The fractions of the shoulder and the fragment peak of mAb-A in acetate pH 4.5 isolated from the SEC column were also analyzed by SDS-PAGE. Each well was then loaded with 0.5g of protein sample. 2b. PubMed Terms of Service. Learn more about Institutional subscriptions, Akers MJ (2002) Excipient-drug interactions in parenteral formulations. m value regardless of buffer species. 2c and the greater part was fragment forms due to the hydrolysis. https://doi.org/10.1016/j.addr.2016.11.004, Ellerby LM, Escobar WA, Fink AL, Mitchinson C, Wells JA (1990) The role of lysine-234 in -lactamase catalysis probed by site-directed mutagenesis. Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species. 22*) values, obtained by dividing the experimental virial coefficient B As a result, the surface accessibility of mAb-A to acidic pH 4.5 solution was increased, which might cause more fragmentation. Table 1. [ 1] Experimental conditions were described elsewhere (12,13). It has been known that buffer species differentially alter the conformational stability of a mAb and cause fragmentation . 1, where R is designated as Rayleigh ratio, K represents an instrument constant, M Although the conformational structure of mAb-A in the buffer species of phosphate pH 6.5, citrate pH 6.5, and acetate pH 6.5 were similar, the potency of the protein-protein interaction was different. 2010;99:296274. This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. Anal Biochem 357:289298. Kellner, R., Malempr, R., Vandenameele, J. et al. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Biochemistry 29:57975806. Salinas B, Sathish H, Shah A, Carpenter J, Randolph T. Buffer-dependent fragmentation of a humanized full-length monoclonal antibody. The value of dn/dc used for the calculation of K was 0.185mL/g. Taken together, the results derived from this study suggested that the attractive protein-protein interaction played the major role in controlling aggregation formation of mAb-A. Figure 2 shows the kinetics of degradation of mAb-A in the four selected formulations. By varying the amount of each salt, a range of buffers can be prepared that buffer well between pH 5.8 and pH 8.0 (please see the tables below). 22 Consequently, the enhanced surface accessibility might facilitate the fragmentation. In addition, a Waters Acquity (UPLC) coupled to a Thermo Fisher LTQ was employed for all tryptic peptide sequence confirmation. These tablets elicit a fast, convenient, and accurate method for the synthesis of phosphate citrate buffer solutions. Add 2.421 g of Citric Acid to the solution. https://doi.org/10.1021/bi00453a018, Waley SG (1975) The pH dependence and group modification of lactamase I. Biochem J 149:547551. But such a pH range causes only a smaller fraction of total pilocarpine . Three phosphate and four citrate formulations, each containing 200 mg/ml of the API and 20 mM buffer, were lyophilized from solutions at pH 6.6-7.35 (phosphate formulations) and 6.4-6.95 (citrate formulations). Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. Biochemistry 29:145153. 3) revealed that the fragment peak fraction isolated from the SEC column contained two components: hydrophilic and hydrophobic components, in comparison with the monomer. PubMed m) and the onset temperature (T In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. 1a) to be used over the complete pH range (i.e. https://doi.org/10.1016/S1074-5521(01)00053-9, Matagne A, Dubus A, Galleni M, Frre JM (1999) The -lactamase cycle: a tale of selective pressure and bacterial ingenuity. 1.97 . Protein Sci 11:500515. Ye S, Strzalka J, Churbanova IY, Zheng S, Johansson JS, Blasie JK. https://doi.org/10.1016/j.addr.2011.06.011, Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E, Carver T, Asel E, Springer BA, Lane P, Salemme FR (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. In contrast, the conformational structure of mAb-A in different buffer species phosphate, citrate, and acetate at pH 6.5 was similar and the aggregation was primarily influenced by the attractive protein-protein interaction. Gomori buffers, the most commonly used phosphate buffers, consist of a mixture of monobasic dihydrogen phosphate and dibasic https://doi.org/10.1007/s11095-018-2398-1, Huynh K, Partch CL (2015) Analysis of protein stability and ligand interactions by thermal shift assay. DSC thermal transition profiles of mAb-A in phosphate pH 6.5, citrate pH 6.5, acetate pH 4.5, and acetate pH 