This document outlines a four phase experimental plan to mitigate monoclonal antibody (mAb) aggregation during downstream processing. Phase 1 will use software to predict aggregation propensity of three mAbs. Phase 2 will assess pH dependence of aggregation rates. Phase 3 will evaluate process performance with respect to elution and hold pH. Phase 4 will vary excipient type, pH, and concentration to optimize buffer conditions and minimize aggregation. The goal is to develop a safe and effective mAb product for clinical trials by reducing aggregation caused by low pH steps like protein A chromatography.
ElogPoct: A Tool for Lipophilicity Determination in Drug DiscoveryBrian Bissett
This document presents a new RP-HPLC method for determining logPoct values, a measure of lipophilicity important for drug discovery. The key advantages of the method are that it is rapid (determinations in 20 minutes on average), uses small amounts of compound (1 mL of a 30-50 μg/mL solution), has a wide dynamic range of 6 log units, and shows good accuracy and reproducibility when tested on 36 drug molecules. A comparison to experimentally determined logPoct values indicates the new RP-HPLC method performs comparably to traditional shake-flask methods.
Analytical purity method development and validation by gas chromatography of ...Alexander Decker
This document describes the development and validation of a gas chromatography method for analyzing the purity of L-valine methyl ester hydrochloride, which is used to produce anti-hypertensive drugs. The method was validated for precision, recovery, linearity, robustness, and solution stability according to ICH guidelines. Gas chromatography with a flame ionization detector was used to separate and detect L-valine methyl ester hydrochloride peaks from potential impurity peaks such as isoleucine methyl ester hydrochloride. The method demonstrated high recovery and low variability, confirming its suitability for analyzing the purity of L-valine methyl ester hydrochloride in pharmaceutical applications.
Validation and method development of Apixaban A research project.Bhavana Gundavarapu
The document presents the development and validation of an HPLC-MS method for the estimation of Apixaban in human plasma. Key points:
- The aim was to develop a simple, precise and accurate HPLC-MS method for quantifying Apixaban levels in plasma using Apixaban-13C D3 as an internal standard.
- The method involved protein precipitation followed by HPLC-MS analysis on a C18 column with mobile phase containing acetonitrile, methanol and ammonium formate buffer at a flow rate of 0.4 mL/min and detection at m/z 459.3 and 462.3 for Apixaban and internal standard.
- The method was
Development and Validation of Novel RP-HPLC method for the estimation of Nalo...Bhavana Gundavarapu
The RP-HPLC method was developed and validated for quantitative determination of Naloxegol in pharmaceutical dosage forms. The method was validated with regard to linearity, accuracy, precision, selectivity, and robustness. The method was applied successfully for the determination of Naloxegol during kinetic studies in aqueous solutions (pH and thermal degradation).
Chromatography: Chromatographic strategies for IVIG purification – Part 2Merck Life Sciences
The document discusses chromatographic strategies for intravenous immunoglobulin (IVIG) purification using anion exchange chromatography. It describes a case study where a Fractogel EMD TMAE (M) resin was able to efficiently separate and purify IgG from a caprylic acid-treated human plasma fraction (worst-case scenario) in a single step in negative mode. Optimization studies showed the resin was robust across a pH range of 5.7-6.3, allowing selective binding of contaminants like IgA and IgM while IgG passed through. Purification trials over 10 cycles demonstrated consistent 94% IgG recovery and 84% removal of IgA and IgM contaminants.
Stability indicating RP-HPLC method for estimation of dapagliflozin in bulk a...SriramNagarajan19
A simple, specific, accurate, precise and stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method is developed for estimation of Dapagliflozin (DGF) in bulk and Pharmaceutical dosage form. The method employed, Hypersil BDS C18 250 mm x 4.6 mm, 5 mm column in isocratic mode with mobile phase of 0.1% Ortho phosphoric acid buffer and acetonitrile 50:50% v/v. The flow rate was 1.0 mL min-1 and effluent was monitored at 245 nm using PDA detector. The injection volume was 10 µl and the total runtime was set as 5min. The retention time for DGF was found to be 2.226min.The method was validated in terms of Linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) etc. in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there was a good linear relationship between response and concentration in the range of 25 - 150 µg/ml respectively. The LOD and LOQ values for HPLC method were found to be 0.04 and 0.121 µg/ml respectively. No chromatographic interference from the tablet excipients was found. The proposed method was successfully used for estimation of Dapagliflozin (DGF) in Bulk and Pharmaceutical dosage form.
Bioanalytical RP-HPLC Method Development and Validation for Estimation of Cur...Sagar Savale
The present study was aimed at developing a reversed phase high performance liquid chromatography (RPHPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal
standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.)
coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid
(40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423
nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation
and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was
validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study
(short and long-term stabilities, Freeze/thaw stability and post-preparative).
Drug discovery library design by biomimetic hplcKlara Valko
The slides explain the early drug discovery process and how the measurements of biomimetic properties can help to design the best ADME properties of compound libraries.
ElogPoct: A Tool for Lipophilicity Determination in Drug DiscoveryBrian Bissett
This document presents a new RP-HPLC method for determining logPoct values, a measure of lipophilicity important for drug discovery. The key advantages of the method are that it is rapid (determinations in 20 minutes on average), uses small amounts of compound (1 mL of a 30-50 μg/mL solution), has a wide dynamic range of 6 log units, and shows good accuracy and reproducibility when tested on 36 drug molecules. A comparison to experimentally determined logPoct values indicates the new RP-HPLC method performs comparably to traditional shake-flask methods.
Analytical purity method development and validation by gas chromatography of ...Alexander Decker
This document describes the development and validation of a gas chromatography method for analyzing the purity of L-valine methyl ester hydrochloride, which is used to produce anti-hypertensive drugs. The method was validated for precision, recovery, linearity, robustness, and solution stability according to ICH guidelines. Gas chromatography with a flame ionization detector was used to separate and detect L-valine methyl ester hydrochloride peaks from potential impurity peaks such as isoleucine methyl ester hydrochloride. The method demonstrated high recovery and low variability, confirming its suitability for analyzing the purity of L-valine methyl ester hydrochloride in pharmaceutical applications.
Validation and method development of Apixaban A research project.Bhavana Gundavarapu
The document presents the development and validation of an HPLC-MS method for the estimation of Apixaban in human plasma. Key points:
- The aim was to develop a simple, precise and accurate HPLC-MS method for quantifying Apixaban levels in plasma using Apixaban-13C D3 as an internal standard.
- The method involved protein precipitation followed by HPLC-MS analysis on a C18 column with mobile phase containing acetonitrile, methanol and ammonium formate buffer at a flow rate of 0.4 mL/min and detection at m/z 459.3 and 462.3 for Apixaban and internal standard.
- The method was
Development and Validation of Novel RP-HPLC method for the estimation of Nalo...Bhavana Gundavarapu
The RP-HPLC method was developed and validated for quantitative determination of Naloxegol in pharmaceutical dosage forms. The method was validated with regard to linearity, accuracy, precision, selectivity, and robustness. The method was applied successfully for the determination of Naloxegol during kinetic studies in aqueous solutions (pH and thermal degradation).
Chromatography: Chromatographic strategies for IVIG purification – Part 2Merck Life Sciences
The document discusses chromatographic strategies for intravenous immunoglobulin (IVIG) purification using anion exchange chromatography. It describes a case study where a Fractogel EMD TMAE (M) resin was able to efficiently separate and purify IgG from a caprylic acid-treated human plasma fraction (worst-case scenario) in a single step in negative mode. Optimization studies showed the resin was robust across a pH range of 5.7-6.3, allowing selective binding of contaminants like IgA and IgM while IgG passed through. Purification trials over 10 cycles demonstrated consistent 94% IgG recovery and 84% removal of IgA and IgM contaminants.
