Decades of chromatography research done with DryLab®.

Journal of Chromatography A · 2021

New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies

Szabolcs Fekete, Amarande Murisier, Alain Beck, Jason Lawhorn, Harry Ritchie, Barry Boyes, Davy Guillarme

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH). These three phases were compared with two commercial RPLC phases. In terms of protein recovery, the new ES-LH stationary phase clearly outperforms the other phases for any type of biopharmaceutical sample, and can already be successfully used at a temperature of only 60°C. In terms of retention, the new ES-LH and C1 materials were the less retentive ones, requiring lower organic solvent in the mobile phase. However, it is important to mention that the stability of C1 phase was critical under acidic, high temperature conditions. Finally, some differences were observed in terms of selectivity, particularly for the ES-LH column. Besides the chemical nature of the stationary phase, it was found that the nature of organic modifier also plays a key role in selectivity.

Magazine Article · 2021

Comparing Multivariate Eluent Design Spaces for Systematic Characterization of (U)HPLC Columns | LCGC International

Arnold Zöldhegyi, Hans-Jürgen Rieger, Imre Molnár

Presenting a novel chromatographic modelling method to establish QbD-compliant comparative testing of eluent design spaces.

Journal of Pharmaceutical and Biomedical Analysis · 2021

Development of an analytical method for the determination of pimavanserin and its impurities applying analytical quality by design principles as a risk-based strategy

Irena Radić, Mislav Runje, Sandra Babić

Pimavanserin is an atypical antipsychotic indicated for the treatment of hallucinations and delusions associated with Parkinson’s disease psychosis. As it is a relatively new drug on the market, limited number of pharmacokinetic information and analytical methods are available. This paper presents an ultra-high performance chromatography for the simultaneous determination of pimavanserin and its four process impurities. The method was developed applying analytical quality by design (AQbD) principles as a risk-based approach. Critical method attributes (CMAs) were selected as a resolution between the worst separated compounds (impurity B and impurity C), a duration of analysis defined by the retention time of the last eluting peak (impurity D), a capacity factor of the first eluted impurity (impurity A), a tailing factor and a theoretical plate number. Risk assessment in the early stage of method development pointed out critical method parameters (CMPs): column temperature, gradient time and pH-value of the mobile phase (water phase, eluent A). Design of experiments (DoE), using DryLab®4 software, was applied to evaluate the influence of CMPs on CMAs and to determine method operable design region (MODR). Based on the risk assessment and the results of robustness and precision tests, a control strategy with system suitability criteria was proposed. Developed method was validated according to ICH Q2 (R1) guideline with respect to the selectivity, LOD, LOQ, linearity, precision, accuracy, robustness and stability. A forced degradation study was performed to provide an evidence of the stability-indicating property of the method. Degradation products of pimavanserin were identified using ultra high-performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-qTOF). Additionally, potential degradation products were assessed in silico with the help of Zeneth® software and good agreement with experimentally identified degradation products was achieved. Main degradation products were formed during acid and base hydrolysis (m/z 223.16 [M+H]+ at RRT 0.37) and under oxidative stress conditions (m/z 444.26 [M+H]+ at RRT 0.57). The results revealed that the pimavanserin undergoes degradation through acid and base hydrolysis of urea and N-oxidation of aliphatic tertiary amine.

Molecules (Basel, Switzerland) · 2021

Development of a Fast and Robust UHPLC Method for Apixaban In-Process Control Analysis

