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Protein structure key for function and safety.

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[Audio] The primary structure of a protein is the amino acid sequence and this needs to be characterized for our product. According to guidlines the amino acid sequence of the desired product should be determined to the extent possible and compared to the gen sequence Amino Acid Composition: Analyzing the overall composition of amino acids within the protein provides valuable insightsSuch as the relative abundance of different amino acids present, which can be indicative of specific structural or functional characteristics. Terminal Amino Acid Sequence: Identifying the amino acids at the ends of the protein chain is essential for understanding its structure and potential functional properties. By determining the terminal amino acid sequence, we can gain insights into post-translational modifications or processing events that might have occurred. Peptide Map: Generating a peptide map involves cleaving the protein into smaller fragments using proteolytic enzymes. The resulting fragments, or peptides, can be separated and analyzed 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 Disulfide Bridges: Proteins often contain cysteine residues that can form intramolecular or intermolecular disulfide bridges. These bridges play a crucial role in stabilizing the protein structure. Analyzing the presence and location of sulfhydryl groups and disulfide bridges helps in understanding the protein's folding pattern and potential functional properties.

[Audio] Proteins exhibit well-defined structural elements known as secondary structures, which is a part of their overall three-dimensional shape and function. Three common types of secondary structures are: Alpha-Helix (α-helix): The alpha-helix is a tightly coiled, right-handed helical structure formed by hydrogen bonding between the amino acid residues in the polypeptide chain. This results in a stable and compact helix-like arrangement. Beta-Sheet (β-sheet): Beta-sheets 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-Barrel: 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, with the polypeptide chain folding back on itself. 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 of left- and right-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. It is important to note that even misfolded or partially denatured proteins can retain elements of secondary structureTherefore, 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, which is crucial for its proper function. 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 guidlines this can be done by adding assays that measure the potency of the protein. Two commonly used assays are: Binding Assays: Here one measures the ability of the protein to bind to a specific 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 catalyze a specific reaction Proteins' three-dimensional structure can be significantly impacted by the surrounding solution conditions. Three key factors include: pH: The acidity or alkalinity of the environment can alter the protein's charge distribution, affecting its folding and stability. Ionic Strength: 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 serie can be used to understand how ions effect protein structure more on this in how to formulate protein drugs choice of excipients. Interaction with Excipients: Excipients, which are additives often used in pharmaceutical formulations, can interact with proteins and affect their folding behavior. The structure of proteins can be disrupted through a range of events. This includes heat denaturation, denaturation by chemical compunds 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.

[Audio] While the guidelines strongly recommends a biological assay for the specification of a biologics. It does not demand that the structure is determinated. 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] This is an example how the. Influencing clearance and uptake– Quaternary structure.