Stability indicating RP-HPLC method for estimation of dapagliflozin in bulk a...SriramNagarajan19
A simple, specific, accurate, precise and stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method is developed for estimation of Dapagliflozin (DGF) in bulk and Pharmaceutical dosage form. The method employed, Hypersil BDS C18 250 mm x 4.6 mm, 5 mm column in isocratic mode with mobile phase of 0.1% Ortho phosphoric acid buffer and acetonitrile 50:50% v/v. The flow rate was 1.0 mL min-1 and effluent was monitored at 245 nm using PDA detector. The injection volume was 10 µl and the total runtime was set as 5min. The retention time for DGF was found to be 2.226min.The method was validated in terms of Linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) etc. in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there was a good linear relationship between response and concentration in the range of 25 - 150 µg/ml respectively. The LOD and LOQ values for HPLC method were found to be 0.04 and 0.121 µg/ml respectively. No chromatographic interference from the tablet excipients was found. The proposed method was successfully used for estimation of Dapagliflozin (DGF) in Bulk and Pharmaceutical dosage form.
Bioanalytical RP-HPLC Method Development and Validation for Estimation of Cur...Sagar Savale
The present study was aimed at developing a reversed phase high performance liquid chromatography (RPHPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal
standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.)
coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid
(40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423
nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation
and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was
validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study
(short and long-term stabilities, Freeze/thaw stability and post-preparative).
Drug discovery library design by biomimetic hplcKlara Valko
The slides explain the early drug discovery process and how the measurements of biomimetic properties can help to design the best ADME properties of compound libraries.
A newly validated HPLC method development for simultaneous estimation of rito...SriramNagarajan19
The aim of the present work was to develop a isocratict RP-HPLC for simultaneous analysis of ritonavir and lopinavir in tablet dosage form. Method: chromatographic system was optimized using a Agilent XDB C18(150 x 4.6mm,5µm) column with potassium dihydrogen phosphate (pH 4.6) and acetonitrile in the ratio of 45;55, as a mobile phase, at a flow rate of 1.0 ml/min. detection was carried out at 215nm by a photodiode array detector. Result: ritonavir and lopinavir were eluted with retention times of 4.821 and 3.814mins respectively. Beer’s lambert’s law was obeyed over the concentration ranges of 12.5 to 50µg/ml and 50 to 200µg/ml for ritonavir and lopinavir, respectively. Conclusion: the high recovery and low coefficients of variation confirm the suitability of the method for simultaneous analysis of both drugs in a tablet dosage form. Statistical analysis proves that the method is sensitive and significant for the analysis of ritonavir and lopinavir in pure and in pharmaceutical dosage form without any interference from the excipients. The method was validated in accordance with ICH guidelines. Validation revealed the method is specific, rapid, accurate, precise, reliable, and reproducible.
This document discusses the use of liquid chromatography (LC) and LC-mass spectrometry (LC-MS) in the analysis of active pharmaceutical ingredients (APIs). LC techniques are required by regulatory agencies and are used for separation, identification, and quantification of APIs and impurities. LC is applied in API development, manufacturing, and analytical method development, including assay development, forced degradation studies, impurity profiling, and stability indicating methods. LC-MS provides advantages like isolation of impurities, spectral peak purity analysis, and analysis of drugs in biological fluids. An example case study on the LC-MS analysis of amlodipine is provided, showing mass fragmentation, degradation pathways, and chromatograms of degradation products.
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
This document describes the development and validation of an RP-HPLC method for the simultaneous estimation of atorvastatin and olmesartan tablets. The method was developed using a BDS hypersil C18 column with a mobile phase of ACN-TEA 40-60-0.1 pH 4.0 and detection at 235 nm. The method was validated for parameters such as linearity, precision, accuracy, robustness and system suitability and found to be suitable for the simultaneous quantification of atorvastatin and olmesartan in combined tablet dosage forms.
Quantification of a Novel Peptide, CPT31 in Rat and Monkey Plasma by LC-MSCovance
ASMS 2019 -- CPT31, a novel D-peptide, is being investigated in the treatment and prevention of HIV by inhibiting the viral entry of HIV. To evaluate the properties of CPT31, an accurate highly reproducible method to quantitate CPT31 in plasma was required. To this end, a robust LC-MS assay for the quantification of CPT31 in rat and monkey plasma samples is reported here. The method follows extraction and clean-up of two plasma matrices, encompasses a range of 90.0 to 45,000 ng/mL, and completes LC-MS analysis in 7.50 minutes.
This document describes a liquid chromatographic method for the simultaneous quantitative determination of candesartan cilexetil and hydrochlorothiazide in pharmaceutical dosage forms. A simple and sensitive RP-HPLC method was developed and validated. The method allows for the separation, identification and quantification of candesartan cilexetil and hydrochlorothiazide in tablet formulations using a C18 column with UV detection. The method was validated in terms of specificity, linearity, precision, accuracy and robustness.
Development and Validation of Reverse Phase Liquid Chromatography Method for ...IOSR Journals
This document describes the development and validation of a reverse phase liquid chromatography method for the estimation of losartan in bulk drug samples. The method utilizes an Acquity BEH C18 column with a mobile phase of buffer and acetonitrile at a ratio of 50:50 delivered isocratically at 0.3 mL/min. Losartan was detected at 230 nm. The method was validated per ICH guidelines and found to be linear, precise, accurate, specific and stability-indicating for the quantification of losartan in the range of 25-75 μg/mL. The method validation shows the method is suitable for the routine analysis of losartan in bulk drug materials.
This document describes the development and validation of a stability-indicating HPLC method for the simultaneous quantification of four active pharmaceutical ingredients: pantoprazole, rabeprazole, lansoprazole, and domperidone. The method was developed using a C18 column with gradient elution of a mobile phase consisting of buffer and organic solvents. The method was validated per ICH guidelines and demonstrated selectivity, linearity, accuracy, precision, sensitivity and robustness. Forced degradation studies subjected the drugs to acid, base, oxidation, heat, and light conditions in order to evaluate the method's ability to separate drugs from degradation products.
1) The document describes the development of a LC-MS/MS method for quantifying highly polar aminoglycoside compounds like gentamicin, kanamycin, and apramycin from rat plasma.
2) It investigates using trichloroacetic acid (TCA) both as a protein precipitation agent during sample preparation and as an ion pairing reagent to improve retention of the polar analytes on the reverse phase column.
3) The results show that TCA concentrations between 25-30% provided the best sample recovery during plasma protein precipitation. TCA was also found to improve analyte retention and sensitivity when used as an ion pairing reagent, without needing to add it to the mobile
This document discusses the use of reversed-phase ultrahigh pressure liquid chromatography to analyze macro molecules. It examines the effect of pressure and hydrophobic mobile phase additives on the retention of small molecules and proteins. The document presents data on the physicochemical characteristics and retention factors of various compounds under different pressure and mobile phase conditions. It finds that increasing pressure and adding liophilic agents like NaClO4 and KPF6 increases the retention of proteins and other compounds. This allows for improved separation selectivity and resolution of mixtures like lysozyme and cytochrome C.
This study investigated the acute toxicity of mixtures of the organophosphate chlorpyrifos and the carbamate carbaryl on Daphnia magna. A preliminary experiment was conducted following ISO guidelines to determine appropriate concentrations for a definitive toxicity test. Daphnia were exposed to serial dilutions of the pesticide mixture for 48 hours, and immobilization was recorded. Results showed immobilization between 1.25-2.5% concentrations, identifying an appropriate range for the definitive test to establish the median lethal concentration and determine if synergistic toxicity occurs.
