No abstract

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.

The time derivative method for deriving the sedimentation coefficient distribution, g(s*), from sedimentation velocity data that was developed by Walter Stafford has many advantages and is now widely used. By fitting Gaussian functions to the g(s*) distribution both sedimentation and diffusion coefficients (and therefore molecular masses) for individual species can be obtained. However, some of the approximations used in these procedures limit the accuracy of the results. An alternative approach is proposed in which the dc/dt data are fitted rather than g(s*). This new approach gives improved accuracy, extends the range to sedimentation coefficients below 1 S, and enhances resolution of multiple species. For both approaches the peaks from individual species are broadened when the data cover too wide a time span, and this effect is explored and quantified.An alternative algorithm for calculating ˆ( *) g s from the dc/dt curves is presented and discussed. Rather than first averaging the dc/dt data for individual scan pairs and then calculating ˆ( *) g s from that average, the ˆ( *) g s distributions are calculated for every scan pair and then subsequently averaged. This alternative procedure yields smaller error bars for g(s*) and somewhat greater accuracy for fitted hydrodynamic properties when the time span becomes large.

No abstract

Protein-based pharmaceuticals exhibit a wide range of aggregation phenomena, making it virtually impossible to fi nd any one analytical method that works well in all cases. Aggregate sizes cover a range from small oligomers to visible " snow " and precipitates, and generally only the smaller species are reversible. It is less widely recognized that aggregates also exhibit a broad spectrum of lifetimes, and the lifetime has important consequences for detection methods. The fact that the measurement itself may destroy or create aggregates poses a major analytical challenge and is a key determinant for method selection. Several examples of some interesting aggregation phenomena and the analytical approaches we have used are presented. In one case, an " aggregate " seen by SEC in stressed samples was shown to actually be a partially denatured monomer using both size-exclusion chromatography with online multiangle laser light scattering (SEC-MALLS) and sedimentation velocity. In a second case, freeze/thaw stress generates transient, metastable oligomers that are extremely sticky and diffi cult to measure by SEC. By using sedimentation velocity as the " gold standard " a much improved SEC method was developed and used to investigate the temperature-dependent dissociation of these oligomers. For problems with visible particulates, dynamic light scattering has been effective, in our hands, at detecting the precursors to the large, visible particles and tracking the source of stress or damage to particular manufacturing steps.

The conformations of the NeuAcα2(I)→3Galβ1(II)→4[Fucα1(III)→3]GlcNAc-O-CH3 tetrasaccharide (sLex), in aqueous solution and bound to E-, P-, and L-selectin have been determined using high resolution NMR spectroscopy. In the free ligand, the conformation of glycosidic linkage I is disordered with {ΦI, ΨI} sampling values close to {−60°, 0°}, {−100°, −50°}, and {180°, 0°}. The trisaccharide portion is rigid and characterized by {ΦII, ΨII; ΦIII, ΨIII} = {46°, 18°; 48°, 24°}. The measured dissociation rates and equilibrium binding constants, {k off, K D}, were {164 ± 24 s-1, 0.72 ± 0.4 mM}, {522 ± 166 s-1, 7.8 ± 1.0 mM}, and {1080 ± 167 s-1, 3.9 ± 0.6 mM} at 300 K for E-, P-, and L-selectin, respectively. The bound conformations of the ligand were calculated from the full relaxation matrix analysis of transferred-NOE spectra for E- and P-selectin or by using a two-spin approximation for the L-selectin complex. Both E- and P-selectin recognize the {−60°, 0°} conformation of sLex while the {−100°, −50°} conformer is probably recognized by L-selectin. The conformation of the branched trisaccharide portion in the bound state remains close to the conformation of the free ligand. In the E-, P-, and L-selectin complexes the GalH4 proton is in the vicinity of protein aromatic protons, most likely Tyr94 and/or Tyr48.

Aggregation or reversible self-association of protein therapeutics can arise through a number of different mechanisms. Five common aggregation mechanisms are described and their relations to manufacturing processes to suppress and remove aggregates are discussed.

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.