6.5 in the presence of 20mM NaCl. Comparative structural studies of Vpu peptides in phospholipid monolayers by X-ray scattering. How Do Buffers Work? However, they have a number of potential disadvantages: Phosphates inhibit many enzymatic reactions and procedures that are the foundation of molecular cloning, including cleavage Correspondence to https://doi.org/10.1016/j.jmb.2008.11.046, Elgundi Z, Reslan M, Cruz E, Sifniotis V, Kayser V (2017) The state-of-play and future of antibody therapeutics. Add 3.394 g of Sodium Phosphate Monobasic Monohydrate to the solution. Ionescu RM, Vlasak J, Price C, Kirchmeier M. Contribution of variable domains to the stability of humanized IgG1 monoclonal antibodies. Phosphates have a very high buffering capacity and are highly soluble in water. Biotechnol J. Microb Cell Fact 13:180. https://doi.org/10.1186/s12934-014-0180-6, Rhazi-Filali F, Zaid A, Ledent P, Vanhove M, Van Beeumen J, Frre J-M (1996) A -lactamase produced by a thermophilic Bacillus. Guest Editors: Jason McConville and Javier Morales, Zheng, S., Qiu, D., Adams, M. et al. The ophthalmic solutions containing pilocarpine (as nitrate or hydrochloride salts) are buffered in the pH range of 4.5-5.0 for reasons of optimum chemical stability. Google Scholar. The different optimal pHs reflect the uniqueness of the amino acid sequence in controlling stability. The authors declare no conflicts of interest or competing interests. m values. https://doi.org/10.1006/jmbi.1998.2148, Vandenameele J, Lejeune A, Di Paolo A, Brans A, Frre JM, Schmid FX, Matagne A (2010) Folding of class a -lactamases is rate-limited by peptide bond isomerization and occurs via parallel pathways. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Drug Product Science and Technology, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, New Jersey, 08903, USA, Songyan Zheng,Difei Qiu,Monica Adams,Jinjiang Li,Rao V. Mantri&Rajesh Gandhi, You can also search for this author in Table 1. The buffer capacity of monoclonal antibodies is practically identical at the process relevant temperatures 5, 25, and 40C. Required components Prepare 800 mL of distilled water in a suitable container. The instrument constant K is defined by \( K=\frac{4{\pi}^2{n}_0^2{\left(\mathrm{d}\mathrm{n}/\mathrm{dc}\right)}^2}{N_{\mathrm{A}}{\lambda}_0^4} \), where n Semin Cell Dev Biol 15:316. 2009;105:51523. FLuc mRNA in the aqueous phase was dissolved in 43-50 mM sodium acetate buffer pH 4 to reach 1 mg/ml, keeping the NP lipid:mRNA ratio (moles amine of the ionizable lipid: moles phosphate of the . 2003;84:42393415. It has been addressed that the degradation behaviors associate with the high specificity of each individual case in terms of kinetics and mechanism (7). Thepeak occurred at the left side of the main peak (monomer peak) shows larger area compared with that occurred at the right side. These tablets elicit a fast, convenient, and accurate method for the synthesis of phosphate citrate buffer solutions. PubMedGoogle Scholar. Since three formulations contained the same concentration of NaCl and the pH values are identical, the apparent differences on the aggregation levels are likely due to the buffer species that induced different attractive forces among three buffer species. Gomori buffers, the most commonly used phosphate buffers, consist of a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate. 22/B volume50,pages 473490 (2021)Cite this article. In general, the knowledge obtained from this study should be useful as a reference for formulation development but it could not be extrapolated to other mAbs. This is generally defined as the pK a or the negative log of the dissociation constant of the acid. . Preparation of Citrate Buffer (pH 3.0-6.2) 4 C. Preparation of Phosphate Buffer (pH 5.8-8.0 at 25C) 4 D. Composition of Additional Buffers and Solutions 4 . 2851 Citrate Phosphate Buffer Concentrate NACRES: NA.47 Recommended Products Sigma-Aldrich P4809 Phosphate-Citrate Buffer View Price and Availability Sigma-Aldrich C2488 Citrate Buffer Solution, 0.09 M View Price and Availability Sigma-Aldrich C9999 Citrate Buffer, pH 6.0, 10, Antigen Retriever View Price and Availability Sigma-Aldrich P4922 As a result, the surface of the double light chain was more hydrophilic than that of the native monomer. Fold Des 3:R9-23. Acta Crystallogr Sect D Biol Crystallogr 67:915919. The samples were prepared in Laemmli sample buffer and heated at 100C for 2min. https://doi.org/10.1128/AAC.45.10.2807, Article The dimerization of haematoferrihaem was studied in phosphate buffer in the pH range 7.02-8.14. 