Chromatography Data System Research Articles

Implementation of a LC-MS based multi-attribute method (MAM) and intact multi-attribute method (iMAM) workflow for the characterisation of a GLP-Fc fusion protein

Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography−mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level.

New HPLC, MS, and CDS Products Introduced in 2023–2024: A Brief Review

This article describes newly introduced high-performance liquid chromatography (HPLC), mass spectrometry (MS), chromatography data systems (CDS), and related products in 2023–2024. It summarizes their technical specifications and significant user benefits.

HPLC, MS, and CDS: The New Products to Watch—from Pittcon and Beyond

This article describes newly introduced high-performance liquid chromatography (HPLC), mass spectrometry (MS), chromatography data systems (CDS), and related products in 2022–2023. It summarizes their unique features and significant user benefits, including relevant technical specifications.

What Are Orphan Data?

The term orphan data is used frequently in the context of data integrity. What does it mean for chromatography data systems? How can we prevent or detect orphan data?

Intact multi-attribute method (iMAM): A flexible tool for the analysis of monoclonal antibodies

The availability of rapid methods that can accurately define and quantify biopharmaceutical critical quality attributes has been the driving force for the implementation of mass spectrometry techniques throughout the development and production pipeline. While the multi-attribute method (MAM) has become widely adopted and developed, some critical information cannot be monitored through this workflow, such as correct chain assembly or the presence of fragments or aggregates. In this study, we combine intact protein mass spectrometry and the multi-attribute method to create an intact multi-attribute method – or iMAM. Using a CFR Part 11 compliant data system, we evaluated the proposed workflow using several intact analysis approaches under both denaturing and native conditions. As for the standard MAM approach, iMAM involves the generation of an intact protein target workbook which is created from the analysis of a reference sample, with ID confirmation obtained from deconvolution results and chromatographic retention times, while quantitation is obtained from the intensities of the m/z of most abundant charge states. The created processing method is then applied to the analysis of subsequent samples. New peak detection can also be performed, monitoring the number of components revealed after each analysis. The entire data process can be automated to generate a report within the chromatography data system software. Three case studies presented herein show the potential of iMAM for implementation at different stages of the production pipeline, from product development to stability testing and batch release.

New HPLC, MS, and CDS Products Introduced in 2021–2022: A Brief Review

This installment describes newly introduced high-performance liquid chromatography (HPLC), mass spectrometry (MS), chromatography data systems (CDS), and related products in 2021–2022 and prior years. It summarizes their unique features and significant user benefits.

Stability Indicating HPLC Method for Estimation of Thymoquinone in Nasal Simulated Fluid: Method Development and Validation

Introduction: A simple rapid and precise HPLC method was developed for estimation of TH in nasal simulated fluid and stability was assessed in various stressed conditions.
 Methods: Chromatographic separation of TH in nasal simulated fluid was done using HPLC AS-4050 coupled with Jasco UV 2075 Plus detector, Jasco LC-Net 11/ADC valve, Jasco PU-2080 pump and hypersil gold C18 (250x6x5 µm) column, ChromNAV 2.0 Chromatography Data System software with mobile phase as acetonitrile: water (65:35) and acetonitrile: NSF (60:40) at a flow rate of 1ml/min and having run time of 10 min with loop volume of 20 µl and detection wavelength of 252 nm. The method was validated for accuracy, precision, linearity, specificity, and sensitivity in accordance with ICH (Q2B) guidelines.
 Results: The results of all the validation parameters were found to be within the acceptable limits. The calibration plots were linear over the concentration ranges from 2 to 14µg/ml. The accuracy and precision were found to be between 97.04±0.112 to101.081±0.0191and ≤2% for three drugs. Developed method was successfully applied for the determination TH in nasal simulated fluid and recovery was found to be >98% for three drugs. The degradation products produced as a result of stress studies did not interfere with drug peak.
 Conclusion: The developed method was found to be simple, specific, economic, reliable, accurate, precise, and reproducible used as a quality control tool for analysis of pure thymoquinone in nasal simulated fluid.

