Fundamentals and Applications of Anion Separations
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During glycoprotein production, an enzymatic post-translational modification process attaches glycans, via a process known as glycosylation, to the proteins affecting the final efficacy and safety of the product . The glycosylation profile depends on many manufacturing factors such as media, cell line, culture conditions and bioreactors . A key step to proving biosimilarity is to ensure proper N-glycosylation such as glycoprotein sialylation using the appropriate analytical technology.
HPAE-PAD provides biosimilar manufacturers a highly effective method for the separation and direct quantification of these non-derivatised carbohydrates. HPAE chromatography at high pH coupled with PAD is one of the most employed techniques for carbohydrate compositional analyses for either routine monitoring or research applications. The compatibility of electrochemical detection with gradient elution allows mixtures of simple sugars and oligo- and polysaccharides to be separated with high resolution in a single run.
This technique has great impact on the analysis of diverse carbohydrates ranging from mono-, di-, and polysaccharides. Direct detection analysis of carbohydrates is quite challenging due to their inherent highly polar structure and the absence of a suitable chromophore. HPAE takes advantage of the weakly acidic nature of carbohydrates to give highly selective separations at a high pH using a strong anion-exchange stationary phase . Coupled with PAD, it permits direct detection and quantification of non-derivatised carbohydrates at high femtomolar concentration levels with minimal sample preparation and cleanup .
HPAE chromatography separates anions under high pH conditions. Carbohydrates typically have pKas in the range of 12— Once the pH rises above the pKa of the carbohydrate, these carbohydrates ionise more specifically, become oxyanions and are then separated. These separations require hydroxide-based eluents. For hybrid or complex carbohydrates, separations are accelerated and improved by using sodium acetate gradients in sodium hydroxide. The analytes are separated using one of an extensive Dionex CarboPac column portfolio developed specifically for carbohydrates ranging from mono-, di-, oligo-, and polysaccharides.
These highly cross-linked, ethylvinyl benzene-divinyl benzene pellicular resins have broad pH stability ranging from 0 to 14, allowing separations at high pH conditions. Silica based stationary phases are not stable in high pH mobile phases, limiting their use in carbohydrate separations using an ion exchange mechanism . Once separated, these non-derivatised carbohydrates are detected on a gold working electrode WE surface by PAD, which is a direct detection technique.
Alternative direct detection techniques available for LC of carbohydrates are short wavelength ultra-violet light UV and refractive index RI , both of which lack the sensitivity required for analysing mono- and oligosaccharides from glycoproteins  . Additionally, the detection of these non-derivatised carbohydrates using UV is limited to the choice of the solvent being used.
As most of the solvents and carbohydrates absorb quite strongly below nm. PAD easily addresses the sensitivity issue, as it is able to routinely detect low picomolar amounts of carbohydrates. This method applies a series of potentials a waveform applied two times per second 2 Hz to a gold working electrode, at high pH, resulting in the oxidation of analytes bound to the working electrode surface.
Fundamentals and Applications of Anion Separations | Bruce A. Moyer | Springer
PAD only detects compounds that contain functional groups that can get oxidised at the detection voltage. Detection is sensitive and highly selective for electroactive species, as many potentially interfering species cannot be oxidised or reduced, and so are not detected. A comprehensive monosaccharide profiling is required for biosimilars to meet FDA regulations. This comprehensive monosaccharide analysis helps in the identification and quantification of the different types of glycosylation occurring in these proteins.
In the biosimilar development process, it is critical to analyse monosaccharides in earlier stages to address any issues related to their manufacturing process. This information will prevent the development of an inappropriate products that would not meet the criteria stipulated by the FDA for biosimilars. Sample Preparation: To prepare glycoprotein samples ahead of injection on the HPAE-PAD system for monosaccharide analysis - a sample with PNGase F were first hydrolysed with slightly varied conditions for neutral sugar or amino sugar analyses Figure 1.
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The acid-hydrolysed samples were dried in a SpeedVac concentrator Thermo Scientific equipped with an acid trap and reconstituted in a small volume of deionised water . Samples were acid hydrolysates of specified proteins. Peak results are shown in pmol.
The Dionex CarboPac PA20 delivers excellent monosaccharide resolution while minimising mobile phase consumption and waste generation. The eluent generator eliminates error associated with hydroxide mobile phase preparation e. Inclusion of a Dionex AminoTrap column Thermo Scientific, Sunnyvale, USA delays the elution of amino acids and small peptides from the glycoprotein acid hydrolysis so that they do not interfere with monosaccharide quantification.
After separation, the monosaccharides are detected on a gold working electrode. The amounts of galactosamine GalN and glucosamine GlcN have been determined as an indicator of O- and N-glycosylation, respectively. The identification of the monosaccharides present in both the treated and untreated PSA were compared with the monosaccharide standards, highlighting the sensitivity of the HPAE-PAD system.
In both cases, glucosamine and galactosamine were present in roughly the same molar ratio of 0. This suggests that for this PSA sample, the galactosamine was not a result of O-glycosylation but is associated with the PSA N-glycans, or that there was a contaminating protein with O-glycosylation that is not removed during the sample preparation to separate protein from released N-glycans.