2008;25:188190. m value are compared in Fig. https://doi.org/10.1007/s00769-002-0470-2, Lejeune A, Vanhove M, Lamotte-Brasseur J, Pain RH, Frre JM, Matagne A (2001) Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 -lactamase. Google Scholar. Biophys J. Protein Sci 10(10):21232130, Kotov V, Mlynek G, Vesper O, Pletzer M, Wald J, Teixeira-Duarte CM, Celia H, Garcia-Alai M, Nussberger S, Buchanan SK, Morais-Cabral JH, Loew C, Djinovic-Carugo K, Marlovits TC (2021) In-depth interrogation of protein thermal unfolding data with MoltenProt. On the other hand, the attractive interaction is different evidenced by B Required components Prepare 800 mL of distilled water in a suitable container. m-onset) of the major transition for each sample were then determined using MicroCal VP-Capillary DSC Automated Analysis, Origin 7. https://doi.org/10.1016/j.pnmrs.2014.05.003, Goldberg DS, Bishop SM, Shah AU, Sathish HA (2011) Formulation development of therapeutic monoclonal antibodies using high-throughput fluorescence and static light scattering techniques: role of conformational and colloidal stability. The confirmed fractions were then further analyzed using liquid chromatography mass spectrometry (LC-MS) and hydrophobic interaction chromatography (HIC). PLoS ONE 13:e0190974. It was found that pH 4.5 caused more fragmentation compared with the same buffer species at pH 6.5. Thus; pH=pKa (+/-)1. Among various factors, the direct environment (e.g. Biotechnol Bioeng. These isotonic buffer solutions containing 1% (w/v . 2. side effects which vary with the tissue type: a. osmolarity so that cells and organelles neither swell nor shrink during fixation. J Biomol Screen 6:429440. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. https://doi.org/10.1007/s40005-017-0341-9, Talley K, Alexov E (2010) On the pH-optimum of activity and stability of proteins. Biotechnol Bioeng. Pharm Res 35:114. They remove the time-consuming and tedious process of weighing individual components in buffer preparation. Proteins 78:26992706. When we added a strong acid or strong base at small . So, for example if we prepare acetate buffer. https://doi.org/10.1002/jps.21955, Hung JJ, Dear BJ, Dinin AK, Borwankar AU, Mehta SK, Truskett TT, Johnston KP (2018) Improving viscosity and stability of a highly concentrated monoclonal antibody solution with concentrated proline. Biochemistry 49:42644275. Citric acid/ Sodium citrate 3.0 - 6.2 . Because phosphates precipitate in ethanol, it is not possible to precipitate DNA and RNA from buffers that contain significant Article Protein aggregation kinetics, mechanism and curve-fitting: a review of the literature. 22* values for formulations of phosphate pH 6.5, citrate pH 6.5, and acetate pH 6.5 are all negative, indicating the attractive interaction. Biochem J 315(Pt 2):537541. For the smaller component, it consisted of a potion of amino acid sequence of the CH2 domain, and this fraction was much more hydrophobic compared with the monomer (Fig. Google Scholar, Dobson CM (2004) Principles of protein folding, misfolding and aggregation. As shown in Fig. Acta Crystallogr Sect Struct Biol Commun 71:13591364. https://doi.org/10.1107/S0907444911036225, El Hajjaji H, Dumoulin M, Matagne A, Colau D, Roos G, Messens J, Collet JF (2009) The zinc center influences the redox and thermodynamic properties of Escherichia coli Thioredoxin 2. PubMed Central The most common buffer systems used in ophthalmic formulations are citrate, phosphate, Tris-HCl (Tris), and borate buffer ( Table 1 ). The primary degradation of acetate pH 4.5 was fragmentation as shown in Fig. A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. J Pharm Sci 99:17071720. European Biophysics Journal Biophys J. Article Int J Pharm 185:129188. Altogether, the results suggesting the conformational structures of those three different types of buffer species at pH 6.5 are comparable. This mass was exactly double of the theoretical mass of the intact light chain. SDS-PAGE was performed using TrisHCl 415% gel (Bio-Rad Cat: # 345-0027) under non-reducing conditions. The major transition temperature (T PubMedGoogle Scholar. Table 1. Comparison of the HIC chromatogram of mAb-A in acetate pH 4.5 with that of the fragment fraction isolated from the SEC column. Differential scanning calorimetry (DSC) has been used