This document discusses recent advancements in impurity profiling. It defines impurity profiling and outlines the importance of identifying impurities. The history of instrumental analysis for impurity identification is reviewed. A systematic approach to impurity profiling is presented, including thresholds for identification, qualification, and reporting. Methods for isolation and identification of impurities are described, including case studies. Both classical and modern methodologies are covered, with examples of separation techniques like HPLC, TLC, and capillary electrophoresis.
This document describes a study that developed a method to determine cabergoline and L-dopa levels in human plasma using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The researchers were able to directly analyze deproteinized plasma samples for both cabergoline and L-dopa. Calibration curves for each drug showed good linearity over specific concentration ranges. The method was then applied to analyze plasma samples from patients taking these drugs, with coefficients of variation ranging from 2.9-10.5% indicating good precision. The LC-MS/MS method proved suitable for routine therapeutic drug monitoring of cabergoline and L-dopa.
This document describes a method for determining chloramphenicol (CAP) and thiamphenicol (TAP) residues in fish, shrimp, and milk using electrospray ionization liquid chromatography-tandem mass spectrometry (ESI-LCMSMS). Samples were extracted with ethyl acetate and analyzed using an Agilent Eclipse C18 column with an isocratic mobile phase of methanol and water. Transitions were monitored for CAP, TAP, and an internal standard. Validation was performed according to EU guidelines, showing good linearity, accuracy, precision, limits of detection, and applicability to real samples. The method provides a simple, selective, and sensitive means for simultaneous analysis of CAP and TAP residues
Biomimetic hplc methods to predict in vivo drug distributionCathe Barty
This document discusses how biomimetic HPLC methods can be used to predict in vivo drug distribution and improve drug candidate quality. It provides three key points:
1) HPLC retention times measured using stationary phases that mimic human serum albumin (HSA), alpha-1-acid glycoprotein (AGP), and artificial membranes can be used to estimate binding to these biomolecules and predict tissue distribution.
2) Chromatographic hydrophobicity indices (CHIs) measured at different pH values reveal a compound's acid/base properties without structural information.
3) HPLC methods allow rapid measurement of various physicochemical properties like lipophilicity, permeability, and solubility which influence drug absorption and efficacy.
Stability indicating analytical method development and validation for estimat...SriramNagarajan18
Stability indicating analytical method development and validation for estimation of Sacubitril and Valsartan in bulk and pharmaceutical dosage form using RP-HPLC
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
This document presents a kinetic-spectrophotometric method for quantifying the drug diclofenac. The method involves measuring the rate of the redox reaction between diclofenac and potassium permanganate in a strong acidic environment. The initial slope of the absorbance over time plot is linearly related to the initial diclofenac concentration. The method was found to have good precision and accuracy for quantifying diclofenac in pure form and pharmaceutical tablets. Validation studies showed the method has a linear range of 5-23 ppm for diclofenac quantification. The method was determined to be simple, rapid and specific for diclofenac analysis without sample extraction steps.
The document summarizes a presentation on the Direct Peptide Reactivity Assay (DPRA) method. The DPRA is an in chemico method that uses HPLC to measure the depletion of synthetic peptides exposed to test chemicals, in order to predict a chemical's ability to bind to epidermal proteins and sensitize skin. The presentation describes the objectives, procedures, advantages and precautions of the DPRA method. Reference chemicals are used to establish the DPRA's ability to distinguish sensitizers from non-sensitizers. Percent peptide depletion is calculated to indicate a chemical's sensitization potential and reactivity.
Reversed-Flow Gas Chromatography (RF-GC) is a relatively new technique to determine the physicochemical properties of solute. So far, RF-GC has been used to determine the diffusion coefficient of various solvents. However, the used of RF-GC in other applications such as environmental studies have not been reported. In this study, RF-GC which is a part of flow-perturbation gas chromatography was used to investigate the rate of evaporation of methanol in the presence surfactant (Triton X-100). Waste methanol is considered as an ignitable hazardous waste by US Environmental Protection Agency (USEPA) when its concentrations is equal to or greater than 24% in water. The aim of this study was to determine the effect of Triton X-100 as non-ionic surfactant to suppress evaporation rate of methanol. The result of this study indicated that the evaporation rate of methanol was found to be retarded by the presence of Triton X-100. The percentage of retardation was found to increase with increasing concentration of Triton X-100.
This document summarizes the validation of a quantitative LC/MSMS method to measure the cleavage of a 17-residue SNAP-25 epitope by botulinum neurotoxin serotype A (BoNT/A). Peptides representing the intact 17-mer epitope and its cleavage products (11-mer and 6-mer) were characterized. The method was validated and used to evaluate the kinetics of 17-mer cleavage, finding near-completion at 40-45 minutes. The validated method will be applied to evaluate potential BoNT/A inhibitors and detect contamination in samples.
Bioanalytical method development and validation .Shubham Bora
1) A bioanalytical method was developed and validated for the quantification of levodopa and carbidopa in rat plasma using LC-MS/MS. Derivatization and ion-pairing chromatography were used to improve the chromatographic retention of the polar analytes.
2) The method was fully validated as per FDA guidelines and demonstrated selectivity, linearity, accuracy, precision, recovery, matrix effects and stability in accordance with acceptance criteria.
3) The validated method was successfully applied to support toxicokinetic studies of levodopa and carbidopa in rats.
Understanding Bioanalytical Method Validation in a Regulatory PerspectiveDr. Ishaq B Mohammed
The document provides an overview of bioanalytical method development and validation. It discusses key aspects of the process including sample preparation techniques, calibration curves and quality control standards, method validation parameters such as selectivity, specificity, carryover, precision and accuracy, and acceptance criteria. The goal of bioanalytical method validation is to demonstrate that the analytical method is reliable and reproducible for its intended use in quantitatively measuring analytes in a biological matrix.
A newly validated HPLC method development for simultaneous estimation of rito...SriramNagarajan19
The aim of the present work was to develop a isocratict RP-HPLC for simultaneous analysis of ritonavir and lopinavir in tablet dosage form. Method: chromatographic system was optimized using a Agilent XDB C18(150 x 4.6mm,5µm) column with potassium dihydrogen phosphate (pH 4.6) and acetonitrile in the ratio of 45;55, as a mobile phase, at a flow rate of 1.0 ml/min. detection was carried out at 215nm by a photodiode array detector. Result: ritonavir and lopinavir were eluted with retention times of 4.821 and 3.814mins respectively. Beer’s lambert’s law was obeyed over the concentration ranges of 12.5 to 50µg/ml and 50 to 200µg/ml for ritonavir and lopinavir, respectively. Conclusion: the high recovery and low coefficients of variation confirm the suitability of the method for simultaneous analysis of both drugs in a tablet dosage form. Statistical analysis proves that the method is sensitive and significant for the analysis of ritonavir and lopinavir in pure and in pharmaceutical dosage form without any interference from the excipients. The method was validated in accordance with ICH guidelines. Validation revealed the method is specific, rapid, accurate, precise, reliable, and reproducible.
This document discusses the use of liquid chromatography (LC) and LC-mass spectrometry (LC-MS) in the analysis of active pharmaceutical ingredients (APIs). LC techniques are required by regulatory agencies and are used for separation, identification, and quantification of APIs and impurities. LC is applied in API development, manufacturing, and analytical method development, including assay development, forced degradation studies, impurity profiling, and stability indicating methods. LC-MS provides advantages like isolation of impurities, spectral peak purity analysis, and analysis of drugs in biological fluids. An example case study on the LC-MS analysis of amlodipine is provided, showing mass fragmentation, degradation pathways, and chromatograms of degradation products.
