Development and Validation of a Reversed Phase High Performance Liquid Chromatography Method for the Assay of Monomeric Human Albumin to Evaluate Recovery for Subsequent Peptide Mapping PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Development and Validation of a Reversed Phase High Performance Liquid Chromatography Method for the Assay of Monomeric Human Albumin to Evaluate Recovery for Subsequent Peptide Mapping PDF full book. Access full book title Development and Validation of a Reversed Phase High Performance Liquid Chromatography Method for the Assay of Monomeric Human Albumin to Evaluate Recovery for Subsequent Peptide Mapping by James M. Sulzberger (Jr.). Download full books in PDF and EPUB format.
Author: James M. Sulzberger (Jr.)
Publisher:
ISBN:
Category : High performance liquid chromatography
Languages : en
Pages : 174
Get Book Here
Book Description
"Analytical characterization of biomolecules such as proteins is inherently more complex than its traditional counterpart, traditional small molecule pharmaceuticals. While it is possible to develop and validate one or two assays with one or two orthogonal methods to assess specificity and stability with regards to product related degradants, proteins require many. Typically, a combination of reversed phase, ion-exchange, gel electrophoresis, and size exclusion chromatography to evaluate charged variants, glycoform variants, and size variant product related impurities in drug substance and drug product. Both top-down and bottom- up approaches for analytical characterization of proteins are critical for ensuring primary, secondary, tertiary, and quaternary structure to be certain of the safety and efficacy of a molecule, as one type of degradation or impurity can be detrimental to either. HPLC and UPLC of intact protein as a top-down approach, as well as a corresponding bottom-up method known as peptide mapping, where digestion of a protein after reduction and alkylation using a specific chemical or enzyme is used to look into the location of post translational modifications, are both employed. When developing a peptide map, a critical parameter is recovery and stability of digested protein. In this study, human serum albumin is uses as a model protein for illustration of sensible approach for evaluation of these two criteria with the development and validation of a reversed phase chromatography assay for evaluation of protein recovery and stability with regards to aggregation formation."--
Author: James M. Sulzberger (Jr.)
Publisher:
ISBN:
Category : High performance liquid chromatography
Languages : en
Pages : 174
Get Book Here
Book Description
"Analytical characterization of biomolecules such as proteins is inherently more complex than its traditional counterpart, traditional small molecule pharmaceuticals. While it is possible to develop and validate one or two assays with one or two orthogonal methods to assess specificity and stability with regards to product related degradants, proteins require many. Typically, a combination of reversed phase, ion-exchange, gel electrophoresis, and size exclusion chromatography to evaluate charged variants, glycoform variants, and size variant product related impurities in drug substance and drug product. Both top-down and bottom- up approaches for analytical characterization of proteins are critical for ensuring primary, secondary, tertiary, and quaternary structure to be certain of the safety and efficacy of a molecule, as one type of degradation or impurity can be detrimental to either. HPLC and UPLC of intact protein as a top-down approach, as well as a corresponding bottom-up method known as peptide mapping, where digestion of a protein after reduction and alkylation using a specific chemical or enzyme is used to look into the location of post translational modifications, are both employed. When developing a peptide map, a critical parameter is recovery and stability of digested protein. In this study, human serum albumin is uses as a model protein for illustration of sensible approach for evaluation of these two criteria with the development and validation of a reversed phase chromatography assay for evaluation of protein recovery and stability with regards to aggregation formation."--
Author: Esar Ghandour
Publisher:
ISBN:
Category : High performance liquid chromatography
Languages : en
Pages : 0
Get Book Here
Book Description
"Human serum albumin (HSA) is the most plentiful protein in human blood plasma, produced by the liver, is multifunctional transport protein in the circulatory system, and acts as a carrier for various kinds of ligands. A rapid and sensitive method was developed and validated for the separation of Human Seium Albumin (HAS) using reversed-phase high-performance liquid chromatography (RP–HPLC) with Diod-array detectoe (DAD). HSA was dissolved in distilled water as a solvent and then separated using reversed-phase HPLC. Throughout the research, various parameters were studied including selection of wavelength, mobile phase solvents, flow rate, buffers with various pH values, column temperature changes, and sample concentration. A gradient elution technieuqe was utilized throughout this investigation. The resulted optimum separation conditions included a reversed-phase column C4, 5.0 μm, 30 mm × 4.6 mm inner diameter; using binary