This work describes an HPAE-PAD method for the determination of ribitol in Hib capsular polysaccharide acid hydrolysis samples. Separation of ribitol from TFA acid hydrolysis products was achieved under isocratic elution conditions using a Thermo Scientific™ Dionex™ CarboPac™ MA1 column. The method proposed here was validated for performance with respect to linearity, precision, sensitivity, and accuracy. The acid hydrolysis samples were also tested using a palladium hydrogen reference electrode.
Tromethamine is commonly used as a buffering agent, alkalizer, and emulsifying agent in pharmaceutical and cosmetic preparations, and as a counterion for acidic drug substances. This work describes an IC method that uses a Thermo Scientific™ Dionex™ IonPac™ CS20 cation exchange column, electrolytically generated MSA eluent, and suppressed conductivity detection to determine tromethamine in pharmaceutical formulations.
The application note describes an efficient approach for identity confirmation of protein-based impurities of a therapeutic protein by multi heart-cut 2D-LC-MS in a top-down experiment. In addition the Thermo Scientific™ Simple Switch™ technology enables the instrument to be used in multi heart-cut 2D-LC-MS, 1D-LC-MS, or Dual 1D-LC mode for the most flexible equipment and lab space utilization and maximum MS productivity.
We demonstrate a workflow combining chemical release of O-linked glycans, followed by sample cleanup and analysis by HPAE-PAD hyphenated to a Q Exactive HF Hybrid Quadrupole-Orbitrap MS. A recently introduced Dionex CarboPac PA300-4μm column, enables simultaneous separation of neutral and charged glycans without the need for derivatization. High-resolution MS data and MS/MS spectra with diagnostic fragment ions provide reliable structural annotations of heterogeneous glycans. We applied HPAE-PAD/MS to analyze O-glycan structures released from four different glycoproteins.
This record has QAR conditions and results for the 2 mm Dionex IonPac CS19-4µm column. The column must be pre-conditioned offline with acetonitrile and methanesulfonic acid (MSA). See instructions in the column manual (attached). The column is optimized for small, hydrophilic amines such as ethanolamines, methylamines, ethylamines, as well as the biogenic amines.
This application note demonstrates that the AU180 method for sialic acid determination of glycoproteins could be successfully modified for use with a Dionex CarboPac PA20-1 mm column and HPAE-PAD in dual eluent generation cartridge mode.
Single-use technologies (SUTs), in particular single use bioreactors (SUBs), represent an important improvement in biopharmaceutical manufacturing due to reduced requirements for cleaning and sterilization while providing increased sterility assurance, reduced manufacturing turnaround times, and the elimination of cleaning validation and its associated costs. The present work explored the performance of ASE as an extraction technique for characterization of the plastic films (inner layers) from SUBs.
Thermo scientific BioLC ultimate 3000RS instrument is used for the analysis of different form of plasmid DNA. The analysis is performed on high resolution anion exchange DNAPac PA200RS column.The result obtain shows well separation of linear and supercoiled form of plasmid DNA.
This technical note (TN) describes how to develop an ion chromatography method to determine an amine in pharmaceutical samples. Two methods were developed in this TN. Method choice depends on the nature of the sample. This record is for the method that uses the Dionex IonPac CS19 column .
This technical note (TN) describes how to develop an ion chromatography method to determine an amine in pharmaceutical samples. Two methods were developed in this TN. Method choice depends on the nature of the sample. This record is for the method that uses the Dionex IonPac CS16 column .
The thermo Scientific ultimate 3000 BioLc system is applied for impurities / isoform analysis of Pertuzumab. The analysis was performed on a thermo scientific MAbPac HIC 10 column.
A method was developed for the determination of nitrite in pharmaceuticals by coupling IC with UV absorbance detection. The LOD of nitrite in a pharmaceutical sample is 0.918 ppm (μg/g API). The method is accurate and precise due to the high reproducibility of the Reagent-Free ion chromatography system. This method should be applicable to the determination of nitrite throughout the manufacturing process of a drug product to assess the likelihood of nitrosamine formation.
