Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties These properties pose difficult stability problems A summary of both chemical and physical decomposition pathways for proteins is given Chemical instability can include proteolysis, deamidation, oxidation, racemization, and β-elimination Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species

Stability of Protein Pharmaceuticals

In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al, Pharm Res 6:903-918, 1989), which has been widely referenced ever since At the time, recombinant protein therapy was still in its infancy This review summarizes the advances that have been made since then regarding protein stabilization and formulation In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability

Stability of Protein Pharmaceuticals: An Update

Glucose reacts nonenzymatically with the NH2-terminal amino acid of the beta chain of human hemoglobin by way of a ketoamine linkage, resulting in the formation of hemoglobin AIc. Other minor components appear to be adducts of glucose 6-phosphate and fructose 1,6-diphosphate. These hemoglobins are formed slowly and continuously throughout the 120-day life-span of the red cell. There is a two- to threefold increase in hemoglobin AIc in the red cells of patients with diabetes mellitus. By providing an integrated measurement of blood glucose, hemoglobin AIc is useful in assessing the degree of diabetic control. Furthermore, this hemoglobin is a useful model of nonenzymatic glycosylation of other proteins that may be involved in the long-term complications of the disease.

https://science.sciencemag.org/content/sci/200/4337/21.full.pdf

The glycosylation of hemoglobin: relevance to diabetes mellitus

Oxygen Carriers (“Blood Substitutes”)Raison d'Etre, Chemistry, and Some Physiology Blut ist ein ganz besondrer Saft1

The electrostatic contribution to the free energy of folding was calculated for 21 salt bridges in 9 protein X-ray crystal structures using a continuum electrostatic approach with the DELPHI computer-program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5 kcal/mol. This is fundamentally different from stability measurements using pKa shifts, which effectively measure the strength of a salt bridge relative to 1 or more charged hydrogen bonds. The calculated effect was due to a large, unfavorable desolvation contribution that was not fully compensated by favorable interactions within the salt bridge and between salt-bridge partners and other polar and charged groups in the folded protein. Some of the salt bridges were studied in further detail to determine the effect of the choice of values for atomic radii, internal protein dielectric constant, and ionic strength used in the calculations. Increased ionic strength resulted in little or no change in calculated stability for 3 of 4 salt bridges over a range of 0.1-0.9 M. The results suggest that mutation of salt bridges, particularly those that are buried, to "hydrophobic bridges" (that pack at least as well as wild type) can result in proteins with increased stability. Due to the large penalty for burying uncompensated ionizable groups, salt bridges could help to limit the number of low free energy conformations of a molecule or complex and thus play a role in determining specificity (i.e., the uniqueness of a protein fold or protein-ligand binding geometry).

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/pro.5560030206

Do salt bridges stabilize proteins? A continuum electrostatic analysis

Identification of residues contributing to the Bohr effect of human haemoglobin.

https://spin.niddk.nih.gov/clore/Pub/pdf/70.pdf

The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices.

Role of the α-Amino Groups of the α and β Chains of Human Hemoglobin in Oxygen-linked Binding of Carbon Dioxide

THE unloading of oxygen in the tissues is facilitated by lowering the oxygen affinity of haemoglobin (Hb) by both CO2 and diphosphoglycerate (DPG)1. This occurs because CO2 and DPG are oxygen-linked, that is, they are bound more firmly to deoxyhaemoglobin than to oxyhaemoglobin2,3. The binding of CO2 to Hb takes place by a reaction of CO2 with the α-amino groups to form carbamino compounds4,5, and binding curves can be measured by rather laborious methods4,6. Perrella et al.7 have modified these methods so that much smaller amounts of Hb, such as the specifically carbamylated Hbs8, can be used. In this method, Hb equilibrated with CO2 is rapidly taken to pH 11 to stabilise the carbamino CO2 and BioRad AG 1×8 resin added to remove carbonate and bicarbonate ions. The carbamino CO2 is displaced from the Hb by acidification and measured in a Van Slyke apparatus. DPG-free human deoxyhaemoglobin gave diphasic CO2 binding curves7 showing that the affinities of the α and β chain α-amino groups for CO2 are different, but it was impossible to establish which group had the higher affinity.

J.H. Fogg

Papers

Identification of residues contributing to the Bohr effect of human haemoglobin.

The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices.

Role of the α-Amino Groups of the α and β Chains of Human Hemoglobin in Oxygen-linked Binding of Carbon Dioxide

Identification of the high and low affinity CO2-binding sites of human haemoglobin.

Mechanism of deamidation of haemoglobin providence Asn.