mobile phases composed of aqueous solution A (0.1% TFA in deionized water) and organic solution B (0.1% TFA in acetonitrile) pumped under gradient scheme in 10 minutes, flow rate 1.2 ml/min, column department temperature 45 °C, and injection volume 5 μl of sample concentration 5mg/ml at temperature 2–8 °C, and UV detection was performed at 251 ± 2. Various forced degradation studies were carried out on HAS including; Thermal, pH, H2O2 and light stress, HSA was most degraded at high temperature (above 70 °C), low pH medium (less pH 7.0), short wavelength and high % H2O2 . The developed method was validated regarding linearity over the range of (0.5mg/ml–20mg/ml) with (R2 = 0.999). Accuracy (% Recovery 97.87%) with relative standard deviation of precision (0.7%) indicating reasonable precision of the developed method. Intermediate precision was confirmed by different analysts, different types of equipment and on different days. Robustness was approved by taking into account five factors; column temperature, flow rate, injection volume, wavelength, percentages of TFA in the mobile phase and considered as robust. Limit of detection and limit of quantitation of the protein were low which enables the determination of the proteins at low concentrations."--
Author: Ante M. Krstulovic
Publisher:
ISBN:
Category : Science
Languages : en
Pages : 316
Get Book Here
Book Description
A comprehensive problem-solving approach to reversed-phase high-performance liquid chromatography covering the theoretical aspects and practical information needed in diverse areas of research. Also reviews RPLC applications in the biomedical/biochemical field.
Author: Gábor Szepesi
Publisher: Wiley-VCH
ISBN:
Category : Science
Languages : en
Pages : 376
Get Book Here
Book Description
Author: Ghada Abusaifan
Publisher:
ISBN:
Category : High performance liquid chromatography
Languages : en
Pages : 0
Get Book Here
Book Description
"Therapeutic proteins, such as monoclonal antibodies (mAb) and antibody-drug conjugates (ADCs), become more important since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune disorders-based diseases. These proteins are susceptible to chemical and enzymatic modifications that can occur during manufacture, formulation, and storage. The large size of mAbs and ADCs and the minor structure diversity between the variants make their separation very challenging. As a result, ion-exchange chromatography is considered the most effective technique for characterizing therapeutic mAbs and ADCs and for monitoring the batch-to-batch process consistency and product stability and purity among several chromatographic modes for their separation such as reversed-phase liquid chromatography, size exclusion chromatography, hydrophobic interaction liquid chromatography. Ion-Exchange Chromatographic approach is based on the electrostatic interaction of the ionic portion of the protein with a cation- or anion- stationary phase. This investigation developed two ion-exchange methods using pH- and Salt- gradients ion exchange modes. A novel, simple and robust method was developed to separate a mixture of OVA, BSA, and HSA proteins on Agilent Technologies 1260 Infinity series HPLC with a diode array detector and Agilent Bio SAX, NP5, SS, 4.6 x 250mm, 5μm, non-porous column controlled at 50oC. Under gradient elution technique, 25 mM Bis-Tris methane at pH 5.8 was found optimum buffer strength and mobile phase acidity. DryLab® modeling software was used to simulate the optimum conditions of NaCl concentration and gradient time. The optimum separation conditions were found under 1ml/min and gradient profile: 0 to 35 min, % B: 0% to 20%, 35 to 35.1 min, % B: 20% to 100%, 35.1 to 50 minutes, %B: 100%, 50.1 minutes %B back to 0% with solvent B = 25 mM Bis-Tris buffer at pH 5.8 + 1 M NaCl. Under the optimum salt-gradient separation conditions, the developed method was validated for OVA protein in terms of system suitability test, specificity, robustness, linearity and range, precision, accuracy, LOD, and LOQ. The validation results fulfilled the U.S. Food and Drug Administration guidelines (FDA). Optimization of separation conditions using pH-gradient ion-exchange chromatography will conclude this presentation."--
Author: Mukesh Maithani
Publisher: Springer
ISBN: 9811387230
Category : Medical
Languages : en
Pages : 101
Get Book Here
Book Description
Reversed-phase high-performance liquid chromatography (RP-HPLC) has become the most widely used method for pharmaceutical analysis, as it ensures accuracy, specificity and reproducibility for the quantification of drugs, while avoiding interference from any of the excipients that are normally present in pharmaceutical dosage forms. This book presents a simple methodology for developing stability-indicating methods and offers a ‘how-to guide’ to creating novel stability-indicating methods using liquid chromatography. It provides the detailed information needed to devise a stability-indicating method for drug substances and drug products that comply with international regulatory guidelines. As such, it is a must-read for anyone engaged in analytical and bioanalytical chemistry: professionals at reference, test, and control laboratories; students and academics at research laboratories, and scientists working for chemical, pharmaceutical, and biotechnology companies.