An HPLC-based approach to monitor the characteristics of polysorbate 80 samples that provides operational simplicity while generating a high degree of information is proposed. The reversed-phase method is highly optimized to ensure sufficient separation of different compound classes within a reasonable run time to permit the fingerprinting of polysorbate 80 samples and detect variability between different suppliers, grades, and production batches.
This record describes the QAR analysis for a Thermo Scientific Dionex CarboPac PA300 column and provides an eWorkflow for running the analysis..
The Thermo Scientific Dionex UltiMate 3000 LC Bio-LC system is applied for the Ion Exchange analysis of ranibizumab sample. The separation was performed on a Thermo Scientific MAbPac SCX-10 with a UV detection at 280 nm.
The Thermo Scientific Dionex Ultimate 3000 system is applied for the peptide mapping analysis of an Pertuzumab (IgG1). The separation was performed on a Thermo Scientific Acclaim RSLC 120 C18 column by using a Thermo Scientific SMART digest kit at UV detection of 214 nm. The SMART Digest kit provides significant improvements in reproducibility, which results in fewer sample failures, higher throughput and the ability to more easily interrogate data..
In this application note, the nitrite determination described in the USP monograph was evaluated with a Thermo Scientific Dionex IonPac AS15 column using an IC system. The method and conditions were exactly as described in the USP Dalteparin Sodium monograph. Key performance parameters were evaluated including the system suitability separation, linearity, limits of detection, accuracy, and robustness. One Dalteparin Sodium sample was analyzed. The percentage of nitrite result was compared with the USP acceptance criterium.
The Thermo Scientific Dionex UltiMate 3000 LC Bio-LC system is applied for the Ion Exchange analysis of teriperatide. The separation was performed on a Thermo Scientific MAbPac SCX-10 and fluorescence detector.
Analysis of protein aggregation and fragment of pertuzumab by size-exclusion chromatography, showing the universal applicability of the Thermo Scientific MAbPac SEC-1 column for aggregate and fragment analysis. Aggregate analysis is major CQA for protein based drugs and requires tight monitoring and control.
Combining the Thermo Scientific™ SMART Digest™ magnetic kits with the Thermo Scientific™ KingFisher™ Duo Prime purification system provides an automated approach to protein digest method optimization. A robust and reproducible, automated digestion time-course protocol to determine the optimal digest time for a biotherapeutic during development of a peptide mapping method.
The Thermo Scientific Dionex Ultimate 3000 system is applied for the peptide mapping analysis of an peptibody (Romiplostim). The separation was performed on a Thermo Scientific Vanquish C18+ column by using a Thermo Scientific SMART digest kit at UV detection of 210 nm. The SMART Digest kit provides significant improvements in reproducibility, which results in fewer sample failures, higher throughput and the ability to more easily interrogate data.
We recently introduced a PdH RE for HPAE-PAD carbohydrate determinations. In this work, we demonstrate two popular carbohydrate applications (honey sugars and glycoprotein monosaccharides) using a PdH RE and compare the results to using an Ag/AgCl RE. Our preliminary data indicate that the PdH RE provides comparable results to the standard Ag/AgCl RE for these two applications that use hydroxide eluents.
Analysis of protein aggregation of Aflibercept i.e fusion protein by size-exclusion chromatography, showing the universal applicability of the Thermo Scientific MAbPac SEC-1 column for aggregate analysis of fusion protein. Aggregate analysis is major CQA for protein based drugs and requires tight monitoring and control.
The Thermo Scientific Dionex Ultimate 3000 system is applied for the peptide mapping analysis of an Fusion Protein (Aflibercept). The separation was performed on a Thermo Scientific Accucore vanquish C18+ column by using a Thermo Scientific SMART digest kit at UV detection of 210 nm. The SMART Digest kit provides significant improvements in reproducibility, which results in fewer sample failures, higher throughput and the ability to more easily interrogate data.
