RESUMEN
Subcutaneous injection of a low volume (<2 mL) high concentration (>100 mg/mL) formulation is an attractive administration strategy for monoclonal antibodies (mAbs) and other biopharmaceutical proteins. Using concentrated solutions may also be beneficial at various stages of bioprocessing. However, concentrating proteins by conventional techniques, such as ultrafiltration, can be time consuming and challenging. Isolation of the dense fraction produced by macroscopic liquid-liquid phase separation (LLPS) has been suggested as a means to produce high-concentration solutions, but practicality of this method, and the stability of the resulting protein solution have not previously been demonstrated. In this proof-of-concept study, we demonstrate that LLPS can be used to concentrate a mAb solution to >170 mg/mL. We show that the structure of the mAb is not altered by LLPS, and unperturbed mAb is recoverable following dilution of the dense fraction, as judged by 1H nuclear magnetic resonance spectroscopy. Finally, we show that the physical properties and stability of a model high concentration protein formulation obtained from the dense fraction can be improved, for example through the addition of the excipient arginine·glutamate. This results in a stable high-concentration protein formulation with reduced viscosity and no further macroscopic LLPS. Concentrating mAb solutions by LLPS represents a simple and effective technique to progress toward producing high-concentration protein formulations for bioprocessing or administration.AbbreviationsArginine·glutamate (Arg·Glu), Carr-Purcell-Meiboom-Gill (CPMG), critical temperature (TC), high-performance size-exclusion chromatography (HPSEC), liquid-liquid phase separation (LLPS), monoclonal antibody (mAb), nuclear magnetic resonance (NMR), transverse relaxation rate (R2).
Asunto(s)
Anticuerpos Monoclonales/química , Extracción Líquido-Líquido/métodos , Estabilidad Proteica , Química Farmacéutica/métodos , Humanos , Soluciones Farmacéuticas/química , Prueba de Estudio ConceptualRESUMEN
Biopharmaceutical proteins are important drug therapies in the treatment of a range of diseases. Proteins, such as antibodies (Abs) and peptides, are prone to chemical and physical degradation, particularly at the high concentrations currently sought for subcutaneous injections, and so formulation conditions, including buffers and excipients, must be optimized to minimize such instabilities. Therefore, both the protein and small molecule content of biopharmaceutical formulations and their stability are critical to a treatment's success. However, assessing all aspects of protein and small molecule stability currently requires a large number of analytical techniques, most of which involve sample dilution or other manipulations which may themselves distort sample behavior. Here, we demonstrate the application of 1H nuclear magnetic resonance (NMR) spectroscopy to study both protein and small molecule content and stability in situ in high-concentration (100 mg/mL) Ab formulations. We show that protein degradation (aggregation or fragmentation) can be detected as changes in 1D 1H NMR signal intensity, while apparent relaxation rates are specifically sensitive to Ab fragmentation. Simultaneously, relaxation-filtered spectra reveal the presence and degradation of small molecule components such as excipients, as well as changes in general solution properties, such as pH. 1H NMR spectroscopy can thus provide a holistic overview of biopharmaceutical formulation content and stability, providing a preliminary characterization of degradation and acting as a triaging step to guide further analytical techniques.
RESUMEN
Proteins frequently exist as high-concentration mixtures, both in biological environments and increasingly in biopharmaceutical co-formulations. Such crowded conditions promote protein-protein interactions, potentially leading to formation of protein clusters, aggregation, and phase separation. Characterizing these interactions and processes in situ in high-concentration mixtures is challenging due to the complexity and heterogeneity of such systems. Here we demonstrate the application of the dark-state exchange saturation transfer (DEST) NMR technique to a mixture of two differentially 19F-labeled 145 kDa monoclonal antibodies (mAbs) to assess reversible temperature-dependent formation of small and large protein-specific clusters at concentrations up to 400 mg/mL. 19F DEST allowed quantitative protein-specific characterization of the cluster populations and sizes for both mAbs in the mixture under a range of conditions. Additives such as arginine glutamate and NaCl also had protein-specific effects on the dark-state populations and cluster characteristics. Notably, both mAbs appear to largely exist as separate self-associated clusters, which mechanistically respond differently to changes in solution conditions. We show that for mixtures of differentially 19F-labeled proteins DEST NMR can characterize clustering in a protein-specific manner, offering unique tracking of clustering pathways and a means to understand and control them.