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
This document describes the development and validation of an RP-HPLC method for the simultaneous estimation of atorvastatin and olmesartan tablets. The method was developed using a BDS hypersil C18 column with a mobile phase of ACN-TEA 40-60-0.1 pH 4.0 and detection at 235 nm. The method was validated for parameters such as linearity, precision, accuracy, robustness and system suitability and found to be suitable for the simultaneous quantification of atorvastatin and olmesartan in combined tablet dosage forms.
Quantification of a Novel Peptide, CPT31 in Rat and Monkey Plasma by LC-MSCovance
ASMS 2019 -- CPT31, a novel D-peptide, is being investigated in the treatment and prevention of HIV by inhibiting the viral entry of HIV. To evaluate the properties of CPT31, an accurate highly reproducible method to quantitate CPT31 in plasma was required. To this end, a robust LC-MS assay for the quantification of CPT31 in rat and monkey plasma samples is reported here. The method follows extraction and clean-up of two plasma matrices, encompasses a range of 90.0 to 45,000 ng/mL, and completes LC-MS analysis in 7.50 minutes.
This document describes a liquid chromatographic method for the simultaneous quantitative determination of candesartan cilexetil and hydrochlorothiazide in pharmaceutical dosage forms. A simple and sensitive RP-HPLC method was developed and validated. The method allows for the separation, identification and quantification of candesartan cilexetil and hydrochlorothiazide in tablet formulations using a C18 column with UV detection. The method was validated in terms of specificity, linearity, precision, accuracy and robustness.
Development and Validation of Reverse Phase Liquid Chromatography Method for ...IOSR Journals
This document describes the development and validation of a reverse phase liquid chromatography method for the estimation of losartan in bulk drug samples. The method utilizes an Acquity BEH C18 column with a mobile phase of buffer and acetonitrile at a ratio of 50:50 delivered isocratically at 0.3 mL/min. Losartan was detected at 230 nm. The method was validated per ICH guidelines and found to be linear, precise, accurate, specific and stability-indicating for the quantification of losartan in the range of 25-75 μg/mL. The method validation shows the method is suitable for the routine analysis of losartan in bulk drug materials.
This document describes the development and validation of a stability-indicating HPLC method for the simultaneous quantification of four active pharmaceutical ingredients: pantoprazole, rabeprazole, lansoprazole, and domperidone. The method was developed using a C18 column with gradient elution of a mobile phase consisting of buffer and organic solvents. The method was validated per ICH guidelines and demonstrated selectivity, linearity, accuracy, precision, sensitivity and robustness. Forced degradation studies subjected the drugs to acid, base, oxidation, heat, and light conditions in order to evaluate the method's ability to separate drugs from degradation products.
1) The document describes the development of a LC-MS/MS method for quantifying highly polar aminoglycoside compounds like gentamicin, kanamycin, and apramycin from rat plasma.
2) It investigates using trichloroacetic acid (TCA) both as a protein precipitation agent during sample preparation and as an ion pairing reagent to improve retention of the polar analytes on the reverse phase column.
3) The results show that TCA concentrations between 25-30% provided the best sample recovery during plasma protein precipitation. TCA was also found to improve analyte retention and sensitivity when used as an ion pairing reagent, without needing to add it to the mobile
This document discusses the use of reversed-phase ultrahigh pressure liquid chromatography to analyze macro molecules. It examines the effect of pressure and hydrophobic mobile phase additives on the retention of small molecules and proteins. The document presents data on the physicochemical characteristics and retention factors of various compounds under different pressure and mobile phase conditions. It finds that increasing pressure and adding liophilic agents like NaClO4 and KPF6 increases the retention of proteins and other compounds. This allows for improved separation selectivity and resolution of mixtures like lysozyme and cytochrome C.
This study investigated the acute toxicity of mixtures of the organophosphate chlorpyrifos and the carbamate carbaryl on Daphnia magna. A preliminary experiment was conducted following ISO guidelines to determine appropriate concentrations for a definitive toxicity test. Daphnia were exposed to serial dilutions of the pesticide mixture for 48 hours, and immobilization was recorded. Results showed immobilization between 1.25-2.5% concentrations, identifying an appropriate range for the definitive test to establish the median lethal concentration and determine if synergistic toxicity occurs.
This document discusses recent advancements in impurity profiling. It defines impurity profiling and outlines the importance of identifying impurities. The history of instrumental analysis for impurity identification is reviewed. A systematic approach to impurity profiling is presented, including thresholds for identification, qualification, and reporting. Methods for isolation and identification of impurities are described, including case studies. Both classical and modern methodologies are covered, with examples of separation techniques like HPLC, TLC, and capillary electrophoresis.
This document describes a study that developed a method to determine cabergoline and L-dopa levels in human plasma using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The researchers were able to directly analyze deproteinized plasma samples for both cabergoline and L-dopa. Calibration curves for each drug showed good linearity over specific concentration ranges. The method was then applied to analyze plasma samples from patients taking these drugs, with coefficients of variation ranging from 2.9-10.5% indicating good precision. The LC-MS/MS method proved suitable for routine therapeutic drug monitoring of cabergoline and L-dopa.
This document describes a method for determining chloramphenicol (CAP) and thiamphenicol (TAP) residues in fish, shrimp, and milk using electrospray ionization liquid chromatography-tandem mass spectrometry (ESI-LCMSMS). Samples were extracted with ethyl acetate and analyzed using an Agilent Eclipse C18 column with an isocratic mobile phase of methanol and water. Transitions were monitored for CAP, TAP, and an internal standard. Validation was performed according to EU guidelines, showing good linearity, accuracy, precision, limits of detection, and applicability to real samples. The method provides a simple, selective, and sensitive means for simultaneous analysis of CAP and TAP residues
Biomimetic hplc methods to predict in vivo drug distributionCathe Barty
This document discusses how biomimetic HPLC methods can be used to predict in vivo drug distribution and improve drug candidate quality. It provides three key points:
1) HPLC retention times measured using stationary phases that mimic human serum albumin (HSA), alpha-1-acid glycoprotein (AGP), and artificial membranes can be used to estimate binding to these biomolecules and predict tissue distribution.
2) Chromatographic hydrophobicity indices (CHIs) measured at different pH values reveal a compound's acid/base properties without structural information.
3) HPLC methods allow rapid measurement of various physicochemical properties like lipophilicity, permeability, and solubility which influence drug absorption and efficacy.
Stability indicating analytical method development and validation for estimat...SriramNagarajan18
Stability indicating analytical method development and validation for estimation of Sacubitril and Valsartan in bulk and pharmaceutical dosage form using RP-HPLC
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
This document presents a kinetic-spectrophotometric method for quantifying the drug diclofenac. The method involves measuring the rate of the redox reaction between diclofenac and potassium permanganate in a strong acidic environment. The initial slope of the absorbance over time plot is linearly related to the initial diclofenac concentration. The method was found to have good precision and accuracy for quantifying diclofenac in pure form and pharmaceutical tablets. Validation studies showed the method has a linear range of 5-23 ppm for diclofenac quantification. The method was determined to be simple, rapid and specific for diclofenac analysis without sample extraction steps.