The Thermo Scientific Dionex UltiMate 3000 LC Bio-LC system is applied for the Ion Exchange analysis of romiplostim i.e peptibody. The separation was performed on a Thermo Scientific MAbPac SCX-10 with a UV detection at 210 nm.
The Thermo Scientific Dionex ultimate 3000 Bio compatible LC system is applied for the oxidised impurities analysis of Denosumab.The analysis was performed on a Thermo Scientific MAbPac HIC 20 column. Please Note: Blank normalized chromatogram is shown in representative profile.
Thermo Scientific ultimate 3000 BioLC system is applied for the impurities analysis of etanercept. Analysis was performed on Thermo MAbPac HIC butyl column.
The Thermo Scientific Dionex UltiMate 3000 LC Bio-LC system equipped with column pre-heater is applied for the Reverse phase analysis of Romiplostim. The separation was performed on a Thermo Scientific MAbPac RP column with a UV detection at 280 nm.
The methods proposed here use a Thermo Scientific™ Dionex™ CarboPac™ PA200 column for glycan separation by HPAE. After the separation the glycans pass through a desalting device and in to the mass spectrometer. Fragmentation of glycans in the negative mode by higher-energy collisional dissociation (HCD) provides information-rich MS2 spectra which enables glycan structure annotation. This allowed correlation of glycan structure with observed elution behavior. Here we show that in some cases, changed elution conditions can be used to resolve different glycan structures.
This application shows a method for HPLC-CAD analysis of polysorbate 80 and insulin in a biopharmaceutical formulation. The characterization and quantification of polysorbates is difficult because these compounds are heterogeneous mixtures with no chromophore. Because polysorbates are used in final formulations, determination of lot-to-lot variability is critical to both the pharmaceutical and biopharmaceutical industries.
Before running any samples, Thermo Scientific recommends that you first confirm the performance of the column by reproducing the lot validation report chromatogram shipped with column. Compare your results with the one reported in the quality assurance report. At least three injections should be made. This record provides an eWorkflow for executing the QAR method on an ICS-5000+ system. An ICS-6000 can be used for this application.
Glycoprotein characterization and glycosylation profiling are important tasks in the development and production of biopharmaceutical proteins. This application note demonstrates Dual EGC capability and performance for profiling N-linked oligosaccharides released from glycoproteins using high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD). This record shows the separation of N-linked glycans from human acid-1 glycoprotein.
Glycoprotein characterization and glycosylation profiling are important tasks in the development and production of biopharmaceutical proteins. This application note demonstrates Dual EGC capability and performance for profiling N-linked oligosaccharides released from glycoproteins using high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD). This record shows the separation for IgG N-linked glycans.
HPLC-UV-CAD method was established in this paper and the consistency of the generic drug for cefepime of injection was evaluated from the impurity profile and excipients. On the basis of UV detection and the charged aerosol detection (CAD) combined with a mixed matrix column Trinity-P1. By adjusting the organic phase and salt concentration, a method for determining the arginine of the auxiliary material was established. The determination can be made simultaneously with the main component of the drug and the salt forming ion.
This application demonstrates reliable verification and quantification of the presence of extraneous compounds in a sample, such as impurities, degradation products or extractables and leachables.
This application update demonstrates improved resolution of sialylated N-glycans on Thermo Scientific™ Dionex™ CarboPac™ PA200 columns. Starting with a recently described method, changes to elution conditions were tested to improve resolution of sialylated N-glycans enzymatically released from four different glycoproteins. Separations were first evaluated on the analytical format (3 × 250 mm column). Next, the possibility that a shorter column, such as a guard column, would allow significantly improved throughput was evaluated. Finally, a narrow bore format 1 × 250 mm column was tested.