Róbert Kormány, Norbert Rácz, Szabolcs Fekete, Krisztián Horváth

In-process control (IPC) is an important task during chemical syntheses in pharmaceutical industry. Despite the fact that each chemical reaction is unique, the most common analytical technique used for IPC analysis is high performance liquid chromatography (HPLC). Today, the so-called "Quality by Design" (QbD) principle is often being applied rather than "Trial and Error" approach for HPLC method development. The QbD approach requires only for a very few experimental measurements to find the appropriate stationary phase and optimal chromatographic conditions such as the composition of mobile phase, gradient steepness or time (tG), temperature (T), and mobile phase pH. In this study, the applicability of a multifactorial liquid chromatographic optimization software was studied in an extended knowledge space. Using state-of-the-art ultra-high performance liquid chromatography (UHPLC), the analysis time can significantly be shortened. By using UHPLC, it is possible to analyse the composition of the reaction mixture within few minutes. In this work, a mixture of route of synthesis of apixaban was analysed on short narrow bore column (50 × 2.1 mm, packed with sub-2 µm particles) resulting in short analysis time. The aim of the study was to cover a relatively narrow range of method parameters (tG, T, pH) in order to find a robust working point (zone). The results of the virtual (modeled) robustness testing were systematically compared to experimental measurements and Design of Experiments (DoE) based predictions.

CRC Press · 2021

Advances in Chromatography: Volume 57

Nelu Grinberg, Peter W. Carr

Journal of Chromatography A · 2021

A methodology employing retention modeling for achieving control space in liquid chromatography method development using quality by design approach

Karthik Jayaraman, Ashok Kumar Rajendran, Gandhi Santosh Kumar, Hemant Bhutani

This study reports the application of retention modeling and quality by design practices for reverse-phase liquid chromatographic method development of a new chemical entity. Prior to the retention modeling, preliminary screening experiments were performed for the selection of stationary phase, organic modifiers, and method parameters. Based on the results of preliminary method conditions, tG-T (gradient time - temperature) 2-D modeling with 4 input runs, and tG-T-tc (gradient time-temperature-ternary composition) 3-D modeling with 12 input runs were designed to build a model for achieving the optimized separation. Modeling of reverse phase separations was based on the measurement of both retention times and peak areas. A design space with appropriate input variables and control strategy was established prior to optimization and robustness evaluation following the quality by design framework. DryLabⓇ was used to predict the optimized gradient profile and separation temperature. The robustness evaluation was carried out using the multiple factors at a time approach and the control space was established. The interdependence of control space and the control strategy was demonstrated by evaluating method robustness using two levels of system suitability criteria. The predictive accuracy of the retention modeling was established through experimental verification of the in-silico predictions. The quality by design based method development approach demonstrated the in-silico optimization as an integral component of reverse-phase chromatographic method development to evaluate the interplay of factors such as organic modifiers, separation temperature and gradient time, which greatly integrated and enhanced method robustness during method development.

CRC Press · 2021

Advances in Chromatography: Volume 38

Phyllis R. Brown

"Volume 38 covers band dispersion in chromatography; structural stochastic concepts, applications to peak shape analysis, and the characteristic function method in the stochastic theory of chromatography; the capabilities and applications of solvating gas chromatography; and more."

Journal of Pharmaceutical and Biomedical Analysis · 2021

Superheated reversed phase chromatography with ultrashort columns for the analysis of therapeutic proteins

Balázs Bobály, Alex Keresztfalvi, Thomas Gräber, Maria Anna Schwarz

Mild or elevated temperatures are routinely used for the analysis of therapeutic proteins by reversed phase liquid chromatography. Generic conditions can be used for the analysis of monoclonal antibodies, and may be adapted for species derived thereof, for instance their immuno-conjugates. Beyond platform monoclonal antibodies, many novel, non-covalent protein complexes are also frequently pursued as protein therapeutics. These complexes, in reverse phased chromatography, may require extremely harsh, superheated conditions to dissociate and elute as interpretable profiles. In order to minimize on-column degradation under superheated conditions, the analysis time has to be reduced as much as possible. Using ultrashort columns and fast gradients is a promising approach in achieving informative separations within a minute, or even faster. Here the applicability of this approach, which supports maintaining levels of degradation products close to the intrinsic sample composition without further on-column degradation is demonstrated. NISTmAb as conventional IgG, a bispecific homodimer and a bispecific homotetramer were used for demonstrating differences in the elution characteristics and the necessity of using the proposed approach. The analysis of the bispecific homodimer was discussed in detail as a case study.