The document summarizes a presentation on the Direct Peptide Reactivity Assay (DPRA) method. The DPRA is an in chemico method that uses HPLC to measure the depletion of synthetic peptides exposed to test chemicals, in order to predict a chemical's ability to bind to epidermal proteins and sensitize skin. The presentation describes the objectives, procedures, advantages and precautions of the DPRA method. Reference chemicals are used to establish the DPRA's ability to distinguish sensitizers from non-sensitizers. Percent peptide depletion is calculated to indicate a chemical's sensitization potential and reactivity.
Reversed-Flow Gas Chromatography (RF-GC) is a relatively new technique to determine the physicochemical properties of solute. So far, RF-GC has been used to determine the diffusion coefficient of various solvents. However, the used of RF-GC in other applications such as environmental studies have not been reported. In this study, RF-GC which is a part of flow-perturbation gas chromatography was used to investigate the rate of evaporation of methanol in the presence surfactant (Triton X-100). Waste methanol is considered as an ignitable hazardous waste by US Environmental Protection Agency (USEPA) when its concentrations is equal to or greater than 24% in water. The aim of this study was to determine the effect of Triton X-100 as non-ionic surfactant to suppress evaporation rate of methanol. The result of this study indicated that the evaporation rate of methanol was found to be retarded by the presence of Triton X-100. The percentage of retardation was found to increase with increasing concentration of Triton X-100.
This document summarizes the validation of a quantitative LC/MSMS method to measure the cleavage of a 17-residue SNAP-25 epitope by botulinum neurotoxin serotype A (BoNT/A). Peptides representing the intact 17-mer epitope and its cleavage products (11-mer and 6-mer) were characterized. The method was validated and used to evaluate the kinetics of 17-mer cleavage, finding near-completion at 40-45 minutes. The validated method will be applied to evaluate potential BoNT/A inhibitors and detect contamination in samples.
Bioanalytical method development and validation .Shubham Bora
1) A bioanalytical method was developed and validated for the quantification of levodopa and carbidopa in rat plasma using LC-MS/MS. Derivatization and ion-pairing chromatography were used to improve the chromatographic retention of the polar analytes.
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Understanding Bioanalytical Method Validation in a Regulatory PerspectiveDr. Ishaq B Mohammed
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This document discusses analytical method development for pharmaceutical drug substances and products. It describes the importance of method development and outlines the key steps involved, including collecting sample information, selecting the chromatographic technique, stationary and mobile phases. It also discusses sources of impurities in drugs, control and qualification of impurities according to ICH guidelines, and pharmacopeial and ICH quality guidelines for impurity testing and thresholds.
This document describes a new approach for fingerprint analysis of Chrysanthemum morifolium Ramat combining ultra-performance liquid chromatography (UPLC) and chemometrics methods. UPLC allowed for chromatographic separation and detection of components in 10 minutes, much faster than conventional HPLC methods. Good precision, reproducibility, and accuracy were obtained for six typical components. Consistent results were seen based on cultivation source. The method provides a more rapid quality control technique for C. morifolium Ramat.
The document discusses optimization of monoclonal antibody purification using protein-A chromatography and precipitation methods. It describes how precipitation of cell culture harvest at low pH can selectively remove impurities. It also discusses how manipulating pH and additives in protein-A chromatography wash buffers can further reduce impurity levels. Optimizing the neutralization pH after protein-A elution can also selectively remove impurities. The optimization strategy reduced process impurities like HCPs and DNA to meet drug substance specifications using just one chromatography column.
This document summarizes the results of an experiment measuring the molecular changes in cells exposed to either acute or gradual environmental changes in hypertonicity. The study found that 1) acute increases in hypertonicity activated caspase signaling and caused cell death, while gradual increases did not activate caspase signaling. 2) Gradual increases instead caused accumulation of proline, which protects cells from death during hypertonic stress. 3) Proline may explain how immune cells can survive in environments within the body where osmolarity changes over time, such as the renal papilla or intestine.
The document describes the development of an enhanced performance test mix for monitoring high-throughput LC/MS systems used in pharmaceutical analysis. The test mix was designed to:
1) Consist of 8 drug-like compounds selected from a pool of 137 compounds to represent typical properties like mass, hydrophobicity, and charge state.
2) Monitor key aspects of LC/MS performance including separation (gradient, flow, pH), detection (UV, ELS, MS signal), and mass accuracy across different conditions.
3) Provide diagnostic information about the likely cause of any errors based on differences observed in test mix results compared to historical data.
Bioanalytical Method Development and Validation for Simultaneous Estimation o...BRNSSPublicationHubI
The document describes the development and validation of a bioanalytical method for the simultaneous estimation of imatinib and its metabolite desmethyl imatinib in human plasma using liquid chromatography-mass spectrometry. Key steps in the method included online enrichment of the analytes followed by separation on a chromatographic column and detection using mass spectrometry. The method was validated in terms of precision, accuracy, selectivity and sensitivity. The developed method was then applied to pharmacokinetic studies of imatinib and its metabolite in patient samples.
The document describes the development and validation of a UPLC-MS/MS method for quantifying terbinafine in human plasma. The method uses a simple liquid-liquid extraction technique, has a run time of less than 2 minutes, and a lower limit of quantification of 15 ng/mL. This high-throughput method requires a small plasma volume of 500 μL and was successfully applied to a bioequivalence study comparing two 250 mg terbinafine tablets in 44 healthy volunteers. The validated method was sensitive, selective, and accurate for quantifying terbinafine plasma concentrations up to 144 hours post-dose to determine pharmacokinetic parameters.
Analysis of bioreactor parameters online and offlinevikash_94
The document discusses various online and offline methods for monitoring cell properties in bioreactors. Online methods discussed include spectrophotometry, fluorometry, and culture fluorescence intensity. Offline analytical methods discussed include measuring medium properties, analyzing cell population composition, analyzing proteins and RNA, and flow cytometry. In situ fluorometry is highlighted as the only continuous monitoring strategy that provides information on the biochemical or metabolic state of cells.
Computational modelling of drug disposition lalitajoshi9
computational modelling of drug disposition is the integral part of computer aided drug design. different kinds of tools being used in the prediction of drug disposition in human body. This topic in the CADD explains the details about the drug disposition, active transporters and tools.
This document presents the development and validation of an RP-HPLC method for the collective analysis of meropenem and amoxicillin. The method was developed to address the need for a cost-effective treatment option for multidrug-resistant tuberculosis using a combination of meropenem and amoxicillin. The objectives were to develop an alternative analytical method for estimating the drugs in combination and to validate the chromatographic method. The developed and validated method can be used to analyze samples containing meropenem and amoxicillin.
Hplc method development for proteins and polypeptides ijsit 2.4.2IJSIT Editor
In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic
purposes. High performance liquid chromatography (HPLC) and its methods of complex peptide or protein
mixtures remains a general method of choice because of the resolution it provides. Unlike small organic
molecules whose chromatographic behavior is described by a finite partitioning equilibrium between the
stationary phase and the mobile phase, proteins and peptides typically do not exhibit such an effect. Instead,
they exhibit an adsorption phenomenon in which the polypeptide is adsorbed onto the stationary phase and
elutes only when the solvent strength of the mobile phase is sufficient to compete with the hydrophobic
forces keeping it there. For this reason, elution of peptides or proteins from reversed-phase supports is by
gradients of increasing solvent strength. There are other differences that one needs to be aware of in order to
develop HPLC methods for separations of proteins and peptides as efficiently as possible.
Indu...impurity profiling of api’s using rp hplc as perhdghcfgfgftf
This document outlines the seminar topic of impurity profiling of active pharmaceutical ingredients (APIs) using reverse phase high performance liquid chromatography (RP-HPLC). It discusses International Conference on Harmonization (ICH) guidelines for classifying and setting limits for impurities. The document describes the process of developing an impurity profile using RP-HPLC, including selecting columns, optimizing the mobile phase pH, organic modifier composition, and gradient slope/temperature. An example application analyzing famotidine impurities is provided.