to evaluate the likelihood of degradation (8), but it has been limited to predict the direction of degradation. Bee JS, Stevenson JL, Mehta B, Svitel J, Pollastrini J, Platz R, et al. https://doi.org/10.1002/prot.22786, Ullah JH, Walsh TR, Taylor IA, Emery DC, Verma CS, Gamblin SJ, Spencer J (1998) The crystal structure of the L1 metallo--lactamase from Stenotrophomonas maltophilia at 1.7 resolution. Among those types of degradation, aggregation has been a major focus since it also causes immunogenic effects during therapy (3). Daugherty AL, Mrsny R. Formulation and delivery issues for monoclonal antibody therapeutics. https://doi.org/10.1016/j.pep.2013.08.002, Boivin S, Kozak S, Rasmussen G, Nemtanu IM, Vieira V, Meijers R (2016) An integrated pipeline for sample preparation and characterization at the EMBL@PETRA3 synchrotron facilities. maleate (pK1) . A model membrane protein for binding volatile anesthetics. The presence of phosphate ions may decrease net charge of the polyamine ligand. m-onset and T The formulation screen allowed automated and fast determination of optimum buffer and pH profiles for the tested proteins. The monomer peak and shoulder fractions isolated from the SEC column were further analyzed by LC-MS spectrometry, which revealed very similar amino acid sequences. McIlvaine buffer is a buffer solution composed of citric acid and disodium hydrogen phosphate, also known as citrate-phosphate buffer. While the SEC chromatograms for each of the four selected formulations were identical at the initial time point (data not shown), the relative level of aggregates and fragments varied after being stressed at 40C for 6weeks. In comparison, the DSC trace of mAb-A of acetate pH 4.5 shows an additional transition occurring at lower temperature. 22* values as displayed in Table II. As indicated in Table I as well, the formulations in acetate buffer at pH 4.5 exhibited the lowest T Google Scholar. https://doi.org/10.1371/journal.pone.0190974, Conrath KE, Lauwereys M, Galleni M, Fre J (2001) -Lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. The results obtained from LC-MS study confirmed that the shoulder isolated from SEC column had a similar amino acid sequence as the monomer itself. Present address: Xpress Biologics SA, Accessia Pharma Site, Avenue du Parc Industriel, 89, 4041, Milmort, Belgium, Laboratory of Enzymology and Protein Folding, Centre for Protein Engineering, InBioS, University of Lige, Building B6C, Quartier Agora, Alle du 6 Aot, 13, 4000, Lige (Sart-Tilman), Belgium, Ruth Kellner,Romain Malempr,Julie Vandenameele,Alain Brans,Marylne Vandevenne&Andr Matagne, Eurogentec S.A., Rue Bois Saint-Jean, 5, 4102, Seraing, Belgium, Anne-Franoise Hennen,Nomie Rochus&Alexandre Di Paolo, You can also search for this author in The observed fragments included the derivative of monomer with a molecular weight of 110kDa, light chain, and the portion of CH2 domain. 1. The principle of this method is that protein molecules in the solution adsorb ultraviolet light with absorbance maxima at 280nm. The protein concentrations of all the samples were determined using a NanoDrop Spectrophotometer (Thermo Scientific, Delaware) by the direct UV A280 method. Therefore, formulation development primarily focuses on identifying key modifications such as aggregation, fragmentation, oxidation, and deamidation since all those types of degradation reduce biological efficacy (2). m values at pH 6.5 were comparable between buffer species. https://doi.org/10.1042/bj1490547, Wang W (1999) Instability, stabilization, and formulation of liquid protein pharmaceuticals. Antimicrob Agents Chemother 38:21432149. PubMed Int J Pharm. Protein Expr Purif 91:192206. Ph range of some buffer systems. Acetate pH 4.5 and acetate pH 6.5 had the same concentration of NaCl, yet different pH values; the effect of solution pH on formulation stability, therefore, was studied. 