Author: Paula Sanchez Garcia
Publisher:
ISBN:
Category : Gamma globulins
Languages : en
Pages : 0
Get Book Here
Book Description
"Separation of a mixture of HSA and GG was perform using Reverse phase liquid Chromatography. Human serum albumin (HSA) is the most abundant protein in the blood and has many vital biological roles [14]. One function of human serum albumin functions is transport hormones, fatty acids, and various compounds through the bloodstream. Gamma Globulin is blood proteins produced by the immune system's lymphocytes and plasma cells. Almost all gamma globulins are known as immunoglobulins, also called antibodies, which are globulins that help with immune responses and immunity. A fast and sensitive method was developed and validated for the separation of Human Serum Albumin (HSA) and Gamma Globulin (GG) using reversed-phase liquid chromatography (RP–HPLC) on Agilent 1100 series system with Diode Array Detector with Jupiter 300 C4 column (250 X 4.6mm, 5 μm). Various parameters were studied during the research, including selecting mobile phase, flow rate, different buffer solutions, column temperature changes, and sample concentration. A gradient elution technique was used for the duration of this research. The final optimum separation conditions were conducted on Jupiter C4, 5.0μm, 250mm × 4.6 mm inner diameter, using binary mobile phases solution. Mobile phase A (0.1%TFA in deionized water) and Mobile phase B organic solution (0.08%TFA in acetonitrile) pumped under linear gradient in 30 minutes, flow rate 0.8 ml/min, column department temperature 45°C, and injection volume 10μl of sample concentration ratio 10:1 mg/ml HSA/GG at temperature 2-8 °C, and UV detection was achieved at 250+2. Forced degradation studies were completed on the mixture of HSA and GG studies: Acidic, basic hydrolysis, oxidation, thermal, and UV light degradation. HSA and GG mixture were taken to those studies at different period times. The method developed was validated according to ICH guidelines validation parameters: linearity with R2 = 0.999 for HSA and R2=0.998 for GG. Accuracy %Recovery 99.337% and 100.33% for HSA and GG, respectively. Repeatability and accuracy were also performed. Robustness was endorsed by considering factors like column temperature, flow rate, and wavelength. The method was considered robust. The limit of detection and limit of quantitation of the protein mixture was 0.719 for HSA and 0.11 GG (LOD), enabling the proteins' determination at low concentrations. Further investigation was performed. The mixture was conducted on a size exclusion liquid chromatography separation where the ionic strength effect was studied—living the door open to continue the research on this field."--
Author: Csaba Horváth
Publisher:
ISBN:
Category : High performance liquid chromatography
Languages : en
Pages : 344
Get Book Here
Book Description
Author: Malvina Haxhiu
Publisher:
ISBN:
Category : Gel permeation chromatography
Languages : en
Pages : 0
Get Book Here
Book Description
"Human serum albumin is a protein in the blood with a molecular mass 66.5 kDa. In this study, was developed a method for determination of human serum albumin protein as monomer and its aggregates using a size exclusion column and HPLC instrument. Optimum conditions of the method were flow rate 0.2 ml/min, injection volume of the sample with concentration 5 mg/ml was 0.2 μl, column temperature 30 °C, wavelength 214 nm. Mobile phase was sodium phosphate at concentration 150 mM and pH 7 using an isocratic elution. The method is validated in term of linearity, precision robustness, specificity, system suitability test and stability."--
Author: Michael E. Swartz
Publisher: CRC Press
ISBN: 9781138402560
Category : High performance liquid chromatography
Languages : en
Pages :
Get Book Here
Book Description
Describes analytical methods development, optimization and validation, and provides examples of successful methods development and validation in high-performance liquid chromatography (HPLC) areas. The text presents an overview of Food and Drug Administration (FDA)/International Conference on Harmonization (ICH) regulatory guidelines, compliance with validation requirements for regulatory agencies, and methods validation criteria stipulated by the US Pharmacopia, FDA and ICH.