1) The co-op student worked at Bristol-Myers Squibb to optimize protein purification chromatography conditions and improve impurity clearance.
2) High-throughput screening was used to evaluate different buffer and resin combinations. Process conditions were optimized to separate variants and improve purity.
3) The student met project goals including developing chromatography methods, characterizing protein populations, and providing insights into improving processes. The work focused on optimizing the initial protein A capture step to reduce downstream purification needs.
ElogDoct: A Tool for Lipophilicity Determination in Drug Discovery. 2. Basic ...Brian Bissett
I received a nice acknowledgement in this paper.
ElogDoct: A Tool for Lipophilicity Determination in Drug Discovery. 2. Basic and Neutral Compounds
Franco Lombardo, Marina Y. Shalaeva, Karl A. Tupper, and Feng Gao
Molecular Properties Group and Mathematical and Statistical Sciences Group, Pfizer Global Research and Development
A poor solubility in water limits in a drastic way the effi cacy of a drug, because the absorption phenomenon requires the drugs be in dissolution. The therapeutic activity of a drug is depending of its acid-base dissociation constant (pKa) and solubility, the knowledge of pKa values being thus of great worth. The solubility method can be very useful in spite of their limitations if an appropriate method is available to carry out the solubility measurements of scarce solubility compounds. Some examples taken from the bibliography whose behaviour is well adapted to conventional acid-base dissociation equilibria without further complications are selected for study: Calcein blue, butaperazine, sulfadiazine, tyrosine, 8-hydroxiquinoleine and nifl umic acid. The pKa values have been recalculated applying the single least squares method and the classic monoprotic acid bilogarithmic model. A slope-intercept procedure is also applied to get the evaluation of acidity constants of overlapping equilibria (pKa2 and pKa3 of tyrosine). Results obtained in all cases are compared with literature data.
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Biopharmaceutical Process Development & Manufacturing Final Project
1. Final Project
~Team #5 on Topic #4~
Mitigation of mAb Aggregation in Drug Substance Through Downstream
Process Optimization
Students: Will Carroll, Jonah Spielman, Kenneth Daniel, Victoria Null
Table of Contents
Problem Description 1
Experimental Overview & Strategy 2
Phase 1: Rank mAb Types for Likelihood of Aggregation (5 Working Days) 2
Phase 2: Assessment of pH Dependence for Aggregation Rates (30 Working Days) 2
Phase 3: Assess Process Performance with Respect to the Elution and Hold pH (55 Working
Days) 3
Phase 4: Vary Excipient Type/Buffer Additives and Concentration (30 Working Days) 3
Experimental Details 4
Phase 1 4
Phase 2 4
Phase 3 7
Phase 4 9
References 12
We pledge on our honor that we have not given or received any unauthorized assistance on this
assignment.
2. 1
Problem Description
There are often multiple sources of aggregation during downstream purification. Protein
aggregation decreases the safety, efficacy, and quality of mAbs. Currently, there is no consensus
on the maximum allowable aggregate levels in protein-based pharmaceutical products because
some proteins may be largely stable and safe despite certain levels of aggregates, while for other
proteins very small changes in aggregate levels may significantly affect protein stability and
efficacy. As such, limits for soluble protein aggregates have to be set on a case-by-case basis as
there are no regulatory limits for aggregates in biotherapeutic preparations. In the absence of
information on clinical relevance and process control, many specifications are instituted with
narrower than necessary acceptance ranges based on manufacturing experience. “The only formal
limit in USP 788 is on limiting sub-visible particles, which says that “Solutions for injection must
be clear and practically free from particles”” (Vázquez-Rey & Lang, 2011).
The issue of protein aggregation is important to address for Phase 1 clinical trials because
the goal of these trials is to find the optimal dose with the fewest side effects. Because aggregates
often have negative side effects, it is crucial that the amount of aggregates is minimized in the
downstream process, thus leading to a safe product for the consumer. This report will focus on the
amount of aggregates formed during Protein A Chromatography.
Protein A Chromatography is the most common mAb capture step that is used in the
downstream process. However, this technique requires the use of pHs around 3 to 4. At low pHs,
the mAb has the potential to undergo structural changes, thus leading to aggregation (Vázquez-
Rey & Lang, 2011). It is important to note that Protein A Chromatography does not remove
aggregates. This is due to the interactions of histidyl residues in Protein A and the binding site of
the mAb (Vázquez-Rey & Lang, 2011). At low pHs, both residues have positive charges. This
leads to the two residues repelling each other and the mAb eluting. As aggregates still contain the
histidyl residue, they are also eluted. Therefore, the challenge of minimizing aggregation formation
during Protein A Chromatography lies within stabilizing the protein at low pHs and finding a
balance between using a strong base and a large amount of base at a lower concentration (Vázquez-
Rey & Lang, 2011).
It is estimated that our proposed experiments will be completed within three months using
two scientists working eight hours per day. This amounts to 60 working days per scientist for a
total of 120 working days, during which time four project phases will be completed.
3. 2
Experimental Overview & Strategy
Phase 1: Rank mAb Types for Likelihood of Aggregation (5 Working Days)
The purpose of using TANGO, which determines aggregation propensity, is to predict
which mAbs are aggregation-prone (Kant, et al., 2017). This experiment will be the first phase of
analysis, since the predictions will be compared to those from the later experiments. This
experiment will not influence the mAbs chosen to be studied, however, it will provide valuable
information for future research and development. Additionally, it is assumed that the primary
sequence of all mAbs tested is available.
Phase 2: Assessment of pH Dependence for Aggregation Rates (30 Working Days)
Low pH methods of purification often lead to increased instances of aggregation among
mAb samples (Vázquez-Rey & Lang, 2011). Due to the many mechanisms of aggregation, the
predominant cause of aggregation remains unclear. In literature, it was hypothesized that a low pH
contributed to an abundance of negative charges that favorably contributes to repulsive interactions
that prevent aggregation, a condition of high colloidal stability (Wälchli, R, 2019). Instead, it was
thought that low pH induces protein denaturation. Typically, denaturing has involved partial
unfolding that exposes hydrophobic residues in the mAb (Vázquez-Rey & Lang, 2011). When this
denatured protein is returned to a neutral pH and the repulsive electrostatic forces are reduced,
aggregation can occur readily (Wälchli, R, 2019). This phase of the experiment will focus on the
pH-dependent effects of protein aggregation. This information will help to uncover a greater
understanding of Protein A Chromatography, Low pH Viral Inactivation, and their effects on mAb
aggregation. To consider the mechanism of aggregation proposed by Wälchli, mAb aggregation
will be evaluated both during a low pH hold and upon titration to a pH of 7. The low pH models
the Protein A elution and viral inactivation while the concluding neutral condition is the likely feed
to downstream polishing. The pH dependence of aggregation will be tested by the variance of the
low pH hold condition. SEC will be used to assess aggregation at neutral pH and 1-
anilinonaphthalene-8-sulfonate (ANS) fluorescence will be used to assess degradation at low pH
(Wälchli, R, 2019). The aggregation values at low pH would then be compared to the result from
neutral pH.