2a, the primary degradation for the three formulations at pH 6.5 is aggregation and the quantity followed an order: phosphate pH 6.5>citrate pH 6.5acetate pH 6.5. The experimental scattering intensities were fitted according to the model presented in Eq. Not only T quantities of phosphate ions. Phosphates have a very high buffering capacity and . Protein concentrations were determined with high accuracy using a Varian Cary 100 Bio Spectrophotometer (Agilent Technologies, Santa, Clara, CA). The fragment peak fraction contains two components which manifests as bands at 44.6 and 22.6kDa as shown in Fig. Adjust solution to final desired pH using HCl or NaOH Add distilled water until the volume is 1 L. Consequently, the loose state of CH2 domain triggered unfolding of whole mAb-A molecule. https://doi.org/10.1111/j.1574-6968.1996.tb08315.x, Ristic M, Rosa N, Seabrook SA, Newman J (2015) Formulation screening by differential scanning fluorimetry: How often does it work? PubMed Central The results obtained from this study were used to direct the formulation design for the late-stage drug product development of mAb-A. Buffer Range alphabetically. Biochemistry 38:87398750. Proteins Struct Funct Bioinforma 33:550557, Wrn A, Plckthun A (1999) Different equilibrium stability behavior of ScFv fragments: identification, classification, and improvement by protein engineering. The comparison between previous studies and the investigation conducted here indicated the high specificity of each individual monoclonal antibody in terms of degradation behaviors. your institution. 2006;837:3543. Furthermore, SDS-PAGE image shows that the shoulder possessed a molecular weight of 110kDa. 2004;87:406574. A is Avogadros number, and Phosphates sequester divalent cations such as Ca2+ and Mg2+. https://doi.org/10.1016/j.ymeth.2015.08.003, Brannigan J, Matagne A, Jacob F, Damblon C, Joris B, Klein D, Spratt BG, Frere JM (1991) The mutation Lys234His yields a class A -lactamase with a novel pH-dependence. The absorbance of dilute . 22 of DNA by many restriction enzymes, ligation of DNA, and bacterial transformation. The B It has been known that buffer species differentially alter the conformational stability of a mAb and cause fragmentation (18). https://doi.org/10.1016/s0378-5173(99)00152-0, Wheeler KA, Hawkins AR, Pain R, Virden R (1998) The slow step of folding of Staphylococcus aureus PC1 -lactamase involves the collapse of a surface loop rate limited by the Trans to Cis isomerization of a non-proline peptide bond. The fractions eluted from the column were collected at retention times of 9.5 to 10.9min for the native monomer, 10.9 to 11.6min for the shoulder of the monomer, and 12.3 to 13.5min for fragments. BioDrugs 34:1126. 1. buffering capacity in the desired pH range with the ability to maintain constant pH during fixation. Hello everyone, How can I prepare a 0.1 M Citrate-Phosphate Buffer for pHs 4.0, 6.0 and 8.0, using the solids Citric Acid (C6H8O7 MW: 192,12 g/mol) and. Intensities were fitted according to the model presented in Eq pH dependence and group modification lactamase... It also causes immunogenic effects during therapy ( 3 ) the least stable structure and a fragmentation. Sequence in controlling stability ) Principles of protein folding, misfolding and aggregation Talley K, Alexov E 2010! Similar amino acid sequence in controlling stability thermal transition peaks related to the model in..., Stevenson JL, Mehta B, Svitel J, Platz R, al! That pH 4.5 with that of the fragment peak fraction contains two components which manifests as bands 44.6. As shown in Fig loaded with 0.5g of protein folding, misfolding aggregation... Through each slide even when adding powerful acids or bases buffer at pH 6.5 fragment! Springer Nature remains neutral with regard to jurisdictional claims in published maps and Institutional affiliations protein.... Cations such as Ca2+ and Mg2+ were comparable between buffer species pH and buffer species due to the stable. So that cells and organelles neither swell nor shrink during fixation bee,! Solution composed of Citric acid and disodium hydrogen phosphate, also known as citrate-phosphate.!: # 345-0027 ) under non-reducing conditions 2 shows the kinetics of degradation, aggregation has a... ] Experimental conditions were described elsewhere ( 12,13 ) range causes only a smaller of! Of proteins antibody Associated with pH and buffer species study confirmed that the additional thermal transition peaks related the! At 100C for 2min prepared in Laemmli sample buffer and heated at 100C for 2min were comparable buffer! Monolayers by X-ray scattering stability of humanized IgG1 monoclonal antibodies is practically at... The confirmed fractions were then further analyzed using liquid chromatography mass spectrometry ( LC-MS ) and hydrophobic interaction (! Environment ( e.g protein concentrations were determined with high accuracy using a Varian Cary 100 Bio Spectrophotometer Agilent. Monobasic dihydrogen phosphate and dibasic monohydrogen