Acta Pharmaceutica Hungarica · 2021

Computer-Assisted Approach for the Development of RP-HPLC Methods for the Separation and Quantification of Bioactive Plant Secondary Metabolites

Mohammed Séghir Daas, Massinissa Faci, Isabella Nicoletti, Malika Douzane, Danilo Corradini

This communication describes the development of computer-assisted RP-HPLC methods for the separation, identification and quantification of phenolic compounds, which are a large class of plant secondary metabolites comprising a great number of heterogeneous structures that range from simple molecules to highly Polymerized compounds. The study has been conducted by a Design of Experiments (DoE) approach that allows the simultaneous optimization of gradient time (tG), column temperature (T), and binary eluent composition on the basis of retention times and peak areas of the analytes of interest, obtained in twelve different experiments. These experiments consist in the linear gradient separations of the investigated compounds performed at two different gradient times and column temperatures, using either the aqueous component of the mobile phase at three different pH values or a combination of two organic solvents at three different volume ratios as the gradient former. The RP-HPLC methods developed by the computer-assisted approach described in this paper were used for the separation and quantification of phenolic compounds occurring in fruits of Olea europaea, extra virgin olive oil (EVOO), and olive mill wastewater (OMWW).

Journal of Chromatography A · 2021

Negative gradient slope methods to improve the separation of closely eluting proteins

Szabolcs Fekete, Amarande Murisier, Jennifer M. Nguyen, Matthew A. Lauber, Davy Guillarme

In the present work, we describe the fundamental and practical advantages of a new strategy to improve the resolution of very closely eluting peaks within therapeutic protein samples. This approach involves the use of multiple isocratic steps, together with the addition of a steep negative gradient segment (with a decrease in mobile phase strength) to "park" a slightly more retained peak somewhere along the column (at a given migration distance), while a slightly less retained compound can be eluted. First, some model calculations were performed to highlight the potential of this innovative approach. For this purpose, the retention parameters (logk0 and S) for two case studies were considered, namely the analysis of a mixture of two therapeutic mAbs (simple to resolve sample) and separation of a therapeutic mAb from its main variant (challenging to resolve sample). The results confirm that the insertion of a negative segment into a multi-isocratic elution program can be a good tool to improve selectivity between critical peak pairs. However, it is also important to keep in mind that this approach only works with large solutes, which more or less follow an “on-off” type elution behavior. Two real applications were successfully developed to illustrate the practical advantage of this new approach, including the separation of a therapeutic mAb from its main variant possessing very close elution behavior, and the separation of a carrier protein from an intact mAb as might be encountered in a quantitative bioanalysis assay. These two examples demonstrate that improved selectivity can be achieved for protein RPLC through the inclusion of a negative gradient slope that selectively bifurcates the elution of two or more peaks of interest.

Analytica Chimica Acta · 2021

Enantioselective multiple heart cutting online two-dimensional liquid chromatography-mass spectrometry of all proteinogenic amino acids with second dimension chiral separations in one-minute time scales on a chiral tandem column

Ryan Karongo, Min Ge, Christian Geibel, Jeannie Horak, Michael Lämmerhofer

In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1D RP-LC (stronger retention) and 2D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1D-RP run and the 68 s 2D chiral separations of 30 heart-cuts (from the 1D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient “smart peak parking” algorithm for the heart cuts and the resulting optimized analysis order thereof. 1D retention time precisions of <0.21% RSD were a requirement for the time-based sampling mode and finally led to a robust, fully automated enantioselective amino acid analysis platform. This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u-13C15N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide.

Journal of Pharmaceutical and Biomedical Analysis · 2021

Computer-assisted UHPLC method development and optimization for the determination of albendazole and its related substances

Elek Ferencz, Éva-Katalin Kelemen, Mona Obreja, Emese Sipos, Sára Vida, Melinda Urkon, Zoltán-István Szabó

Computer-aided ultrahigh performance liquid chromatographic (UHPLC) method development and optimization was undertaken in order to replace an underperforming European Pharmacopoeia method for the determination of albendazole and its related substances. In the preliminary screening, a temperature-gradient time bidimensional model was chosen to aid selection of the proper stationary phase. Hereinafter temperature-gradient time-ternary composition and temperature-gradient time-pH tridimensional models were applied for the optimization of critical method parameters. The simulation and in silico robustness testing were realized using DryLab modeling software. The final method was validated for quantification of impurities and assay of the active substance according to the current ICH guidance. The validated methods were tested on a real, commercial tablet formulation. The experimental design-based and software-assisted method development proved to be a fast and reliable way of replacing a method with inadequate selectivity and long runtime with a robust UHPLC-based method, which offers baseline separation for all monitored impurities in 10 min. Results confirm that software-based chromatographic modelling can not only speed up the analytical method development process, but also improve the reliability of the developed method.