4. 3
Phase 3: Assess Process Performance with Respect to the Elution and Hold pH (55 Working
Days)
While phase 2 provides a clear understanding of the pH dependence of mAb degradation
rates, these results do not indicate the performance of Protein A chromatography or the low-pH
viral inactivation. The predominant roles of Protein A chromatography include, among other
functions, the removal of Host Cell (HC) proteins and DNA. Meanwhile, viral inactivation aims
to destroy any viral particles and bacterial contaminants. Given the diversity of potential
contaminants, designing an experimental approach requires substantial restrictions in the interest
of time. All tests are standardized and restricted to a hypothetical condition. Notably, it is assumed
that all mAb stock solutions are essentially pure, lacking both HC materials and viral contaminants.
Hence, all analyses will be restricted to the evaluation of (1) the mAb %
purity/aggregates/fragments in correspondence with Phase #2, (2) the HC-DNA presence in the
protein A eluent, and (3) the presence of select viral particles spiked into the mAb stock. These
observations are made with respect to a set eluent and hold pH obtained with the same stock buffers
tested in phase 2’s analysis. The effects of surfactants and excipients are not of interest in this
study and their presence is not varied.
Phase 4: Vary Excipient Type/Buffer Additives and Concentration (30 Working Days)
The low pH conditions of the elution buffer used during protein A purification is a potential
source of aggregation as described above. Based on an experiment conducted by A.A. Shukla et
al, sucrose, NaCl, and urea buffer additives will be tested for each mAb type (Shukla, 2007).
Variables tested in this experiment include additive type, pH, and additive concentration. The
objective of this experiment is to optimize elution buffer conditions to minimize aggregation
during low pH conditions of Protein A purification. This experiment is conducted last to utilize
pH and aggregation data found in the earlier experimental phases.
5. 4
Experimental Details
Phase 1
The problem at hand pertains to a specific mAb undergoing downstream process
development yet failing to maintain stability and readily aggregating in the drug substance. The
objective of phase 1 is to predict the inherent tendency of a mAb to aggregate. This analysis is
achieved with predictive simulations via a software called TANGO. TANGO observes the mAbs
known amino acid sequence and structure to identify aggregation-prone regions (APRs) in the
protein (Kant, et al., 2017). Additionally, it is important to note that TANGO distinguishes between
APRs that are protected in the thermodynamically stable hydrophobic core and those that are more
easily exposed (Kant, et al., 2017). While the stability issues under question pertain to a singular
mAb (mAb #1) undergoing downstream development, the versatility of this study is expanded by
the inclusion of two additional mAbs (mAb #2 and mAb #3) that are similar in function and
behavior yet have successfully established processing techniques. By the inclusion of these
reference mAbs, a robust analysis of mAb #1’s present issue may be provided in reference to the
specific amino acid sequence and the challenges it may pose. The results of this phase will be
compared to the results of phases 2-4. This will gauge the accuracy of TANGO and determine if
the predictive approach can be implemented in the initial selection process to eliminate
aggregation-prone mAbs, thus speeding up future research and development.
Phase 2
The objective of this phase is to determine when and how the majority of mAb aggregates
develop to focus on future mitigation efforts. A full factorial design is tabulated in Table 1. It
includes 24 experiment conditions, two control conditions, and three baseline conditions. The
experiment samples differ in two parameters: pH and mAb type. As discussed previously, three
different mAbs are evaluated. Meanwhile, the eight pH conditions that are evaluated are 3.0, 3.2,
3.4, 3.6, 3.8, 4.0, 4.5, and 5.0. These pH values are selected upon the consideration of standard
protein A elution conditions (pH 3-4). A pH of 4.5 and 5.0 observe alternative conditions that may
prove less harmful to mAbs during alternative forms of viral inactivation (e.g. via surfactants).
Meanwhile, the time-dependent nature of aggregation during the low pH hold is observed by the
sampling process over a 7-hour period. The 2 control conditions are formed with mAb #2 held at
6. 5
a moderate pH of 4.0 (samples #25 and #26) to measure data precision. The baselines, one of each
mAb at neutral pH, serve as the base values for SEC and DLS analysis.
Table 1. All variable conditions tested in phase 3 and control samples
Sample # pH mAb used
1 pH 3.0 #1
2 pH 3.0 #2
3 pH 3.0 #3
... .... ...
25 pH 4.0 #2
26 pH 4.0 #2
27 Neutral pH #1
28 Neutral pH #2
29 Neutral pH #3
To observe the mAb condition over the course of the hold, one sample will be collected
from the bulk at the start of the hold. This sample will be split into a triad and transferred to a
microplate-well, treated with ANS, and measured for its fluorescence with respect to time
(Wälchli, R, 2019). ANS is used to measure mAb degradation, showing modifications in surface
hydrophobicity associated with denaturation and aggregation (Wälchli, R. 2019). An increase in
ANS fluorescence shows an increase in protein denaturing. At the end of the 7-hour hold period,
the bulk solution will undergo titration to natural pH, another ANS sample is taken and split into
a triad. The fluorescence of these samples are to be observed for an additional 5 hours and
compared to that of the baseline neutral sample of the same mAb (Wälchli, R, 2019). Lower pHs
and longer holds are expected to yield higher fluorescence values. In Figure 1 below, the result of
a similar experiment shows an increase in ANS fluorescence over time at low pH.
7. 6
While ANS is underway, samples are extracted from each condition’s bulk once every 30
minutes for the first 2 hours of the low pH hold, which marks the typical time required for
inactivation. Additional samples are collected once every hour for 5 hours, a total of 7 testing
hours. Each sample is split into two tests, one at the hold-pH and on titrated back to the neutral
pH. Subsequent evaluation is achieved via Size Exclusion Chromatography (HP-SEC). At the end
of the hold, a sample is collected from the titrated bulk solution, allowed to stand for 1 hour, and
run through the HP-SEC. Peaks below the elution time of the mAb’s baseline indicate high
molecular weight aggregates and peaks above this baseline indicate low molecular weight
fragments. Figure 2 shows how SEC data corresponds to different aggregate sizes (Wang, 2018).
The amount of aggregation and fragmentation at low pH is compared to the results post-titration
to confirm or deny the proposed mechanism. If the aggregation rates are noticeably higher post-
titration, the focus of the experiment will turn towards preventing degradation at low pH.
Figure 1. ANS fluorescence
intensity of mAb 1 (a) and
mAb 2 (b) at different solution
pHs, over time. A lower pH
there is a sharper increase in
fluorescence. Adapted from
Ruben Wälchli et al.
Figure 2. Different
aggregate sizes
correspond to
different elution
times. The area
under the curve
corresponds to
particle abundance.
Adapted from
Shunhai Wang et al.
8. 7
Phase 3
The objective of this study is to couple phase 2 results with the observed pH dependence
of protein A elution performance and subsequent viral inactivation. With this interest in mind, each
of the tested eluent and hold conditions are generated with buffers, concentrations, and pHs that
are identical to those of the previous study. Hence, 8 Citrate/Phosphate elution buffers are prepared
with each differing only in their pH: 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.5, 5.0. These buffers serve in the
elution step of the Protein A and set the pH condition for subsequent viral inactivation.
Due to the complexity of potential contaminants faced in downstream processing, the
extent of this experiment is made uniform by the use of a purified mAb stock when formulating
the load-condition solution. This stock is one that is of known mAb type and concentration, free
of undesirable contaminants, and set to the standard loading condition of pH 7 with a
citrate/phosphate buffer. To test the system’s performance, clearly defined stocks of contaminants
are to be generated and spiked into the load-condition solution at known concentrations. One such
class of contaminant stocks will be high concentration HCP-DNA solutions. Three HCP-DNA
stock solutions will be generated; one stock solution per each mAb type, each utilizing the specific
DNA of the mAb’s associated host cell. Accounting for variation in the HCP-DNA among mAbs
enables robust analysis of the elution pH and its impact on eluted mAb purity. Furthermore, a
singular viral stock solution is generated with a mixture of 16 common viral strains, provided in
Table 2, in accordance with standard testing methods (Gombold et al., 2014). Each virus will be
obtained from stock seed solutions, incubated in fetal bovine serum, purified, and placed in a
neutral pH citrate/phosphate buffer. In conclusion, 3 contaminant-spiked neutral-pH load-
condition solutions are generated, varying only in the present mAb type: mAb 1, mAb 2, mAb 3.