AMINO ACID PROFILING IN EDIBLE SHOOTS OF SOME BAMBOO SPECIES BY USING RP-UHPLC AND THEIR THERAPEUTICAL ASPECTS

Objective: This study was conducted to assess the amino acid profiles of edible shoots of five species of bamboos growing in various geographical regions of India. Methods: Qualitative and quantitative estimation of free amino acids was done by Reverse Phase-Ultra High Performance Liquid Chromatography (RP-UHPLC). Separation of amino acids was carried out using Zorbax eclipse plus C18 Reverse Phase (1.8 μm), 50 mm × 2.1 mm column on quaternary UHPLC system using modified gradient elution. Derivatization was done by using auto-sampler Agilent’s 1290 infinity. Data assortment and processing were accomplished by using Open Lab Chromatography Data System EZchrome edition, version A.04.06 (Agilent technologies, USA). Results: In the study, 19 free amino acids were identified, eight essential, five conditionally essential, and six non-essential amino acids (EAA). The principal amino acids were tyrosine (29.41–74.64 μg/mg d.w.) and asparagine (12.81–111.04 μg/mg d.w.). The amount of EAAs ranged from 0.46 to 21.11 μg/mg d.w. For the 1st time, taurine and ornithine are reported in bamboo shoots. The analysis exhibited significant variation (p<0.05) in free amino acids content among the five studied species. Of the five species, Phyllostachys mannii was best compared to the other species in amino acid content. Conclusion: Results showed superior amino acid composition of bamboo shoots that can be utilized to improve nutritional value of functional foods. Amino acids have various health and therapeutic properties can contribute to the basis for further expansion of these bamboos and imminent potential in applications of food and pharmaceuticals.

Consolidating and Managing Data for Drug Development within a Pharmaceutical Laboratory: Comparing the Mapping and Reporting Tools from Software Applications

We present a perspective on drug development for the synthesis of an active pharmaceutical ingredient (e.g., agomelatine) within a commercial technology called Luminata and compare the results to the current method of consolidating the reaction data into Microsoft Excel. The Excel document becomes the ultimate repository of information extracted from multiple sources such as the electronic lab notebook, the laboratory information management system, the chromatography data system, in-house databases, and external data. The major needs of a pharmaceutical company are tracking the stages of multiple reactions, calculating the impurity carryover across the stages, and performing structure dereplication for an unknown impurity. As there is no standardized software available to link the different needs throughout the life cycle of process development, there is a demand for mapping tools to consolidate the route for an API synthesis and link it with analytical data while reducing transcription errors and maintaining an audit trail.

Double entry method for the verification of data a chromatography data system receives

Abstract. The importance of software validation increases since the need for high usability and suitability of software applications grows. In order to reduce costs and manage risk factors, more and more recommendations and rules have been established. In the field of pharmacy the vendors of so-called chromatography data systems (CDSs) had to implement the guidelines of the Code of Federal Regulations Title 21 (CFR 21) during the last few years in order to fulfill the increasing requirements. The CFR 21 part 11 deals with electronic records and signatures. This part is binding for each company in the regulated environment that wishes to create, edit and sign electronic information instead of printing them on paper. Subsection CFR 21 part 11.10(h) explains how to perform an input check for manual user entries as well as for data that will be collected from an external device. In this article we present an approach performing the double entry method on data provided by the hardware instrument in order to investigate possible influences on the raw data by the handling CDS. A software tool has been written which allows us to communicate with a high-performance liquid chromatography (HPLC) detector and acquire data from it. The communication is completely independent of a CDS which is started separately and connected to the same system. Using this configuration we made a parallel data acquisition of two instances at the same time possible. Two CDSs have been tested and for at least one of them it has been shown that a comparison of the acquired data can be done as with the double entry method for the data verification. For the second CDS we checked whether it would be applicable after a few modifications. The given approach could be either used for a live data verification of produced raw data or as a single test during a software operational qualification to verify the data acquisition functionality of the software.

