Protein loss, surface adsorption

There are several formulation concerns related to delivery of proteins from an implantable pump. Because this pump is placed within the body, it requires a formulation that is stable at body temperature (37°C) over the length of time the pump will deliver the protein until it is refilled. Motion of the body results in agitation of the solution contained within the pump therefore it is necessary to ensure that the formulation is stable to shaking. The high surface-to-volume ratio of the catheter used for delivery may result in losses of protein because of surface adsorption. Each of these potential pitfalls may be overcome with the appropriate selection of excipients for most proteins. 

Surfactants may be added at low levels (i.e., 0.05% w/w in solution) for several purposes. Surfactants may aid reconstitution if the drug does wet well, and surfactants are often added to low dose products to minimize losses due to surface adsorption. Surfactants may also be effective as stabilizers in low dose protein systems. 

Inert carrier proteins, such as bovine serum albumin, are commonly added to the assay media at high concentrations for screening of isolated targets in order to saturate the potential protein binding surfaces and reduce the loss of target proteins due to adsorption onto the surfaces of the containers, such as plates and vials. The added benefit of inert proteins is that they can bind to lipophilic compounds and increase the solubility of insoluble compounds in the assay buffer. The presence and absence of serum protein can cause difference in solubility and cause a discrepancies between the two assays. 

It has been observed for several proteins that the intermediate structures are formed as the protein unfolds from N state to D state [26]. As the protein unfolds, protein loses tertiary structure and, frequently, secondary structure. In some instances, the secondary structure remains intact while the tertiary structure is lost [12], which is clear from spectral studies that measure loss of secondary and tertiary structural changes. One spectroscopic technique that is sensitive to tertiary structure (e.g., fluorescence) would detect changes, whereas other techniques that are sensitive to secondary structures (e.g., far UV CD) do not show any spectral changes. This molecular property is defined as molten globule or structured intermediate [12]. These intermediates expose hydrophobic domains, and thus promote aggregation or surface adsorption. 

The presence of glycosyl moieties reduced the sensitivity of protein to pH effects (41), Changes in the hydrodynamic volume of proteins would be expected to reduce the rate of diffusion of the modified protein e.g, G-b-Lg to the interface thereby slowing the rate of surface adsorption and, finally, loss of conformational energy during modification i.e. less secondary structure may result in a decreased gain in free energy of these proteins upon adsorption at the interface. 

All chemicals are at least reagent grade and may be purchased from any convenient source. Carrier-free Na[ I] is obtained from ICN (Cat. No. 63034), but the equivalent radioisotope from any source is acceptable. Iodobeads are purchased from Pierce (Cat. No. 28665 G) and kept at 4°C. Solutions of protein and radioiodine are contained in 5-ml snap-cap polypropylene vials (Bio-Rad, Cat. No. 223-9820) and are transferred from one vial to another using 1.5-ml polypropylene transfer pipettes. These minimize the loss of protein due to adsorption to glass or other surfaces. Nitrocellulose (25 X 25-cm NC sheets with a 045- m pore size. Cat. No. 00850) is from Schleicher and Schuell. Washed iodobeads are blotted on Whatman No. 540 paper (Cat. No. 1540N321). Prepacked columns for desalting solutions of macromolecules are from Pierce (2- or 5-ml Excellulose gel columns. Cat. No. 20439 G or 20449 G, respectively) or Amersham (NAP 5 column. Cat. No. 17-0853-01). Any available gamma detector can be used to quantitate radioiodine. All operations are carried out at room temperature in a fume hood. 

Sodium dodecyl sulphate readily inactivates rotavirus, an enteric pathogen belonging to the Reoviridae it also causes loss of poliovirus infectivity-by disruption of virion proteins [83, 84]. Non-infective rotaviral particles lack the outer protein shell associated with infectivity. The initial step in infection of a cell by a virus is the adsorption of the virus to receptors on the surface of the cell. The outer layer of the viral particle must be obviously involved in this process and alterations to the capsid, say by detergent, may lead to a loss of adsorptive capacity and thus a loss in activity. NaDS causes such a loss in the ability of the rotavirus to adhere to CV-1 cells. However, most of the proteins of the outer shell seem to remain associated with the virions and the decreased adsorption may be an electrostatic effect due to adsorption of NaDS molecules on the virus surface. 

To conclude, a strong correlation was found to exist between the net charge of the proteins in solution, the net charge of the SUM surface, and the extent of protein adsorption, which was expressed in terms of flux losses measured after filtration of the different protein solutions. Moreover, in the case of charge-neutral SUMs, flux losses increased with the hydrophobicity of the nucleophiles bound to the S-layer lattice. All proteins caused higher flux losses on SUMs modified with HDA than on those modified with GME or... 

Human serum albumin (HSA) may be used as a protectant against adsorptive loss of proteins present at low concentrations. HSA is present at higher concentration than the active substance and is preferentially adsorbed, coating the surface of interest and preventing adsorption of the drug. For example, insulin is subject to adsorptive loss to hydrophobic materials. Addition of 0.1-1.0% HSA has been reported to prevent this adsorptive loss [9],... 

A change in the environment of a protein molecule, e.g. adsorption from aqueous solution onto a sorbent surface, may lead to a partial breakdown of its ordered structure, resulting in an increase of conformational entropy. This is a fundamental difference between protein adsorption and the adsorption of flexible polymers, for which attachment to a surface implies a loss of conformational entropy. 

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