Each load solution contains known concentrations of HCP-DNA, all 16 viral strains, and one of
the three mAbs.
9. 8
Table 2. Viral strains for system spiking and performance testing adapted from Gombold et al.
Viral Class Viral Strain Viral Class Viral Strain
Adenoviridae:
Adenovirus 5
Herpesviridae:
Simian
Cytomegalovirus
Adenovirus 41
Herpes Simplex Virus
Type 1
Polyomaviridae: Simian Virus 40
Paramyxoviridae:
Bovine Parainfluenza
Virus Type 3
Flaviviridae:
Bovine Viral Diarrhea
Virus (NY-1)
Coxsackie Virus A16
Orthomyxoviridae:
Influenza A Coxsackie Virus B3
Measles Rhabdoviridae:
Vesicular Stomatitis
Virus
Mumps Togaviridae: Rubella Virus
Picornaviridae: Echovirus 11 Picornaviridae: Rhinovirus 2
The experiment itself will be conducted as a factorial design, testing all possible
combinations of load solution mAb type and elution buffer pH; 24 experimental conditions. All
additional formulation parameters and column chromatography system parameters are to remain
constant at some preselected platform condition. Two additional control studies will be utilized in
an effort to evaluate the natural variance of the measurement techniques. MAb type #2 at elution
pH 4 will serve as the control and the experimental variance is set to the standard deviation of the
triad at this condition. For each test condition, a sample is collected from the load solution pre-
chromatography and from the eluent, low-pH hold, material immediately post-chromatography.
Following the experimental methods of Phase #2, samples are collected from the low-pH hold
every 30 minutes for the first 2 hours and every hour for an additional 5 hours.
All samples are to undergo three assays. The first of which is HP-SEC, discussed
previously, in the interest of evaluating the % purity pre and post chromatography in addition to
measuring the product yield. Furthermore, quantitative PCR (qPCR) will evaluate the presence of
residual HC-DNA in the post-elution samples as a measure of the system’s purification
performance (). This metric is provided in terms of the Cq, Quantitative Cycle, in which a larger
10. 9
value pertains to a lesser presence of HC-DNA and a greater quality of separation (Ichihara, 2018).
Lastly, all samples will receive a viral presence test with in vitro techniques. In this test, each
sample undergoes serial dilutions, creating a subsample array varying only in the dilution factor
(Gombold et al., 2014). Each subsample is split three ways and inoculated onto three distinct test-
cell plates; MRC-5, HeLa, and Vero (Gombold et al., 2014). Following the 14-day incubation and
analysis protocol set forth by Gombold et al., the presence of viral particles is deduced. Observing
the maximum dilution factor of observed virality, and the associated test-cell’s known limit of
detection, the viral concentration can be deduced. The cumulative data of phase 2 and phase 3 will
support the selection of an optimal pH condition.
Phase 4
The objective of Phase 4 experimentation is to optimize protein A elution buffer conditions
to minimize protein aggregation caused by low pH conditions. Three buffer additives will be tested
including sucrose, NaCl, and urea. These three buffer additives have shown to reduce aggregation
during low pH elution in previous studies (Shukla, 2007). The variables tested in this experiment
include additive type, additive concentration, pH, and mAb type.
A full factorial design will be used to assess each of the buffer condition variables. Each
additive will be tested at three concentrations for each mAb type at three well-performing pH
values found in phase 2 and 3. A base or platform buffer control will be tested at each of the pH
values. One trial will be conducted three times to assess the variability of the experiment. This
results in 32 trials per mAb for a total of 96 trials. Conducting a full factorial will enable the study
to include possible variable interactions. Trial condition variables are listed in Table 3 below.
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Table 3. All variable conditions of the full factorial design per mAb.
pH Buffer Additive Additive Concentration (M) mAb type
No. 1 Sucrose 0.5 mAb #1
No. 2 NaCl 1 mAb #2
No. 3 Urea 2 mAb #3
- Platform Buffer -
Percentage of high molecular weight species, HMW%, versus time will be measured for
each trial by SEC. This will indicate the fraction of the elute that is aggregated versus the stable
mAb fraction. An example of the SEC results are shown in Figure 3a below. HMW% versus
elution time will be plotted for each trial to analyze the trend in aggregated species. An example
of this graph is shown below in Figure 3b. The trial with the lowest HMW% trend over time will
indicate the best buffer conditions for each specific mAb type.
Figure 3. a) Absorbance units
versus time in minutes of a
protein A mAb elution adapted
from b) Percentage of HMW
species versus time in hours of
protein A elution fractions under
varying buffer conditions.
Adapted from A.A. Shukla et al.
12. 11
Each lab-scale protein A purification run is expected to be around 10 minutes. 10 additional
minutes will be needed for column cleaning between runs. Therefore, each trial is expected to
require 30 minutes to complete. This requires a total of 48 working hours for all 96 trials. Including
the time needed for data analysis and sample preparation, the total time for experiment phase 4
will be approximated to 30 working days. The results of this study will aid in both present and
future mAb aggregation mitigation efforts.
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References
1. A. A. Shukla et al. (2007) Protein Aggregation Kinetics during Protein A
Chromatography. Journal of Chromatography, 1171, 22-28.
https://doi.org/10.1016/j.chroma.2007.09.040
2. Gombold, J., et al. (2014) Systematic Evaluation of In Vitro and In Vivo Adventitious
Virus Assays for the Detection of Viral Contamination of Cell Banks and Biological
Products. Vaccine, 32(24), 2916-2926. https://doi.org/10.1016/j.vaccine.2014.02.021
3. Ichihara, T., ITO, T., Galipeau, K., Gillespie, C., (2018) Integrated Flow-Through
Purification for Therapeutic Monoclonal Antibodies Processing. MABS, 10(2), 325-334.
https://doi.org/10.1080/19420862.2017.1417717
4. Kant RVD, Karow-Zwick AR, Durme JV, et al. (2017) Prediction and Reduction of the
Aggregation of Monoclonal Antibodies. Journal of Molecular Biology, 429(8),1244-
1261. doi:10.1016/j.jmb.2017.03.014.
5. Vázquez-Rey, M., & Lang, D. A. (2011, July). Aggregates in Monoclonal Antibody
Manufacturing Processes. Biotechnology and Bioengineering, 108(7), 1494-1508. doi:
10.1002/bit.23155.
6. Wälchli, R., et al. (2019, December 11). Understanding mAb Aggregation During Low
pH Viral Inactivation and Subsequent Neutralization. Biotechnology and Bioengineering,
117(3). https://doi-org.proxy-um.researchport.umd.edu/10.1002/bit.27237
7. Wang, S., Liu, A. P., Yan, Y., Daly, T. J., & Li, N. (2018, March 16). Characterization of
Product-related Low Molecular Weight Impurities in Therapeutic Monoclonal Antibodies
Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry.
Journal of Pharmaceutical and Biomedical Analysis, 154, 468-475. https://doi-org.proxy-
um.researchport.umd.edu/10.1016/j.jpba.2018.03.034