Journal of Chromatography B · 2021

Integrated analytical workflow for chromatographic profiling and metabolite annotation of a cytotoxic Phorbas amaranthus extract

Bruno S. do Amaral, Fernanda B. da Silva, Gabriel Mazzi Leme, Letícia S. S. Schmitz, Paula C. Jimenez, Roberto Carlos Campos Martins, Quezia B. Cass, Alessandra L. Valverde

Phorbas is a widely studied genus of marine sponge and produce structurally rich cytotoxic metabolites. Still, only few studies have assessed metabolites present in Brazilian species. To circumvent redundancy, in this work, we applied and herein report the use of a scouting liquid chromatographic system associate to the design of experiment produced by the DryLab® software to obtain a fast and efficient chromatographic separation of the active hexane fraction, further enabling untargeted high-resolution mass spectrometry (HRMS) data. To this end, a crude hydroalcoholic extract of the sponge Phorbas amaranthus collected in Brazilian coast was prepared and partitioned. The cytotoxicity of the crude extract and the fractions was evaluated using tumor cell culture models. Fragmentation pathways assembled from HRMS data allowed the annotation of 18 known Phorbas metabolites, while 17 metabolites were inferred based on Global Natural Product Social Molecular Networking (GNPS), matching with a further 29 metabolites annotated through molecular subnetwork. The workflow employed demonstrates that chromatographic method development can be accelerated by the use of automated scouting systems and DryLab®, which is useful for profiling natural product libraries, as well as data curation by molecular clusters and should be incorporated to the tools of natural product chemists.

Journal of Pharmaceutical and Biomedical Analysis · 2020

Updating the European Pharmacopoeia impurity profiling method for terazosin and suggesting alternative columns

Dániel Enesei, Imre Kapui, Szabolcs Fekete, Róbert Kormány

This work was motivated by the demand of European Directorate for the Quality of Medicines and HealthCare (EDQM). A new liquid chromatographic (LC) method was developed for terazosin impurity profiling to replace the old European Pharmacopoeia (Ph. Eur.) method. This new method is published as part of the new Ph. Eur. monograph proposal of terazosin in Pharmeuropa issue 32.2. The aim of the method renewal was to cut the analysis time from 90 min (2 × 45 min) down to below 20 min. The Ph. Eur. monograph method is based on two different chromatographic separations to analyze the specified impurities of terazosin. The reason for the two methods is that two of the impurities are not sufficiently retained in reversed phase (RP) conditions, not even with 100% water as eluent. Therefore, next to RP, an ion-pair (IP) chromatographic method has to be applied to analyze those two impurities. With our new proposed method it was possible to appropriately increase the retention of the two critical compounds using alternative stationary phases (instead of a C18 phase which is suggested by the Ph. Eur. method). Applying a pentafluoro-phenyl (PFP) stationary phase, it was feasible to separate and adequately retain all the impurities. The detection wavelength was also changed compared to the Ph. Eur. method and is now appropriate for the detection and quantification of all impurities using perchloric acid in the mobile phase at low pH. Another goal of the present study was to develop a generic workflow and to evaluate the chromatographic resolution in a wide range of method variables and suggest some replacement columns for terazosin impurity profiling. Retention modeling was applied to study the chromatographic behavior of the compounds of interest and visualize resolution for the different columns, where a given criterion is fulfilled. A zone (set of chromatographic conditions) of a robust space could be then quickly identified by the overlay of the individual response surfaces (resolution maps). It was also demonstrated that two columns from different providers (Kinetex F5 and SpeedCore PFP) can be used as replacement columns, providing sufficient resolution at the same working point and a high degree of robustness.