Proving the Preclusion of Data Manipulation Using Parallel Data Acquisition in Chromatography

Traceability has an enormous value for companies, but especially for those working in the regulated environment. It plays a special role in the field of pharmacy with respect to manufacturing, controlling and distributing batches of drugs. Through the guidance of Good Manufacturing Practice (GMP) traceability should be ensured. An increasing number of pharmaceutical companies are member of one of the global pharmacopoeias (United States Pharmacopeia, European Pharmacopeia and Japanese Pharmacopeia). The specifications of these pharmacopoeias describe the best practice in documentation, control, qualification and risk management. But however, the pharmacopoeias are written very generally and do not distinguish between the vendors of the analytical instruments. Here, we analyze how chromatographic analyses and data acquisition rely on a specific vendor of the device and the chromatography data system (CDS), the controlling software. We present a way to compare the data acquisition of different CDSs communicating with HPLC instruments. A newly developed software called Data Collector allows the acquisition of data from a HPLC detector parallel to the controlling CDS in the same run. Two HPLC systems and two different CDSs using a well defined sample standard have been tested. The direct comparison of the acquired data precludes unexpected data manipulations of both tested CDSs and shows that there are primarily deviations between the CDSs due to time variations only which depend on the sampling rate. All in all the Data Collector can be used for the traceability of data acquisition.

Functional risk assessment as part of the validation in the implementation of chromatography data system

A Chromatography Data System (CDS) is a complex software that can be configured to the specific needs of the user’s business process. As such it falls into the Good Automated Manufacturing Practice (GAMP) 5 Category 4 – Configured Products. The validation process is planned and follows along the phases proposed by GAMP 5 for configured products. The Risk assessment stage of the CDS validation process is to carry out a risk assessment of each function of the User Requirements Specification (URS) determined on if the function is regulatory risk critical or not. The functional risk assessment is made according to the method- Failure Mode and Effects Analysis (FMEA). The Overall Risk resulting from the Risk Assessment has identified all potential failures requiring mitigating actions/controls. Mitigating actions and testing controls during the PQ phase is implemented. The final Overall Risk after implementation of Mitigating actions and testing controls during the PQ phase is not more than Medium. Keywords: chromatography data system, validation of the CDS Software, risk assessment, laboratory data integrity

Data integrity aspects of reporting results for the specification parameter related and degradation products in chromatography data system

The most convenient way to implement the requirements of Good Manufacturing Practice and the data integrity principles in a control analytical laboratory is to automate the entire analytical process as fully as possible. Obtaining a final reliable result from an analytical testing as part of a formal analysis is one segment of the process of providing Good Manufacturing Practice. In this paper, we describe the steps by which one control analytical laboratory can formalize and automate the entire process of obtaining and issuing analytical results through creating Report Template for calculation of the specification parameter Related and Degradation Products as part of a Chromatography Data System. The process is described in several steps from designing and structuring of the report so that it would respond best to the end-user's requirements, through identifying eventual oversights in the creation phase, to the final phase of validation, which once again confirms the integrity of its content and justifies its use for formal analysis. The use of such validated Report Templates for calculation and reporting of the results from analytical testing eliminates the human error factor and increases the confidence in the issued results as well as in the entire laboratory’s workflow. Their application facilitate compliance with regulations and minimizes manual input of data which in turn meets one of the most crucial requirements of Good Manufacturing Practice. Keywords: data integrity, chromatography data system, report template, related and degradation products

Direct Determination of Gas pH and Carbon Dioxide Concentration with pH Electrodes

A simple apparatus to measure gas pH and carbon dioxide (CO2) concentration was designed for undergraduate students in the analytical chemistry laboratory. Gas passed through a flow meter and a mixer, then reached the homemade measuring chamber and caused the potential change of a pH combination electrode. Poly(ethylene glycol)-20000 aqueous solutions with hygroscopicity and nontoxicity were used as reaction media by forming absorption liquid films around the junction point and the bulb of the pH combination electrode. A laboratory-made voltage follower and a N2000 chromatography data system were used to transform impedance and record data, respectively. Gas samples containing 10–100% CO2 were measured with the apparatus, and fast potential responses for different concentrations of CO2 were obtained. This apparatus is more cost-effective and eco-friendly as compared with other conventional methods.

