The mixture of the two buffering salts allows us to control ph well in the range of 4 8. The imidazole ring of histidine can exist in three conformations that almost always exchange rapidly in aqueous buffer solutions 51. Expression and purification of proteins using 6x histidinetag. Apr, 2017 since prp protein stability is heavily influenced by ph and because histidine pka is in the physiologically relevant range, histidine pka values for hamster and rabbit prp were measured by. Priority date the priority date is an assumption and is not a legal. To assay metalinduced structural changes, protein samples 10m were preincubated with 1mm of different metal ions as. Herein we describe the development of several types of affinity ligands which enable the purification of antibodies under much milder conditions. Dec 29, 2016 histidine is widely used as a buffer in formulation of mabs and other recombinant protein products in both the liquid and solid lyophilized and frozen states. Data file 28404105 ab affinity chromatography imac. The state of the iron atom may also influence t m fig. If interaction a is weak, there is no binding of the 6xhistidinetag protein.
This is the primary step in histidine degradation that has been studied in. Why is trehalose an exceptional protein stabilizer. Dual effect of histidine on polysorbate 20 stability. A combinatorial histidine scanning library approach to. Mar 01, 20 furthermore, selective crystallization of buffer components histidine in this case during freezing is expected to also cause ph shifts and thereby lead to the denaturation of phsensitive proteins. Results staphylococcal protein a variants were engineered by using both. Effects of histidine and sucrose on the biophysical properties of a. Aug 03, 2012 the protein was then concentrated using a centrifugal filter device amicon with a molecular mass cutoff of 3 kda, purified by size exclusion chromatography hiload 1660 superdex 75 prep grade column, ge healthcare by eluting the proteins with 10 m m sodium phosphate buffer, ph 7. The stability of biologics can be highly dependent. Increasing protein stability by improving betaturns. Buffer capacity of biologicsfrom buffer salts to buffering. The amyloid precursor protein copper binding domain. Key buffer exchange parameters were user defined in the big tuna software.
Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freezedried state. Histidine tagged decrease concentration of imidazole and verify ph range of 78. Since control of ph is widely provided by added buffer salts, the current study summarizes the buffer characteristics of acetate, citrate, histidine, succinate, and phosphate buffers. Investigation of histidine stabilizing effects on ldh. This suggests that histidine deprotonation has little effect on the rate of im7 folding from the intermediate state. Investigation of histidine stabilizing effects on ldh during freeze. Gear up your histidinetagged protein purification today. Cold shock protein b cspb, histidine containing phosphocarrier protein, ubiquitin, ribonucleases sa2, sa3, t1, and hi, tryptophan synthetase alphasubunit, and maltose binding protein. A protein drug substance or drug product is usually dissolved in a formulation buffer, for which histidine is commonly used. Histidine is a commonly used buffer in formulation of monoclonal antibodies mab, often with excipients like sucrose.
The effect of buffers on protein conformational stability aws. The pk, values of two histidine residues in human haemoglobin. Pdf hyperthermal stability of neuroglobin and cytoglobin. Desalting has been performed to remove imidazole and nacl. The aim of the study was to investigate the effect of histidine on the stability and physical properties of a fully human antiil8 monoclonal antibody abxil8 in aqueous and solid forms. Nmr spectroscopy has become an important tool for studies of histidine side chains through the measurement of sitespecific pk. Impact of freezing on ph of buffered solutions and. Furthermore, selective crystallization of buffer components histidine in this case during freezing is expected to also cause ph shifts and thereby lead to the denaturation of phsensitive proteins. Fluorescence spectroscopy in peptide and protein analysis.
An understanding of protein stability is essential for optimizing the expression, purifn. Uncle sets a new benchmark for protein characterization two additional formulations. Impact of an nterminal polyhistidine tag on protein thermal. Protein quantification in formulation buffer using a. Semiautomated sample preparation for protein stability and. High stability of histidine tagged protein captured on the htg chip the use of trisnta instead of traditional nta mononta enables high ligand stability, a requisite for analysis of interaction kinetics. Doe design of experiments analysis of the effects of ph, buffer type. The concentration range of histidine for elution of.
Pdf measurement of histidine pk a values and tautomer. Novel approaches for stabilization and characterization of. The chemical stability of biologics such as monoclonal antibodies is ph. Purification and characterization of a histidinebinding protein from. Protein stability can also be improved using excipients, including.
The objective of this study was to examine the effects of both histidine and sucrose on the biophysical characteristics of a mab. Interplay of buried histidine protonation and protein. In antibody purification processes, the acidic buffer commonly used to elute the bound antibodies during conventional affinity chromatograph, can damage the antibody. Background in antibody purification processes, the acidic buffer commonly used to elute the bound antibodies during conventional affinity chromatograph, can damage the antibody. Protein stability a study of the stability of hen eggwhite lysozyme exposed to chemical and thermal denaturation at ph 4, ph 7, and ph 10 authors anders sobye asger kolding marielouise knop lund mia dalgaard jensen school of engineering and science aalborg university 2015 group 4. Resultsa strategy for studies of histidine side chains in excited protein states. Improved stability of a model igg3 by doebased evaluation of.
Kinetic control of histidinetagged protein surface density. Impact of an nterminal polyhistidine tag on protein. Histidine is a commonly used buffer in formulation of monoclonal antibodies. Investigation of a degradant in a biologics formulation.
The amyloid precursor protein copper binding domain histidine. Protein preparation a therapeutic igg1type mab was dialyzed into the formulation buffers listed in table 1. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins. Uncle sets a new benchmark for protein characterization. Of the 15 single proline mutations, 11 increased stability average 0. Degradation of polysorbates 20 and 80 catalysed by. Role of buffers in protein formulations request pdf researchgate. Lightinduced covalent buffer adducts to histidine in a. Dec 20, 2012 protein stability was evaluated both functionally by measuring the activity recovery of the model protein ldh after freeze. Jun 26, 2019 the same concentration of buffer was used but differing amounts of salt were added to change the ionic strength. The goal of this work is to understand if covalent buffer adducts on his residues can be generated by light through similar mechanisms in nucleophilic buffers such as tris and his.
Histidine binds to ninta and competes with histidine can be used at low concentrations 12 mm to inhibit non specific binding and at higher concentrations 20 mm to elute the histagged protein from the ninta matrix. Other techniques adapted to the analysis of proteinprotein interactions. Second, the possibility of using histidine as both buffer and stabilizer cryoprotectant and lyoprotectant in the protein formulations was evaluated with focus on protein stability and the. The present application describes antibody formulations, including monoclonal antibodies formulated in histidine acetate buffer, as well as a formulation comprising an antibody that binds to domain ii of her2 for example, pertuzumab, and a formulation comprising an antibody that binds to dr5 for example, apomab.
Histidine tagged protein is buried within protein structure. Sep 23, 2015 different buffer substances had some effect at ph stability than histidine, citrate, or succinate buffers. Because loss of the disulfide bond in human ngb increases the affinity of the distal histidine table 1, the protein stability may depend more directly on the hexacoordination rather than the disulfide bond. Rapid formulation development for monoclonal antibodies. However, no such trend was observed when studying the effect of ph on adsorbed hbsag. Role of buffers in protein formulations request pdf. The histidine imidazole side chain plays a critical role in protein function and stability. If desired, save supernatant for downstream analysis. Analysis of these sigmoidal curves to determine the free energy of. Wo2004055164a2 system and method for stabilizing antibodies. The type of buffer has also been reported to influence the stability of mabs. Excipients use in parenteral and lyophilized formulation. Protein stability can also be improved using excipients, including both. Ldh showed improved stability in histidine buffer in comparison with other buffers.
Mulkerrin steven m chamow original assignee abgenix, inc. Prepare sample by mixing the protein extract with an equal volume of equilibrationwash buffer. Protein stability was assessed by visual inspection, sub. Using a fractional factorial design, we tested many excipients, including histidine, sucrose, and other commonly used excipients, on the stability and physical properties of the antibody in. Influence of histidine on the stability and physical properties of a. This would suggest that ngb without the disulfide bond is the most stable form.
Since histidine is a very common buffer for therapeutic proteins, especially for mabs 37, an explanation with the low physical stability of mab1 in this buffer is. I will need this protein for downstream assays so its activity is important. Protein stability is a topic of major interest for the biotechnol. The protonation state of an evolutionarily conserved. The stability of the adsorbed antigen was very low across the whole ph range between 5 and 9. Automated platform buffer screening for multiple proteins on. Data file 18117440 af tagged protein purification ni.
The size of the c terminal amino acid retards the formation of the cyclic imide. Effects of histidine and sucrose on the biophysical. Role of buffers in protein formulations sciencedirect. Along these lines, mechanisms for protein stabilization using buffers have previously been. Ligand drift can lead to distorted kinetic results or make the fitting process to a kinetic model inaccurate or even impossible. Histagged proteinsproduction and purification thermo. Keywords protein, formulation screening, stability, automation, biologics, buffer exchange. Kinetic control of histidine tagged protein surface density on supported lipid bilayers jeffrey a. The capture of 6xhistidinetag proteins by metalchelate affinity matrices relies on two interactions. Ep1802344b1 antibody formulation in histidineacetate. However, it was reported that cold denaturation of protein can be minimized by the use of buffer 55,58.
Resultsin our efforts to develop and improve methods for the analysis of histidine kinases and histidine phosphorylated proteinsmedzihradszky et al. All protein concentrations were verified with a280 measurements collected on a lunatic. The crosslinking is resulted from the nucleophilic attack of a his to a photooxidized his from another protein. Stability testing of therapeutic proteins using dls and. Automated platform buffer screening for multiple proteins. Development of a highthroughput formulation screening. Histidine tagged protein does not elute from the column. First, histidine was used as a buffer in the protein formulations and compared with. Engineered protein a ligands, derived from a histidine. Pnas plus measurement of histidine pk values and tautomer. Add the prepared protein extract to the tube and mix on an endoverend rotator for 30 minutes. The decrease of intracellular ph inhibits glycolysis and the buffering capacity of.
Investigation of histidine stabilizing effects on ldh during. Histidine has a pk a of ph 6 and is thus very effective at controlling ph under weakly acidic conditions, which are attractive for mab formulations due to the high isoelectric point. Up to 2 m can be used, at least 300 mm should be used. Jun 01, 2020 effect of the formulation condition on the kcr. To assay phdependent stability, protein 10m was measured in 20mm mops, ph 7. Staphylococcal protein a variants were engineered by using both structurebased. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in. Your histidine tagged histagged purification and analysis workflow fig 1 includes sample preparation, filtration, purification, purity check and western blotting for protein identification andor quantitation. Influence of histidine on the stability and physical. The result is expression of a recombinant protein with a 6xhis or polyhistag fused to its n or cterminus. Protein stability uncle was used to analyze the stability of each of the four stock mabs and each of the 24 formulations that resulted from buffer exchange on big table 2. Apr 01, 2019 in this study, we explore the impact of each histidine residue in the dimerization and folding stability of the forkhead domain of foxp1 using both wild type protein wt and the h59a mutant. Groves, department of chemical engineering and department of chemistry, university of california, berkeley.
1271 847 349 984 733 1190 496 366 533 1347 992 1488 959 1552 1209 1006 742 1172 1459 360 180 939 715 148 983 685 239 236 78 758 1570 434 1133 527