ABSTRACT
The stabilization of antibodies in aqueous solution against physical stress remains a problematic issue for pharmaceutical applications. Recently, protein-polyelectrolyte complex (PPC) formation using poly(amino acids) was proposed to prepare antibody formulation in a salt-dissociable precipitated state without protein denaturation. Here, we investigated the stabilization effect of PPC of therapeutic antibodies with poly-l-glutamic acid on agitation and thermal stress as forms of mechanical and non-mechanical stress, respectively. The precipitated state of PPC prevented the inactivation and aggregation induced by agitation. Similar results were obtained using the suspension state of PPC, but the stabilizing effects were slightly inferior to those of the PPC precipitate. PPC precipitate and PPC suspension prevented heat-induced inactivation of the antibodies, but showed little effect on heat-induced aggregation. Thus, PPC is a new candidate as a simple storage method for antibodies in aqueous solution, as an alternative state for freeze-drying.
Subject(s)
Antibodies, Monoclonal/chemistry , Models, Chemical , Pharmaceutical Preparations/chemistry , Polyglutamic Acid/chemistry , Preservatives, Pharmaceutical/chemistry , Adalimumab/chemistry , Adalimumab/metabolism , Animals , Anti-Asthmatic Agents/chemistry , Anti-Asthmatic Agents/metabolism , Antibodies, Monoclonal/metabolism , Antirheumatic Agents/chemistry , Antirheumatic Agents/metabolism , Chemical Precipitation , Chemistry, Pharmaceutical , Drug Stability , Drug Storage , Hot Temperature/adverse effects , Humans , Omalizumab/chemistry , Omalizumab/metabolism , Particle Size , Pharmaceutical Preparations/metabolism , Polyglutamic Acid/metabolism , Preservatives, Pharmaceutical/metabolism , Protein Aggregates , Protein Stability , Solubility , Stress, Mechanical , SuspensionsABSTRACT
Long-term storage in aqueous solution has been demanded for the practical application of therapeutic proteins. Recently, a precipitation-redissolution method was proposed to prepare salt-dissociable protein-polyelectrolyte complex (PPC). To elucidate the utility of the complex for storage of proteins, we investigated the stress tolerance of PPC precipitates containing l-asparaginase (ASNase) and poly-l-lysine (polyK). PPC precipitate containing ASNase and polyK was prepared by precipitation-redissolution method. The sample was treated to three types of stress, i.e., heat, shaking, and oxidation. The protein concentration, enzyme activity, and CD spectrum of the supernatants of samples were measured after stressed. PPC precipitate consisting of ASNase and polyK showed tolerance against thermal and shaking stress compared to the native solution. In addition, PPC precipitate protected ASNase from inactivation by oxidation. PPC precipitate of ASNase/polyK complex successfully stabilized ASNase against physicochemical stresses. These results suggest that the PPC precipitate has great potential as a storage method in aqueous solution for unstable proteins.
Subject(s)
Asparaginase/chemistry , Chemical Precipitation , Drug Storage/methods , Polylysine/chemistry , Asparaginase/metabolism , Electrolytes/chemistry , Enzyme Activation , Hot Temperature , Motion , Oxidation-Reduction , Protein Stability , Solubility , SolutionsABSTRACT
Development of high-concentration antibody formulations for subcutaneous administration remains challenging. Recently, a precipitation-redissolution method was proposed to prepare suspensions or precipitates of salt-dissociable protein-poly(amino acid) complexes. To elucidate the utility of this method for protein therapy, we investigated the feasibility of a precipitation-redissolution method using poly(amino acid) for high-concentration antibody formulation. Omalizumab and adalimumab formulations of 150 mg/mL could be prepared using poly-l-glutamic acid (polyE) from low-concentration stock solutions. Enzyme-linked immunosorbent assay, circular dichroism, and size-exclusion chromatography revealed that the formation of antibody-polyE complex and precipitation-redissolution process did not significantly affect the immunoreactivity or secondary structure of the antibodies. The precipitation-redissolution method was less time-consuming and more effective than lyophilization-redissolution, evaporation-redissolution, and ultrafiltration from the viewpoint of final yield. Scalability was confirmed from 400 µL to 1.0 L. The general toxicity and pharmacokinetic profiles of the antibody-polyE complex formulations were similar to those of conventional antibody formulations. These results suggested that the precipitation-redissolution method using poly(amino acid) has great potential as a concentration method for antibody formulation and medicinal use.
Subject(s)
Adalimumab/chemistry , Anti-Asthmatic Agents/chemistry , Anti-Inflammatory Agents/chemistry , Omalizumab/chemistry , Polyglutamic Acid/chemistry , Adalimumab/blood , Adalimumab/pharmacology , Animals , Anti-Asthmatic Agents/blood , Anti-Asthmatic Agents/pharmacology , Anti-Inflammatory Agents/blood , Anti-Inflammatory Agents/pharmacology , Chemical Precipitation , Humans , Omalizumab/blood , Omalizumab/pharmacology , Polyglutamic Acid/blood , Polyglutamic Acid/pharmacology , Protein Structure, Secondary , RatsABSTRACT
A method for concentration of protein solutions is required for high-dosage protein formulation. Here, we present a precipitation-redissolution method by poly(amino acid) for proteins, including therapeutic enzymes, antibodies, and hormones. The proteins were fully precipitated by the addition of poly-L-lysine or poly-L-glutamic acid at low ionic strength, after which precipitate was dissolved at physiological ionic strength. The activities and secondary structures of redissolved proteins, especially antibodies, were almost identical to the native state. The precipitation-redissolution method is a simple and rapid technique for concentration of protein formulations.
