audio] The transport of insulin over cell membranes are an example of how Quaternary structure can affect the uptake and clearance of proteins. The monomeric form of insulin has a rather fast transport over the membrane while the hexamer cannot passe over the membrane at all. This is used for designing insulin products that either has a fast uptake and are used in connection with meals or to design insulin that can give a baseline concentration in the blood for the whole day..