RESUMEN
Therapeutic potential of immunoconjugates has opened a new window for antibody-based biopharmaceuticals. Greater tissue penetration and hence enhanced cell toxicity are obtained with a smaller version of antibodies. While the whole antibody can be readily produced via mammalian expression system, antibody fragments often require refolding of insoluble proteins. Here we report a new refolding method for antibody fragments using a novel amino acid-based detergent as a solubilizing agent and arginine-assisted refolding. Inclusion bodies of antibody fragments were solubilized by 2.5% lauroyl-L-Glu (C12-L-Glu) and successfully refolded by multi-step dilution into a buffer solution containing arginine hydrochloride and thiol/disulfide-exchange reagents. Adjustment of temperature was found to be critical for increase in the refolding yield. Although each protein requires appropriate optimization, solubilization by C12-L-Glu and dilution refolding assisted by arginine can generate the native, functional antibody fragments. The procedure should enable us to utilize bacterial expression systems for the large-scale manufacturing.
Asunto(s)
Arginina/química , Detergentes/química , Ácido Glutámico/análogos & derivados , Lauratos/química , Anticuerpos de Cadena Única/metabolismo , Animales , Pollos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Fluoresceína/química , Ácido Glutámico/química , Cuerpos de Inclusión , Modelos Químicos , Muramidasa/química , Replegamiento Proteico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/química , Espectrometría de Fluorescencia , TemperaturaRESUMEN
More than 50 detergents, including acylated amino acid derivatives, were screened for their ability to solubilize and refold recombinant proteins expressed as inclusion bodies. Two model proteins, human interleukin-6 and microbial transglutaminase, were solubilized by these detergents and the solubilized proteins were rapidly diluted for testing their solubilization and refolding effectiveness. Long chain-acylated amino acid derivatives having dicarboxylic acid moieties were found to be superior to others under the conditions tested. In particular, lauroyl-l-glutamate (C12-l-Glu) showed the highest recovery of the native proteins. The effectiveness of dilution refolding was greatly improved by adding aggregation suppressive arginine into the refolding solvents. To gain understanding how this detergent works, interactions between detergents and proteins were examined using spectroscopic and native gel electrophoretic analyses, showing ideal properties for C12-l-Glu as a solubilzing agent, i.e. highly reversible nature of the detergent binding to the model globular proteins and of the conformational changes. These properties most likely have contributed to the effective protein solubilzation and refolding of inclusion bodies using C12-l-Glu and arginine.
Asunto(s)
Arginina/metabolismo , Detergentes/metabolismo , Glutamatos/metabolismo , Interleucina-6/metabolismo , Replegamiento Proteico , Transglutaminasas/metabolismo , Humanos , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Interleucina-6/química , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Transglutaminasas/químicaRESUMEN
Loss of protein aggregates due to matrix protein interaction during SEC often causes underestimation of aggregate content in the quality control of pharmaceutical proteins. Since new columns especially show stronger tendency to bind proteins, an effort should be made to suppress protein adsorption. We examined the effects of arginine on protein binding to SEC columns and found that loss of aggregates was reduced in the presence of arginine, leading to a reliable estimate of aggregate content even with newly used column and small protein load. This is not due to increased ionic strength, as NaCl resulted in increased protein loss.
Asunto(s)
Arginina/química , Cromatografía en Gel/métodos , Tampones (Química) , Cromatografía Líquida de Alta Presión , Inmunoglobulina G/química , Indicadores y Reactivos , Cloruro de Sodio/química , Espectrofotometría UltravioletaRESUMEN
Mildly acidic arginine solution is highly effective in elution of bound proteins from Protein-A columns. Although Protein-A is specific in antibody capture, it does bind other proteins, which must then be removed before elution by aqueous arginine solution. If they are not removed, a strong elution property of aqueous arginine solutions will elute the contaminating proteins along with antibodies. Here we have examined various salt solutions as a column rinse solvent. We screened various solvents for their effects on binding of purified antibodies to Protein-A, instead of their effectiveness to elute the bound contaminants. Those solvents that result in a slight flow-through of the antibodies during loading should be effective in eluting non-specifically bound proteins that have weaker affinity for Protein-A than antibodies: namely, if a particular solvent reduces antibody binding to Protein-A, it is expected to be effective in reducing binding of contaminants and hence eluting them. Such screening showed a few compounds, including arginine and sodium acetate, as potential column rinse agents. A combination of arginine and sodium acetate was tested for a few crude materials containing antibodies.
Asunto(s)
Anticuerpos/aislamiento & purificación , Cromatografía/métodos , Proteína Estafilocócica A/química , Anticuerpos Monoclonales/aislamiento & purificación , Arginina/química , Unión Proteica , Acetato de Sodio/farmacología , SolventesRESUMEN
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.
Asunto(s)
Ácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Humanos , Concentración de Iones de Hidrógeno , Ratones , Conformación Proteica , Temperatura , VolumetríaRESUMEN
A major problem in gel permeation chromatography (GPC) or size exclusion chromatography is non-specific binding of applied proteins to the column matrix (stationary phase). We have tested an aqueous arginine solution as the GPC mobile phase on silica-based and polymer-based columns, using mouse monoclonal antibody and recombinant human activin, interleukin-6, basic fibroblast growth factor, and interferon-gamma as model proteins. We observed that addition of arginine to the mobile phase improves separation of the proteins and their soluble aggregates from the GPC columns, which suggests that arginine is an effective additive for the GPC mobile phase.
Asunto(s)
Arginina/química , Cromatografía en Gel/métodos , Activinas/aislamiento & purificación , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Factor 2 de Crecimiento de Fibroblastos/aislamiento & purificación , Humanos , Interferón gamma/aislamiento & purificación , Interleucina-6/aislamiento & purificación , Ratones , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.
Asunto(s)
Anticuerpos/química , Anticuerpos/aislamiento & purificación , Arginina/química , Cromatografía de Afinidad , Animales , Anticuerpos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Ácido Cítrico/química , Guanidina/química , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/inmunología , Ratones , Proteína Estafilocócica A/químicaRESUMEN
Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatography.