Impact of freezing on ph of buffered solutions and consequences for monoclonal antibody aggregation. A combinatorial histidine scanning library approach to. Buffer screening with the fdaplate quick, simple and. Overall, this relationship assumes independent, noninteracting, protonation events. To address this question we examined the conformational stability of the mab in both buffer systems. 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. 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. Desorption of the histidine tagged protein may be accomplished in one of three ways. Histidine s side chain allows it to act as both a base and an acid, both donating and accepting protons, which can be of considerable importance in its role as part of proteins. Buffer capacity of biologicsfrom buffer salts to buffering by antibodies.
This is a major reason for the difficulty of quantitative computational calculation of protein stability. The statistical design, experimental randomization, and analysis were. In the context of protein structure, the term stability can. This booklet is designed to help answer several basic questions about the use of buffers in biological systems. A quantitative analysis of the effects of ph and buffer system. Jul 10, 2012 one of the key factors in stabilising proteins is determining the optimal ph and buffer system to provide adequate solubility and stability. In a recent analysis of the factors contributing to the stability of rnase t1, the stabilizing and destabilizing interactions were estimated at 271 and 286 kcalmol, respectively pace et al. Histidine tags are small and generally less disruptive to the properties of the protein than other tags. Protein therapies are entering a new era with the in. Histidine is reported to be the preferred buffer for longterm stability of antibodies against programmed cell death protein 1. Stability of proteins in aqueous solution and solid state.
Wo2004055164a2 system and method for stabilizing antibodies. The chemical stability of biologics such as monoclonal antibodies is ph. Investigation of histidine stabilizing effects on ldh during. The buffer systems in and outside cells help maintain that ph. Histidine and acetate as single buffer systems were eliminated in early rounds due to extensive opalescence in all doe test articles see figures 1a and 2. Pdf approaches for capture of histidinetagged proteins in. Arginine, even at a ph close to the antibody isoelectric point, provided better results relative to the other two buffer systems, and stability correlated with higher arginine concentrations. 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. Increased stability of thiophosphohistidine michael lasker,1 cuong d. Investigation of histidine stabilizing effects on ldh. These include a pair of neutral tautomers, denoted as. Or use the plate to determine important parameters for regulatory approval of your antibodies like colloidal stability. Effects of histidine and sucrose on the biophysical. The imidazole ring of histidine can exist in three conformations that almost always exchange rapidly in aqueous buffer solutions.
Protein oxidation by buffer components, antioxidants and trace. Oxidation can alter a protein s physiochemical characteristics e. Currently, three buffers, citrate, phosphate and acetate, make up the majority of buffers used in parenteral pharmaceuticals approved by the fda, but less precedented excipients are certainly available to use in commercial dosage forms. An unknown uv 280 nm absorbing peak was observed by sec for protein stability samples formulated in l histidine during a stress stability study. Stabilized antibody formulations containing histidine are described. Furthermore, this project includes a state of the art chapter, which concerns with fatal protein misfoldings. Role of buffers in protein formulations request pdf researchgate. Before removal of histidine, a histidine gap in the adc sample and a histidine dip in the mab sample are observed around pi 7.
Biopharmaceutical product stability considerations, part. While you need to eat protein to be able to make protein, you need protein for more than just production of buffers. Altering buffer salts, ph, ionic strength, excipients and surfactants may. We have carried out a systematic study by measuring protein protein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mm at 4 ph values 5. Histidine is a commonly used buffer in formulation of monoclonal antibodies mab, often with excipients like sucrose. Investigation of a degradant in a biologics formulation.
Met and cys because of their sulfur atoms and his, trp, and tyr because of their aromatic rings. Even though histidine presented the higher b 22 value and enhanced stability of igg1 at a concentration of 20 mgml, histidine buffer did not better stabilize igg1 at high concentration than the. Because freezethaw cycles decrease protein stability, samples for frozen storage are best dispensed and prepared in singleuse aliquots so that, once thawed, the protein solution will not have to be refrozen. Concentration limits for buffers in protein assays. The stability of the antibody in each buffer formulation was assessed by uvvis. Pdf approaches for capture of histidinetagged proteins. To date, such studies have been confined to observable protein ground states. Histidine removal from formulation buffers using hissep.