Asunto(s)
Anticuerpos Monoclonales/análisis , Resonancia Magnética Nuclear Biomolecular , Análisis por Conglomerados , Flúor/química , TemperaturaRESUMEN
PURPOSE: Anti-drug antibodies can impair the efficacy of therapeutic proteins and, in some circumstances, induce adverse health effects. Immunogenicity can be promoted by aggregation; here we examined the ability of recombinant mouse heat shock protein 70 (rmHSP70) - a common host cell impurity - to modulate the immune responses to aggregates of two therapeutic mAbs in mice. METHODS: Heat and shaking stress methods were used to generate aggregates in the sub-micron size range from two human mAbs, and immunogenicity assessed by intraperitoneal exposure in BALB/c mice. RESULTS: rmHSP70 was shown to bind preferentially to aggregates of both mAbs, but not to the native, monomeric proteins. Aggregates supplemented with 0.1% rmHSP70 induced significantly enhanced IgG2a antibody responses compared with aggregates alone but the effect was not observed for monomeric mAbs. Dendritic cells pulsed with mAb aggregate showed enhanced IFNγ production on co-culture with T cells in the presence of rmHSP70. CONCLUSION: The results indicate a Th1-skewing of the immune response by aggregates and show that murine rmHSP70 selectively modulates the immune response to mAb aggregates, but not monomer. These data suggest that heat shock protein impurities can selectively accumulate by binding to mAb aggregates and thus influence immunogenic responses to therapeutic proteins.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas HSP70 de Choque Térmico/farmacología , Animales , Anticuerpos Monoclonales/metabolismo , Formación de Anticuerpos , Femenino , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/inmunología , Fenómenos Inmunogenéticos , Ratones , Ratones Endogámicos BALB C , Agregado de Proteínas , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Estrés MecánicoRESUMEN
Peptide therapeutics have the potential to self-associate, leading to aggregation and fibrillation. Noncovalent PEGylation offers a strategy to improve their physical stability; an understanding of the behavior of the resulting polymer/peptide complexes is, however, required. In this study, we have performed a set of experiments with additional mechanistic insight provided by in silico simulations to characterize the molecular organization of these complexes. We used palmitoylated vasoactive intestinal peptide (VIP-palm) stabilized by methoxy-poly(ethylene glycol)5kDa-cholane (PEG-cholane) as our model system. Homogeneous supramolecular assemblies were found only when complexes of PEG-cholane/VIP-palm exceeded a molar ratio of 2:1; at and above this ratio, the simulations showed minimal exposure of VIP-palm to the solvent. Supramolecular assemblies formed, composed of, on average, 9-11 PEG-cholane/VIP-palm complexes with 2:1 stoichiometry. Our in silico results showed the structural content of the helical conformation in VIP-palm increases when it is complexed with the PEG-cholane molecule; this behavior becomes yet more pronounced when these complexes assemble into larger supramolecular assemblies. Our experimental results support this: the extent to which VIP-palm loses helical structure as a result of thermal denaturation was inversely related to the PEG-cholane:VIP-palm molar ratio. The addition of divalent buffer species and increasing the ionic strength of the solution both accelerate the formation of VIP-palm fibrils, which was partially and fully suppressed by 2 and >4 mol equivalents of PEG-cholane, respectively. We conclude that the relative freedom of the VIP-palm backbone to adopt nonhelical conformations is a key step in the aggregation pathway.
Asunto(s)
Colanos/química , Ácido Palmítico/química , Polietilenglicoles/química , Polímeros/química , Péptido Intestinal Vasoactivo/química , Humanos , Lipoilación , Conformación ProteicaRESUMEN
We report a novel method for the extraction of alpha-1 antitrypsin (AAT) from plasma. This method uses an anion-exchange column and two metal chelate columns. The AAT is recovered in a 60% yield and a purity of over 90%. The AAT manufactured by this method was stable at 4 degrees C for 12 months without any stabilisers. Other proteins of therapeutic value and commercial interest are recoverable in good yields. The simplicity of this process makes it a suitable alternative to the traditionally employed ethanol precipitation method where high ethanol levels inactivate AAT.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , alfa 1-Antitripsina/aislamiento & purificación , Animales , Cromatografía en Agarosa , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Elastasa Pancreática/antagonistas & inhibidores , Elastasa Pancreática/metabolismo , Reproducibilidad de los Resultados , Solventes/química , Porcinos , alfa 1-Antitripsina/sangre , alfa 1-Antitripsina/farmacologíaRESUMEN
VanXY(C), a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His(6)-VanXY(C) were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K(m) was 3.6 mm and k(cat), 2.5 s(-1); for UDP-MurNAc-L-Ala-D-Glu-L-Lys-DAla-D-Ala (UDP-MurNAc-pentapeptide[Ala]), K(m) was 18.8 mm and k(cat) 6.2 s(-1); for D-Ala-D-Ser, K(m) was 15.5 mm and k(cat) 0.35 s(-1). His(6)-VanXYC was inactive against the peptidoglycan precursor UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser (UDP-MurNAc-pentapeptide[Ser]). The rate of hydrolysis of the terminal D-Ala of UDP-MurNAc-pentapeptide[Ala] was inhibited 30% by 2 mm D-Ala-D-Ser or UDP-MurNAc-pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in D-Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in D-Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan. Mutation of an aspartate residue (Asp59) of His-tagged VanXY(C) corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant (k(cat)/K(m)) for D-Ala-D-Ala. This situation is in contrast to VanX in which mutation of Asp68-->Ala produced a greater than 200,000-fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.