Chapter 1: Downstream Processing of Biotechnology Products Primary Structure of Proteins Polypeptide chain of amino acids only L-isomers (not

t D-isomers) found in natural proteins Mw,avg of the 20 amino acid component = 109 Da can use this as an approximate for number of amino acids in polypeptide chain Side chains can be: o Charged acids and bases of differing pKa determining net charge of protein as a function of pH o Hydrophobic determine hydrophobic character of primary structure; determinant of folding pattern of polypeptide chain Cysteine (Cys, C) can go through oxidation of form intra/inter-molecular disulfide bonds (reversible in reducing conditions) often stabilizes proteins folded structure; can also form covalently bonded multimeric protein structures. Reversibility of disulfide linkage utilized in covalent chromatography which uses sulfhydryl ligands to separate IgG heavy and light chains Proline (Pro, P) cyclic imino acid that can exist in cis and trans forms isomeric forms are in equilibrium in free solution but in polypeptide, interconversion of these forms is slow and can be rate-limiting step in establishing the folded protein structure Secondary Structure of Protein -Helices spiral arrangement of polypeptide chain comprising of 3.6 amino acid residues per turn; stabilized by hydrogen bonds; may be hydrophilic/phobic/amphipathic depending on the sequence -Sheets very stable structure resulting from hydrogen bonding planar which can be parallel, antiparallel or a mixed depending on directional alignment of polypeptide chains o Often observed during irreversible protein aggregation Urea (a strong H-bond breaker, denaturing agent) can be used to disrupt the H-bonds but can also cause complete destabilization and unfolding of protein. Loops flexible parts of protein connecting other secondary structure elements critical in artificial fusion of different proteins as in the case of single chain antibodies Circular dichroism (CD) and infrared spectroscopy can be used to measure relative number of secondary structure Tertiary Structure of Protein Refers to 3D-arrangement when secondary structures are folded together stabilized primarily by disulphide and hydrophobic bonds

Fluorescence spectroscopy provides info on location of highly hydrophobic residues, Phe, Tyr and Trp. Extent of unfolding can be known by calculating the fluorescence spectra of native and unfolded forms

Quaternary Structure of Protein Superstructure formed by two or more polypeptide chains association which in many cases is essential for biological functions (e.g. hemoglobin which has 4 polypeptide units held together by H-bonds and hydrophobic interactions flexibility of quaternary structure in response to oxygen binding is critical for oxygen uptake and release in lungs and capillaries; antibodies with their 2 heavy and light chains) Post-Translational Modifications can dramatically impact downstream processing Occur after primary structure is formed; highly cell specific, varying with the physiological status of the cell; further modifications can occur following expression results in high heterogeneity in protein, influencing its biological and pharmacological activity as such, biologicals are defined by their manufacturing process, rather by unique molecular entities. Represents a productivity bottleneck high expressions rates; modifications often altered or incomplete when modification lags behind protein translation results in protein variants with varying biological activity, stability, and biophysical properties (e.g. solubility, charge, hydrophobicity and size) Hence, improved cell culture/fermentation titers have to be balanced against the increased heterogeneity of product. Two especially relevant post-translational modifications to chromatographic properties of protein: o Glycosylation O-linked oligosaccharides bound to Ser and Thr; N-linked oligosaccharide bound to Asp renders protein more hydrophilic (i.e. more soluble); Also influences net charge of protein as terminal carbohydrates of such oligosaccharides are often neuraminic acids (sialic acid) which are ve charged > pH 3 o Deamidation Chemical transformation of asparagine and glutamine (both uncharged) into aspartic acid and glutamic acid (both -ve chaged > pH 4) respectively, with the former being more frequently observed but also depending on residues location (exposed surface ones are affected while those buried within protein core are protected). facilitated by higher pH values and higher temperatures Removal of deamidated variants is a challenge; Ion-exchange possible but often difficult as net charge difference between native and deamidated forms can be small Oligonucleotides and Polynucleotides (RNAs/DNAs) either contaminants or constitute the product (e.g. genomic DNA as a contaminant in production of plasmid DNA for gene therapy; genomic and plasmid DNA as contaminants in protein production) Nucleotides (composed of a phosphate group, sugar group, and a nitrogenous nucleoside group) are rather hydrophilic and negatively charged due to acidic phosphate group.

DNA and RNA are chemically very stable unless DNAse and RNAse enzymes are present; sensitive to mechanical shear Upon cell lysis, DNA/RNA are released into culture SN and alter viscosity of fermentation broths due to their size and filamentous structure

Genomic DNA present in nucleus of eukaryotic organisms always associated with very basic proteins known as histones