Leveraging UV detection for the determination of the impurity levels and mass detection for identification of the active pharmaceutical ingredient (API) and other product related impurities using a rapid method for synthetic peptide impurity profiling
To demonstrate the rapid quantitation of a low-level human IgG in animal plasma by LC/MS/MS using the Thermo Scientific™ SMART Digest™ Immunoaffinity (IA) kits (including magnetic and non-magnetic versions of the Streptavidin kit, Protein A kit, and Protein G kit), which combine the IA capture and digestion process into a single well. The assay is required to be both selective and accurate.
This application note demonstrated the robustness, reproducibility, accuracy, and precision of quantification of IgG N-glycans using the Thermo Scientific™ Vanquish™ Horizon UHPLC integrated biocompatible system and Thermo Scientific™ Accucore™ 150 Amide HILIC column.
To demonstrate the robustness, reproducibility, carry-over, recovery, accuracy, and precision of quantification of the NIST monoclonal antibody IgG1K (NISTmAb) using the Thermo Scientific™ Vanquish™ Horizon UHPLC integrated biocompatible system and Thermo Scientific™ MAbPac™ RP LC columns
This study demonstrates use of a commercially available glycopeptide standard as a control to determine glycoprotein hydrolysis reaction efficiency. Because the monosaccharide composition of glycopeptide is known, hydrolysis efficiency under a set of conditions can be easily calculated. The monosaccharide yield for the glycopeptide was compared with that of two glycoproteins under identical conditions. This comparison revealed efficient hydrolysis using the conditions tested here. Minimally the glycopeptide standard serves as a positive control for glycoprotein monosaccharide determination.
To demonstrate the effectiveness of a simple pH gradient/ion-exchange chromatography workflow approach to the characterization of charge variant profiles of an innovator molecule (cetuximab) and a candidate biosimilar. To show the assay is simple, reproducible, easily optimized and resolves variants effectively.
To demonstrate the CX-1 pH buffer system is capable of charge variant determination for the majority of therapeutic monoclonal antibody species. To show the ease of optimization and improved reproducibility of this buffer system when compared to salt-based gradient systems used for charge variant analysis.
Before running any samples, Thermo Scientific recommends that you first confirm the performance of the column by reproducing the lot validation report chromatogram shipped with column. Compare your results with the one reported in the quality assurance report. At least three injections should be made. This record provides an eWorkflow for executing the QAR method on an ICS-6000 system using Dual EGC mode.
This application note demonstrates the ability of the Thermo Scientific iCAP 7000 Plus Series ICP-OES to determine trace elements in materials of plant origin that are used for health benefits.
This record describes method for reproduction of column Quality Assurance Report (QAR). Analyzing a standard sample using method conditions described in QAR is recommended in order to validate column performance prior to use. This record also includes a one-click eWorkflow for executing the QAR method on a ICS 5000+ system.
This work demonstrates an improved HPAE-PAD method using a recently introduced Thermo Scientific™ Dionex™ CarboPac™ PA20 column with 4 μm particle size to simultaneously quantify six major monosaccharides present in glycoprotein acid digests. The total run time is 20 min as compared to 32 min with the method using a 6.5 μm particle size column. The smaller particle size of this column offers higher peak efficiencies, leading to high-resolution separations and allowing a shorter column format and, ultimately, significantly shorter run times. An ICS-6000 can be used for this application.
The biopharmaceutical industry requires reliable high throughput analysis of N-linked glycans. Many of the commonly used analytical approaches are based on derivatization, which is known to introduce differential loss of sialic acid and incomplete labeling. Preparation of native glycans is not subject to these concerns. This approach starts with a fast in-solution glycoprotein digestion to obtain oligosaccharides. These native glycans are then analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection coupled to MS. This record is for neutral and IgG glycans.
The biopharmaceutical industry requires reliable high throughput analysis of N-linked glycans. Many of the commonly used analytical approaches are based on derivatization, which is known to introduce differential loss of sialic acid and incomplete labeling. Preparation of native glycans is not subject to these concerns. This approach starts with a fast in-solution glycoprotein digestion to obtain oligosaccharides. These native glycans are then analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection coupled to MS. This record is for charged glycans.