Developing A Strategy for A Regulated Electronic Bioanalytical Laboratory

This perspective article considers the strategy, design and implementation of an electronic bioanalytical laboratory working to GLP and/or GCP regulations. There are a range of available automated systems and laboratory informatics that could be implemented and integrated to make an electronic laboratory. However, which are the appropriate ones to select and what is realistic and cost-effective for an individual laboratory? The answer is to develop an overall automation strategy that is updated periodically after each system or application has been implemented to assess if the strategy is still valid or needs to be changed. As many laboratory informatics applications have functional overlap or convergence, for example, Laboratory Information Management System, Electronic Laboratory Notebook, and Instrument and Chromatography Data Systems, the decision of which application performs a specific task needs to be carefully considered in the overall strategy. Ensuring data integrity and regulatory compliance, especially in light of a number of recent falsification cases, is a mandatory consideration for the overall strategy for an electronic bioanalytical laboratory submitting data to regulatory authorities.

Determination of paraquat and diquat in drinking water and environmental water by high performance liquid chromatography coupled with on-line clean-up and solid phase extraction

An on-line solid phase extraction (SPE) system was used to eliminate the interferences sufficiently and fulfill the simple and sensitive determination of diquat and paraquat in tap and pond water. This on-line SPE system used two SPE cartridges. One was an Acclaim Mixed-Mode WAX-1 cartridge for the elimination of anionic interferences; the other one was an Acclaim Mixed-Mode WCX-1 cartridge for the enrichment of diquat and paraquat and the elimination of co-enriched cationic interferences. The baseline separation of diquat and paraquat was achieved on an Acclaim Trinity P1 column. A dual-gradient high performance liquid chromatographic (HPLC) system provided an efficient platform to fulfill the on-line SPE and separation, and the system operated under automatic control of chromatography data system software. The complete analysis only required 16 min, and the detection limits of the method were 0.12 microg/L for diquat and 0.10 microg/L for paraquat. The method is simple, rapid and sensitive, and can be applied to the determination of diquat and paraquat in drinking water and environmental water.

Electronic Laboratory Notebooks Progress and Challenges in Implementation

Electronic laboratory notebooks (ELNs) are increasingly replacing paper notebooks in life science laboratories, including those in industry, academic settings, and hospitals. ELNs offer significant advantages over paper notebooks, but adopting them in a predominantly paper-based environment is usually disruptive. The benefits of ELN increase when they are integrated with other laboratory informatics tools such as laboratory information management systems, chromatography data systems, analytical instrumentation, and scientific data management systems, but there is no well-established path for effective integration of these tools. In this article, we review and evaluate some of the approaches that have been taken thus far and also some radical new methods of integration that are emerging.

On-line solid phase extraction for desalting coupled with mass spectrometric analysis

The work shown here describes a simple, fast and effective on-line solid phase extraction (on-line SPE) method for facilitative high-throughput sample desalting before the detection of mass spectrometry (MS) or liquid chromatography-mass spectrometry (LC-MS). This method includes single SPE column mode and dual SPE column mode. It accomplishes the on-line desalting with an Ultimate 3000 HPLC system equipped with a dual pump system (loading pump/analytical pump), an autosampler, a column oven equipped with a 2p-10p valve, controlled by a chromatography data system. In the single SPE column mode, sample loading and desalting were performed on the SPE column using the loading pump. The analytes were retained on the SPE column, and the salt in the sample solution was flushed out of the SPE column. After desalting, the retained analytes were eluted from the SPE column with the analytical pump. In the dual SPE column mode, two same SPE columns were used. Firstly, the sample loading and desalting were performed on the SPE column 1. The analytes were retained and the salt was flushed out. After desalting on the SPE column 1, the sample loading and desalting were performed on the SPE column 2. Meanwhile, the retained analytes were eluted from the SPE column 1 with the analytical pump. As both of the processes on the SPE columns 1 and 2 described above completed, the sample loading and desalting on the SPE column 1, and the elution of the analytes from the SPE column 2 started again. As the SPE columns 1 and 2 worked in turn, the on-line SPE desalting system was efficient. The eluted analytes from the SPE columns may be determined by MS directly, or separated on the analytical column and then determined by MS/UV.