Journal of Chromatography A · 2020

Simultaneous determination of loading capacity and selectivity in preparative off-line two-dimensional separation: An application for purification of corilagin from Pomegranate flower extracts

Guliqire Adili, Guangying Sun, Munire Abuduaini, Yongxin Zhao, Rahima Abdulla, Haji Akber Aisa

This work describes the development of capacity orthogonal chromatography (COC), a new technique for simultaneously determining the loading capacity and orthogonality during the construction of two-dimensional (2D) separations. Three steps were required for the construction of a COC based on the correlation between the selectivity factor (α) and both orthogonality and loading capacity. (1) α values of the impurities-target compound were used to normalize the retention of the impurities around the target compound. (2) α values were input into four quadrants of a coordinate system to identify correlations between orthogonality and loading capacity. α values of the impurities must be greater in the first dimension than the second dimension, with iterated analyses performed until an αmax is obtained for the two purification methods. (3) Touch-peak separation using the first-dimensional αmax was performed and the target compound was collected. The co-eluted impurities are further separated in the second dimension. To test the efficiency of this technique, a COC using two methods on a standard C18 column was developed to purify corilagin from pomegranate flower extract. Despite its low abundance, 288 mg of corilagin was obtained by COC and further purified by LH-20 gel chromatography to obtain the compound with an 80.0% recovery and 98.4% purity. Compared to COC, the purity of corilagin independently obtained using the same purification methods and identical loading capacity was poor (60.1% and 61.6%). These results indicate that COC is a useful tool for extending loading capacity in the development of preparative 2D separations.

Advanced Materials Technologies · 2020

A Novel Approach to Glycan Method Development for Biotherapeutics using Superficially Porous Particle Technology

Joseph L. Glajch, Andrew Harron, Benjamin P. Libert

In this white paper, Advanced Materials Technology presents a novel method for separating and identifying glycans. Glycans are instrumental in cellular signaling, therefore a specific protein’s glycosylation can influence the effectiveness of the biotherapeutic. Given the importance of glycosylation to the overall performance of a glycoprotein, there is a need to completely characterize and then monitor these glycans for both research and manufacturing purposes.

Science of The Total Environment · 2020

High-performance liquid chromatography (HPLC)-fluorescence method for determination of bisphenol A diglycidyl ether (BADGE) and its derivatives in canned foods

Mingming Guo, Mingfeng He, Jianjun Zhong, Qiao He, Balarabe B. Ismail, Guoying Chen, Donghong Liu

Bisphenol A diglycidyl ether (BADGE) is used as a raw material for the production of epoxy resins and PVC organosols, which are commonly applied as inner coatings for food cans. BADGE and its derivatives can migrate from coatings to foodstuffs during processing and storage thereby creating adverse health issues. In this work, a method based on high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) method was developed for the rapid determination of BADGE and its five derivatives in canned foods. Modeling software DryLab® was applied for the optimization of separation conditions. An adequate separation was achieved in 5 min including equilibration time, using a core–shell particle column; such application has not been reported so far. Also, the results showed that LOD varied from 0.01 to 0.20 ng/g, while LOQ varied from 0.03 to 0.66 ng/g, and RSD was found to be <8.64%. The analytical recoveries ranged from 70.46 to 103.44%. Excellent validation data revealed that this method is suitable for the investigation of can coating-to-food migration of BADGE and its derivatives. The HPLC-FLD method is rapid, inexpensive and highly efficient, which could be applicable for safety inspection of food contact materials involving BADGE and its derivatives.

