Method Development and Validation for Quantitation of Human Serum Albumin Using Reversed-phase Liquid Chromatography 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 Method Development and Validation for Quantitation of Human Serum Albumin Using Reversed-phase Liquid Chromatography PDF full book. Access full book title Method Development and Validation for Quantitation of Human Serum Albumin Using Reversed-phase Liquid Chromatography by Esar Ghandour. Download full books in PDF and EPUB format.
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: 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: 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: 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: Sussan Oladipo
Publisher:
ISBN:
Category : Chymotrypsin
Languages : en
Pages : 0
Get Book Here
Book Description
"Reversed-phase liquid chromatography (RPLC) has been demonstrated to be a promising separation technique in separating proteins and peptides. The efficiency obtained with the RPLC is generally superior to other separation techniques such as size-exclusion chromatography, ion-exchange chromatography, hydrophilic interaction chromatography, and hydrophobic interaction chromatography. Another advantage of RPLC is its ability to couple with mass spectrophotometry detection and use in forced-degradation studies. A simple reversed-phase liquid chromatographic method was developed to separate a sample of chymotrypsin, lysozyme, bovine serum albumin, and ovalbumin. The method was developed on a 25 mm Phenomenex Column C8, Particle size: 5 μm, I.D. 4.6 mm under 50oC column temperature. Mobile phase used consisted of 1.0% trifluoroacetic acid in deionized water and Acetonitrile at a flow rate of 1.0 ml/min with UV-Vis detection at 210, 220 and 280 nm using DAD detector. The separation was conducted under gradient elution with 30 to 70% ACN gradient rate for 20 min. Forced Degradation studies was done on the BSA sample with resulting significant degradation effect from acid, basic, hydrogen peroxide, UV Light and Heat incubations stress test on the BSA sample. Mass spectroscopic analysis of the degradants identifies amino acid fragments. The developed method was validated for robustness, linearity, accuracy, precision, detection limit and quantitation to quantify bovine serum albumin. The method proved simple, accurate and precise with over 97% average recovery of bovine serum albumin."--
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: Shyam Dedaniya
Publisher:
ISBN:
Category : High performance liquid chromatography
Languages : en
Pages : 0
Get Book Here
Book Description
"The goal of this investigation is to develop a reversed-phase high-performance liquid chromatographic method to separate ovalbumin (OVA) and bovine serum albumin (BSA) proteins and to quantify BSA protein in its raw material. This technique is considered one of the most promising analytical techniques for the quantification of peptides and intact proteins, including monoclonal antibodies (mAbs). Two stationary phases, C4 and C3 with 300 Å pore size columns, were tested in this investigation. The C4 column is in 250 × 4.6 mm dimension, while the C3 column is in 75 × 2.1 mm dimension. A 0.08 % TFA was added to the mobile phase as an ion-pairing reagent to control its acidity and column efficiency. Proteins standard solutions of BSA and OVA were prepared in 6 M urea as a denaturing agent to improve chromatographic efficiency. The optimum separation conditions were found under a flow rate 1.0 mL/min, injection volume 20 μL, column temperature 50°C, and a gradient profile: 0 to 10 min, % B: 25% to 80%, 10 to 20 min, % B: 80% to 100% with solvent B = 100% acetonitrile + 0.08% trifluoroacetic acid. Under these separation conditions, the percentage recovery of BSA protein from the stationary phase was found 100%, while only 90- 95% of OVA was recovered. A short alkyl chain column (30 x 4.6 mm) helped improve the recovery of OVA protein and reduced the separation run time of the method. Forced degradation studies were conducted under the optimum separation conditions to determine the stability of these two proteins under sodium hydroxide, hydrochloric acid, hydrogen peroxide, heat, and light. In addition, the developed method was validated to verify its specificity and robustness and confirm its high accuracy and precision. The validation results fulfilled the U.S. Food and Drug Administration guidelines (FDA).--
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: Satish Y. Gabhe
Publisher: Anchor Academic Publishing (aap_verlag)
ISBN: 395489307X
Category : Business & Economics
Languages : en
Pages : 114
Get Book Here
Book Description
This book details: 1. Development and validation of a HPTLC-densitometric method for concurrent estimation of metformin hydrochloride, pioglitazone hydrochloride and gliclazide in combined dosage form. 2. Development and validation of a HPTLC method for simultaneous estimation of moxifloxacin hydrochloride and dexamethasone sodium phosphate in combined pharmaceutical dosage form. 3. Development and validation of a RP-HPLC method for simultaneous estimation of ciprofloxacin hydrochloride and dexamethasone in combined dosage form, which is a better alternative to existing ones. The developed analytical methods are simple, selective, accurate, robust, and precise with shorter analysis time for the analysis of drug/s in combined pharmaceutical dosage forms. All the developed HPTLC and HPLC methods have been validated as per ICH Q2 (R1) guideline. Developed analytical methods could boost analytical researchers to work more efficiently in the field of analytical method development and validation of Pharmaceutical dosage forms.
Author: Ariunaa Nordog
Publisher:
ISBN:
Category : High performance liquid chromatography
Languages : en
Pages : 0
Get Book Here
Book Description
"Reversed-phase chromatography used to separate four proteins human serum albumin (HSA), ovalbumin (Ova), lysozyme, and chymotrypsin. The method is widely used, inexpensive, and robust. Trial and error were employed to determine the optimal conditions for the experiment. A column temperature of 50°C, an injection volume of 10 uL, a flow rate of 1.0 mL per minute, a solvent strength of 30% to 70% B, and a gradient time of 13 minutes were the optimal conditions for the separation. A forced degradation test was conducted using lysozyme. The lysozyme stability was tested using sodium hydroxide, hydrochloric acid, hydrogen peroxide, heat, and light. The method was further validated to confirm its precision and accuracy to determine its specificity and robustness under optimal conditions. The method validation results were per FDA guidelines, with a measured value of 99.76%, close to the true value."--
Author: Michael E. Swartz
Publisher: CRC Press
ISBN: 9780824701154
Category : Science
Languages : en
Pages : 100
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.