Request permission export citation add to favorites track citation. Effect of polysorbate 20 and polysorbate 80 on the higher. Turck1,2,3 1howard hughes medical institute, university of california san francisco, san francisco, california 941430724 2department of medicine, university of california san. This project will introduce the topic of protein folding. A typical purification scheme for monoclonal antibodies involves protein a affinity.
An insight into the fundamental understanding of the mechanism by which protein stabilizes, will help in the formulation of protein based pharmaceuticals. The chemical stability of biologics such as monoclonal antibodies is. Besant,2 kiyoshi sugawara,2 philip thai,2 gergely medzihradszky,2 and christoph w. It can do this by abstracting a proton with its basic nitrogen to make a positively charged intermediate and then use another molecule, a buffer, to extract the proton from its acidic nitrogen. The discussion presented here is by no means complete, but we hope it will help in the understanding of general. Improved stability of a model igg3 by doebased evaluation. Understanding the source would enhance the confidence in the sec results. Rapid formulation development for monoclonal antibodies. Frozen at 20c or 80c is the more common form of cold protein storage. Among the 20 natural amino acids histidine is the most active and versatile member that plays the multiple roles in protein interactions, often the key residue in enzyme catalytic reactions. Apr 29, 2014 an application to an invisible folding intermediate of the im7 protein is provided, where sitespecific pka values of histidine residues have been determined that explain the strong phdependent stability differences between native and intermediate states. The effect of buffers on protein conformational stability.
Impact of an nterminal polyhistidine tag on protein thermal. Better predictive ability of salt and buffer effects on protein protein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion binding, and saltinginout behavior. Concentration series for capture of histidine tagged proteins on sensor chip nta. Posttranslational phosphorylation of proteins is an important event in many cellular processes. The stability of this structure is mediated by the presence of the. A critical question is why the mab is more susceptible to the hydrolysis of the hinge region in phosphate as opposed to histidine buffer systems. Addition of buffers can result in increased conformational stability of proteins. As high concentrated protein formulations become more and more. Protein purification methods process development forum. Keywords protein, formulation screening, stability, automation, biologics, buffer exchange. Protein samples 1 mgml mab in histidine buffer without and with polysorbates were subjected to sedimentation velocity analytical ultracentrifugation svauc experiment on a beckman xla analytical centrifuge at 25 c. This gene encodes a member of the group ii decarboxylase family and forms a homodimer that converts l histidine to histamine in a pyridoxal phosphate dependent manner. In competitive elution, a step or gradient elution with ligands such as imidazole, histidine, histamine, or glycine may be carried out.
Jan 01, 2011 methionine, cysteine, histidine, tryptophan, and tyrosine are most susceptible to oxidation. Histamine regulates several physiologic processes, including neurotransmission, gastric acid secretion,inflamation, and smooth muscle tone. In conformity with the proposition that nature designs the optimum molecules,buffers should mimic the antidenaturant properties of nature exhibited by osmolytes 15 that are independent of the evolutionary history of the proteins 6, 7. There is growing interest in the fast and robust engineering of protein phsensitivity that aims to reduce binding at acidic ph, compared to neutral ph. A theoretical and comprehensive study on the structural features and interaction properties of histidine. 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. Investigation of a degradant in a biologics formulation buffer containing l histidine. 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. Within the tested range 1050 mm, higher concentrations of histidine provided better protein stability. Buffer capacity of biologicsfrom buffer salts to buffering. Better predictive ability of salt and buffer effects on proteinprotein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion binding, and saltinginout behavior. What is the effect of immidazole on activity and stability of. So we selected a histidine containing formulation and performed a 24month realtime stability study.
In a histidine proton shuttle, histidine is used to quickly shuttle protons. Methionine, cysteine, histidine, tryptophan, and tyrosine are most susceptible to oxidation. Load condition, wash salt concentration, temperature, and residence time each affects the doublepeak profile. Pdf impact of freezing on ph of buffered solutions and. C, structure of surface bound mononta molecule left and trisnta molecule right. We have carried out a systematic study by measuring proteinprotein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mm at 4 ph values 5. The objective of this study was to examine the effects of both histidine and sucrose on the biophysical characteristics of a mab. Dec 29, 2016 histidine is a commonly used buffer in formulation of monoclonal antibodies mab, often with excipients like sucrose. Role of buffers in protein formulations sciencedirect. Interplay of buried histidine protonation and protein. 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. Improved stability of a model igg3 by doebased evaluation of. The scope of the present study was to investigate the influence of histidine on the stability of a model protein ldh for concentrations relevant for use as a buffer and as a stabilizer. Use the fdaplate for buffer screening, analysis of protein and antibody stability, buffer optimization, and pre formulation development.