Does chronic alcohol use by HIV-infected patients on d4T/3TC/NVP drug regimen effect the HIV viral load and what is the therapeutic window of the drugs, CD4+ count and WBC count in patients with high viral load during the 9 months period of follow up?

The study investigated the effects of chronic alcohol use on HIV viral load in HIV-infected patients on d4T/3TC/ NVP drug regimen during 9 months follow up period. It also determined plasma drug concentrations of d4T, 3TC and NVP; CD4 + and WBC counts for patients with high HIV viral load. A case-control study using repeated measures with serial measurements was used. A total of 41 patients (20 alcohol group and 21 control group) were screened for alcohol use using WHO AUDIT tool and chronic alcohol use biomarkers. Blood sampling was done at 3 month intervals for a period of 9 months. HIV viral load was determined using Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor). The d4T, 3TC and NVP concentrations were determined by Shimadzu Class-VPTM HPLC Chromatography data system version 6.1. The CD4 + cell count was determined using FACSCalibur flow cytometer. T he WBC was determined using automated hematological Coulter CBC-5 Hematology Analyzer system. Results show that % patients with HIV viral load ≥400 copies/ml in control group was highest (23.8%, n=5) at 3 month while in chronic alcohol use group, it was at 0 month (35%, n=7) for both WHO AUDIT tool and chronic alcohol-use biomarkers groups. Generally patients with high viral load ≥400 copies/ml was observed in chronic alcohol use as compared to control group in both WHO AUDIT tool and biomarkers group despite of patients having high steady state d4T, 3TC and NVP plasma drug concentrations in circulation that is available to suppress HIV virus. The high viral load could be associated with the emergence of resistance of the HIV virus and these patients generally had a low CD4 + cell count. Some of these patients had no detectable d4T plasma drug concentrations in circulation and most of them with high viral load had sub-therapeutic NVP plasma drug concentrations in their blood circulation. Chronic ethanol use by HIV-infected patients on d4T/3TC/NVP drug regimen increased HIV viral load and the patients with high viral load had sub-therapeutic NVP plasma drug concentrations and some with undetectable d4T drug concentrations in their blood circulation.

Chronic alcohol use affects therapeutic steady state plasma drug concentrations of stavudine, lamivudine and nevirapine in HIV-infected patients during 9 months follow up period: WHO AUDIT tool application

Chronic alcohol consumption is a common problem among the HIV-infected patients on HAART. The study determined the effect of chronic alcohol use on steady state plasma drug concentrations of stavudine (d4T), lamivudine (3TC) and nevirapine (NVP) in HIV-infected patients during the 9 months follow up period. It also determined whether there were some patients with undetectable plasma drug concentrations in their plasma during the follow up. A case control using repeated measures design with serial measurements model, where plasma drug concentrations were measured at 3 month intervals was used. Chronic alcohol-use using WHO AUDIT tool was used to screen patients. A total of 41 patients (21 alcohol group and 20 control group) were followed up for 9 months with blood sampling done at 3 month intervals. The Shimadzu Class-VPTM HPLC Chromatography data system version 6.1 equipment with UV detector was used to measure the plasma drug concentrations. Data was analyzed using SAS 2003 version 9.1 statistical package with repeated measures fixed the model and means were compared using the student t-test. The mean steady state plasma concentration of both d4T and 3TC in chronic alcohol use group were lower than in the control group all throughout the 9 months period of follow-up. The mean steady state plasma drug concentrations of NVP were higher in the alcohol group at 0 and 3 months and lower in the 6 and 9 months as compared to the control group. The mean total plasma NVP concentration was higher in the chronic alcohol group as compared to the control group and the difference was statistically significant (p≤0.05). However some patients had undetectable plasma drug concentrations despite of having ≥ 95 % adherence rate. Chronic alcohol use by the HIV-infected patients lowers the steady state plasma drug concentrations of d4T, 3TC and NVP in patients.