phosphate Waters Acquity ( UPLC ) coupled to a Fisher. The end to navigate through each slide of Sodium phosphate Monobasic Monohydrate to the hydrolysis ions decrease... Stable structure and a greater fragmentation these tablets elicit a fast, convenient, and accurate for! In Fig # 345-0027 ) under non-reducing conditions mAb-A of acetate pH exhibited! Ph range ( i.e, stabilization, and 40C ) coupled to a Thermo Fisher LTQ was employed all... Light with absorbance maxima at 280nm highly soluble in water the conformational structures of three! A mixture of Monobasic dihydrogen phosphate and dibasic monohydrogen phosphate SG ( 1975 ) the dependence... Institutional affiliations comparative structural studies of Vpu peptides in phospholipid monolayers by X-ray.!, Pollastrini J, Randolph T. Buffer-dependent fragmentation of a mAb and fragmentation! Immunogenic effects during therapy ( 3 ) high specificity of each individual antibody... H, Shah a, Carpenter J, Price C, Kirchmeier M. Contribution of variable domains to stability... Of Monobasic dihydrogen phosphate and dibasic monohydrogen phosphate Buffer-dependent fragmentation of a and. ( SEC ) and hydrophobic interaction chromatography ( HIC ) were employed to separate degradation products shows! Acetate buffer at pH 4.5 caused more fragmentation compared with the same buffer species to be used over complete! Navigate the slides or the negative log of the theoretical mass of the fragment fraction isolated from the column! Very high buffering capacity and are highly soluble in water elsewhere ( 12,13.... The phosphate citrate buffer range scattering intensities were fitted according to the hydrolysis the tissue type: osmolarity... Described elsewhere ( 12,13 ) K, Alexov E ( 2010 ) On the pH-optimum of activity and of... At 44.6 and 22.6kDa as shown in Fig phosphate and dibasic monohydrogen phosphate ) Cite this article in formulations. Species differentially alter the conformational stability of proteins g of Sodium phosphate Monohydrate! Even when adding powerful acids or bases Morales, Zheng S, Johansson JS, Blasie JK complete range... Method for the tested proteins, misfolding and aggregation navigate the slides or the controller... Employed to separate degradation products interest or competing interests the stability of humanized IgG1 monoclonal antibodies practically! M values at pH 6.5 were comparable between buffer species at pH 6.5 pH... Antibody therapeutics the DSC trace of mAb-A of acetate pH 4.5 caused more compared! With that of the fragment peak fraction contains two components which manifests as bands at and! % gel ( Bio-Rad Cat: # 345-0027 ) under non-reducing conditions lactamase I. phosphate citrate buffer range J 149:547551 HIC chromatogram mAb-A! 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Fragmentation compared with the tissue type: a. osmolarity so that cells and organelles neither swell shrink!: //doi.org/10.1042/bj1490547, Wang W ( 1999 ) Instability, stabilization, and phosphates sequester divalent cations as! ) On the pH-optimum of activity and stability of proteins as well, the most commonly used phosphate,! Individual components in buffer preparation part was fragment forms due to the solution adsorb ultraviolet with! Suggesting the conformational structures of those three different types of buffer species at pH 4.5 more! Prepared in Laemmli sample buffer and pH profiles for the tested proteins a. Chromatography ( HIC ) were employed to separate degradation products or competing interests the column! Tryptic peptide sequence confirmation values at pH 4.5 shows an additional transition occurring lower! 345-0027 ) under non-reducing conditions mcilvaine buffer is a buffer solution has the function of resisting in... With that of the acid X-ray scattering the enhanced surface accessibility might facilitate the fragmentation of lactamase I. J. Design for the tested proteins hydrophobic interaction chromatography ( HIC ) were employed to separate degradation.... Species at pH 6.5 6.5 were comparable between buffer species citrate-phosphate buffer total pilocarpine 415 gel... Phosphate and dibasic monohydrogen phosphate navigate through each slide the presence of ions... Four selected formulations the tissue type: a. osmolarity so that cells and organelles neither swell nor during! Of each individual monoclonal antibody so that cells and organelles neither swell nor shrink during fixation practically identical the...