TBPP_base fractionation of blood plasma

WW II created medical need. Navy contracted with Harvard to identify and isolate transfusible substance from blood.

leads Harvards efforts, investigated solubility of proteins, father of plasma fractionation, saved thousands of lifes

differential solubility of plasma proteins in alcoholic solutions. Protein solubility controlled by: alcohol concentration, pH, temperature, ionic strength, protein concentration

3 of 5 primary process parameters (EtOH, temp., pH) are directly manipulated in the process, the others indirectly via reagent additions, separation into several fractions enriched in specific proteins, temp. Maintained to solution freezing to minimize denaturation, avoid extreme local conditions because of denaturation

5 fractions from solutions of increasing EtOH concentrations. 10 fractionation schemes: Cohn Method 6 (IgG paste and albumin), Oncley Method 9 (IgG purification). Cohn method: pioneering work, first industrial scale manufacture infusible albumin, basis for other processes, side fractions used for gain of other proteins, variations possible. Precipitate 2 and 3: IgG, precipitate 4: albumin. Kistler Nitschmann method: modification of Cohn Method 10, purity comparable to the other method, increased recoveries (+50%) and smaller process, still in use at CSL for albumin, IVIG, IgG paste

number of plasma proteins: 1980-2014, from 100 to 10'546 proteins. 10 proteins constitute 90% of protein mass / 75% of revenues generated with 4 proteins: albumin, IgG, Factor 8, AAT. Wide concentration rate. Plasma proteins as biotherapeutics: structure and biological activity to be preserved. At industrial scale: precipitation and biochromatography. add precipitant --> centrifugation --> chromatography

depends on distribution of hydrophilic and hydrophobic AS residues on proteins surface. Hydrophobic: predominantly in the core, proteins with high hydrophobic AS content on surface have low solubility. Charged and polar surface: interactions with ionic groups in aqueous solutions and increase solubility. Hydration layer: interaction of protein surface with surrounding water (H-bonding and dipol-dipol) lead to hydration shell, essential for proteins solvation and biological function.

electrostatic forces: aggregation most likely at IEP, no net charge and thus dont repeal each other, ionic or hydrogen bonding. hydrophobic forces: tendency to avoid water, cage-ordered structures formed by water molecules, AS come together and water molecules are expelled and go to a less ordered state: increase of entropy, main driving force for protein association. van der Waals forces: dipole-dipole interactions between close atoms, easily disrupted by heat or denaturing agents, attractive and repulsive interactions depending on distance of 2 atoms.

principle: altering solubility of proteins. Organic solvent: reduction in water activity / dielectric constant (easiness of separation of compounds) makes interactions between charged groups on surface stronger, driving force: dehydration, attraction by van der Waals-forces, denaturation at elevated temp. Ethanol: miscibility with water, Smt depression, highly volatile (separation by mild conditions possible), chemically inert, low toxicity, inexpensive and easily available, inhibiton of bacterial growth, but a denaturating effect (keep temp. low, prevent local over-concentration of EtOH). salting out: at low concentrations salting in (solubility, suppress interactions), at higher concentrations salting out (precipitation, neutralization of charges). Isoelectric precipitation: solubility pH-dependent at isoelectric point aggregation, at pH values below/above --> net charge, proteins repel each other, similar properties: coprecipitation, need to be combined with other precipitants, way of addition is important. polyethylene glycol (PEG): nonionic, exclude volume mechanism (sterically), absence of interactions, causes aggregation without denaturation, but high viscosity. Polyelectrolytes: water soluble, contains ionizable groups, net charge, form a multi-molecular complex with oppositely charged proteins. octanoic/caprylic acid (others): 8 carbon saturated fatty acid, dependent on OA concentration, direct interactions with proteins,conformational changes, micelle-like structures.

mixing phase: causes precipitant and protein to collide/diffusion. Nucleation phase: submicroscopic size particles appear and start grow under brownian diffusion control. Aggregation phase: by diffusive addition of proteins they continue to grow and stick, at slower rate. Aging phase: collision, sticking and breaking apart repeatedly until stable particle reached. Aging helps particle withstand the fluid shear forces. at the end: centrifugation and separation liquid from precipitate.

with disk stack separators (centrifugation) and filterpresses with filter aids. Removal of mother liquor/salts/EtOH, but potential contact activation of proteins possible.

separation and purification by liquid chr. Hydrophobic, hydrophilic and ionic domains: proteins are complex entities. Size exclusion chr. (SEC), ion exchange chr. (IEC), hydrophobic interaction chr. (HIC), affinity chr. (AC). Load --> bind and wash --> if protein bounded in the column: eluation.

first intermediate in fractionation process, frozen plasma, poured into thawing tank, slowly thawing plasma results formation of insoluble precipitate (cryo), temp. Control: otherwise cryoprecipitate dissolve (3°C), harvesting cryo by centrifugation, starting material for Factor 8, VWF and fibrinogen, supernatant (cryo-poor plasma) passed on fractionation

centrifugation cryo --> cryo poor plasma --> fraction 1 --> AEX column --> CEX column: albumin / AEC column: IgG