Sialic acids are critical to therapeutic glycoprotein efficacy, bioavailability, function, stability, and metabolism. When present, they occupy terminal positions in glycosylated proteins, providing charged points of interaction essential in many biological pathways. Any unexpected sialylation in biosimilars can cause immunogenicity, so it is critical to monitor total glycoprotein sialylation, and the identification of the sialic acids when changing process and or conditions.
Although over 50 natural sialic acids have been identified, two forms are commonly determined in therapeutic glycoprotein products: N-acetylneuraminic acid Neu5Ac and N-glycolylneuraminic acid Neu5Gc. Due to the potential immunogenicity of Neu5Gc, it is considered undesirable in therapeutic proteins and must be tested for . Sample Preparation: Neu5Ac and Neu5Gc, are negatively charged at pH 7 and acid-labile so require acetate in the eluent and weak acid conditions for hydrolysis. Before injection into the HPAE—PAD system, the samples were subjected to acid hydrolysis or treated with neuraminidase to release the sialic acids, dried to remove the acid, and reconstituted in deionised water.
This information is critical in protein expression experiments for clonal selection for cell line development for therapeutic glycoproteins as well as optimising and monitoring protein production methods. Biosimilars are strong candidates for leveraging themselves as alternative drugs for intended use at a much-reduced healthcare cost. However, FDA regulations must first be satisfied. Comprehensive glycosylation profiling, including sialic acid compositional analysis, is required to determine the efficacy and safety of biosimilars.
HPAE-PAD is the most direct, sensitive, and selective method for monosaccharide, sialic acid, and other carbohydrate analyses. Grabowski, H. Demain, A. Angus, M. The first known chemical reaction used in the analysis was developed by Caius Plinius Secundus A. In the second half of the 17th century, Robert Boyle studied the application of chemical reactions for the identification of several substances also introducing new analytical chemicals. As pointed out by Benedetti-Pichler and co-workers, some chemistry teachers already recognized the superiority of qualitative analysis as a teaching instrument Benedetti-Pichler et.
In addition, the course would comprise instrumental techniques, ion separation and the investigation of the composition of unknown samples. Nowadays, the qualitative analysis lab classes, in many Brazilian universities, comprise separation and identification of cations and anions, by means of individual or group tests.
The cations are divided into five groups according to their precipitation reactions and the group reagent. Apart from of group V, the cation analysis comprises some procedure sequences which aim to separate the interfering ions and obtain the desired species, which is then confirmed by means of specific tests Abreu et al. On the other hand, despite the existence of proposals for grouping the anions according to their volatility or silver salt reactions, there is a great difficulty in determining the composition of anionic mixtures due to chemical incompatibility or similarity of these species Vogel, Thus, the anion analysis is frequently carried out by means individual tests.
The systematic approach previously described for the five cation groups allows a fruitful discussion between teacher and students concerning some Chemistry fundamentals concepts, as equilibria of precipitation, acidity and basicity, and solubility. In order to conduct an investigative laboratory the teacher must lead the class to work the chemical knowledge based on macroscopic observation, what can be visualized during the experiment; microscopic interpretation such as scientific theories that explain the studied phenomena; and representational expression, the use of scientific language such as chemical reactions, formulas, graphs and models that represents the phenomena observed Silva, et.
In this sense, our research group developed recently a didactic approach concerning the systematic analysis of the chloride Cl - , bromide Br - and iodide I - anions Souza et al. Our methodology has been satisfactorily applied in Chemistry undergraduate courses at University of Lavras, in Brazil, since In the present work, we report a new laboratory investigative experiment for separation and identification of the carbonate CO 3 2- , phosphate PO 4 3- and chromate CrO 4 2- anions.
We believe that the proposed experiment can improve the study of anions in undergraduate courses through the discussion of essential topics in chemistry what also contribute to students training. In addition, questions to the students were suggested in order to encourage their active participation in the experimental work.
For acidity measurements, we recommend J Prolab pH indicator strips with 4 colors. For separations, a centrifuge such as the QTM model Quimis. Use a pipette to add 0. To the stirred resulting mixture add 0. Add 1. Discard the supernatant into appropriated residue flask.
Then use the centrifuge for another 3 minutes at RPM. Into the tube B slowly add NaOH 3. At this point, a white solid is formed.
Into tube A, add 1. The pH will reach 2 after solubilization. At this point, observe the color change. Then add 1. This results in a system with two phases. To such system, add the H 2 O 2 3. At first, the teacher should ask the students about the possibility of qualitative separate carbonate, phosphate, and chromate anions in an aqueous solution. For teaching aims, the incomplete flowchart depicted in Figure 1 is presented and discussed. On the basis of this flowchart, the first point introduced corresponds to the precipitation equilibrium and solubility.
In Walther Nernst showed that the equilibrium between an ionic solid and its aqueous solution is governed by a solubility-product constant Ksp. Such product determines a limit to the ionic concentration in aqueous media.
Above such limit, the ions present in the solution will interact to form the solid salt, the precipitate. Butler, When the salts present the same stoichiometry their solubility can be compared by the Kps value. However, for salts of different stoichiometry, it is necessary to calculate molar solubility to determine the species that will precipitate first. The addition of this reagent results in the formation white and yellow precipitates.