The unknown peak was fractionated by preparative size exclusion. The hydrodynamic radius of the mab was determined by dynamic light scattering and confirmed by size exclusion chromatography. Ninta binding to histidine occurs after activation with nickelii. An understanding of protein stability is essential for optimizing the expression, purifn. Conformational stability has been shown to significantly affect the rates of chemical degradation of proteins, as was shown in studies of oxidation of subtilisin. The charge variants of the adc and the mab were analyzed using icief on ice280. Protein distribution between the two elution peaks correlates with protonation equilibrium.
Because of this, tag removal after purification of a histidine tagged protein is not always of high priority. Semiautomated sample preparation for protein stability and. The project includes theoretical chapters describing proteins, amino acids, protein stability, circular dichroism spectroscopy, and steadystate. Here, we describe a novel strategy for the incorporation of phsensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface. Examples of injections of varying concentrations of histidine tagged proteins over sensor chip nta and anti histidine antibody are shown in figures 3 and 4. Currently, three buffers, citrate, phosphate and acetate, make up the majority. Impact of an nterminal polyhistidine tag on protein. The pka of a buffer is commonly perceived as the ph of the said buffer when the concentrations of the two buffering species are equal, and where the maximum buffering capacity is achieved. Buffers, protein or otherwise, work by consuming small amounts of acid or base added to a solution. While you need to eat protein to be able to make protein, you need protein for more than just production of buffers it helps you maintain all your cells. Generally, protein drugs are effective at low concentrations with less side effects relative to small molecule drugs, even though, in rare cases, protein induced antibody formation could be serious.
Buffer screening with the fdaplate quick, simple and effective. Good buffers generally have a low degree of temperature sensitivity, and carboxylic acid buffers citrate. 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. Key buffer exchange parameters were user defined in the big tuna software. What is the effect of immidazole on activity and stability of enzyme. The presence of such excipients might complicate the identification of the effect of histidine on protein stability during freezedrying. Pnas plus measurement of histidine pk values and tautomer. Expression and purification of proteins using 6xhistidinetag 5 1 introduction 1. Fluorescence spectroscopy in peptide and protein analysis.
Dual effect of histidine on polysorbate 20 stability. The buffer capacity of a mixed weak acidbase buffer system is greater, the closer the individual pka values lie. Protein stability is a topic of major interest for the biotechnol. Gel electrophoresis has become one of the most important methods in the analysis of nucleic acids and proteins. Bufferdependent fragmentation of a humanized fulllength. 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. An adc and a therapeutic mab were formulated in a formulation buffer containing 20 mm histidine. A strategy for studies of histidine side chains in excited protein states. Protein stability and storage indiana university bloomington.
A generic approach to engineer antibody ph switches using. System and method for stabilizing antibodies with histidine. Automated platform buffer screening for multiple proteins. Like all other amino acids, l histidine also acts as a substrate for protein synthesis during translation process. Stability of protein in cores in frozen storage 10, 20, 40c.
Doublepeak elution profile of a monoclonal antibody in. Turck1,2,3 1howard hughes medical institute, university of california san francisco, san francisco, california 941430724 2department of medicine, university of california san francisco, san. We are frequently asked questions on the use of buffers that we offer to research laboratories. Expression and purification of proteins using 6x histidinetag. Each buffer has been approved by fda or ema for use in antibody formulations.
The protonation state of an evolutionarily conserved. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids. In addition, methods of stabilizing liquid formulations of antibodies using histidine are also described. Jul 15, 2011 where k int is the proteinprotein binding constant for the unprotonated species, k and k are the protonation binding constants k p 10 for the ionizable groups in the complex and free states, respectively, and n is the number of ionizable histidine groups. Measurement of histidine pka values and tautomer populations. Most common used buffers for ab formulation, are acetate, histidine, citrate. 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. By plotting a suitable graph, the pka values of histidine can be observed. The objective of this experiment is to determine the buffering region of histidine monohydrochloride by titrating histidine with a base, naoh. By plotting a suitable graph, the pka values of histidine. The multiple roles of histidine in protein interactions. We identified the unknown peak, studied the cause, and evaluated ways to eliminate it. However, it is often forgotten, that when defined as above, pka depends on buffer concentration and temperature.
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