Journal of Pharmaceutical and Biomedical Analysis · 2020

A validated UHPLC-MS method for tryptophan metabolites: Application in the diagnosis of multiple sclerosis

Ferenc Tömösi, Gábor Kecskeméti, Edina Katalin Cseh, Elza Szabó, Cecília Rajda, Róbert Kormány, Zoltán Szabó, László Vécsei, Tamás Janáky

The simultaneous quantitative estimation of tryptophan (TRP) and its metabolites represents a great challenge because of their diverse chemical properties, e.g., presence of acidic, basic, and nonpolar functional groups and their immensely different concentrations in biological matrices. A short ultra high-performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS) method was validated for targeted analysis of TRP and its 11 most important metabolites derived via both kynurenine (KYN) and serotonin (SERO) pathways in human serum and cerebrospinal fluid (CSF): SERO, KYN, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, anthranilic acid, kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), xanthurenic acid, melatonin, picolinic acid (PICA), and quinolinic acid (QUIN). After selecting the “best” reversed-phase column and organic modifier, DryLab®4 was used to optimize the gradient time and temperature in chromatographic separation. To achieve absolute quantification, deuterium-labeled internal standards were used. Among all compounds, 3 were analyzed in derivatized (butyl ester) forms (3-HK, PICA, and QUIN) and the remaining 9 in underivatized forms. Validation was performed in accordance with the ICH and FDA guidelines to determine the intraday and interday precision, accuracy, sensitivity, and recovery. To demonstrate the applicability of the developed UHPLC–MS/MS method, the aforementioned metabolites were analyzed in serum and CSF samples from patients with multiple sclerosis (multiple sclerosis group) and those with symptomatic or noninflammatory neurological diseases (control group). The concentration of QUIN dramatically increased, whereas that of KYNA slightly decreased in the multiple sclerosis group, resulting in a significantly increased QUIN/KYNA ratio and significantly decreased PICA/QUIN ratio.

Journal of Chromatography A · 2020

Improving selectivity and performing online on-column fractioning in liquid chromatography for the separation of therapeutic biopharmaceutical products

Szabolcs Fekete, Harald Ritchie, Jason Lawhorn, Jean-Luc Veuthey, Davy Guillarme

A novel column-coupling approach is suggested to improve both the selectivity and efficiency of protein separations in liquid chromatography. Protein separations often suffer from limited selectivity or not appropriate resolving power. For a new biopharmaceutical product, the identification of the main and minor variant species is required. For that purpose, often offline collection fractioning is applied which is time consuming and regularly dilute the samples to an unacceptable extent. By serially coupling columns in the order of their increasing retentivity and applying “multi-isocratic” elution mode, indeed any (arbitrary) selectivity can be attained. Moreover, if a protein peak is trapped at the inlet of a later column segment – of a coupled system -, its band will be refocused and elute in unprecedented sharp peak. Furthermore, it becomes possible to perform online on-column fractioning of protein species within a very short analysis time (∼ 1 min) and without sample dilution. Two-, three- or multiple column systems can be developed and applied for complex sample separations (such as antibody mixtures). This new methodology can be particularly useful to improve the analysis (and therefore, safety and quality) of therapeutic mAbs and related products and offers benefits compared to offline fractionating. It is also demonstrated in this proof of concept study, that methyl (C1) modified RP phase has a great potential for protein separations despite it is not commercially available in state-of-the-art wide pore superficially porous particle format..

Journal of Pharmaceutical and Biomedical Analysis · 2020

Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles

Julian Kopp, Florian B. Zauner, Andreas Pell, Johanna Hausjell, Diana Humer, Julian Ebner, Christoph Herwig, Oliver Spadiut, Christoph Slouka, Reinhard Pell

Biopharmaceutical drug substances are generally produced using fermentation technology and are subsequently purified in the following downstream process. For the determination of critical quality attributes (CQAs), such as target protein titer and purity, monitoring tools are required before quality control analysis. We herein present a novel reversed phase liquid chromatography method (RPLC), which enables facile and robust protein quantification during upstream and downstream processing of intracellularly produced proteins in E. coli. The overall goal was to develop a fast, robust and mass spectrometry compatible method which can baseline resolve and quantify each protein of interest. Method development consisted of three steps, oriented on an Analytical Quality by Design (AQbD) workflow: (i) the stationary phase as primary parameter was chosen based on state-of-the art technology thus minimizing protein on-column adsorption and providing high efficiency, (ii) secondary parameters (i.e. gradient conditions and column temperature) were optimized applying chromatographic modeling, and (iii) the established Method Operable Design Region (MODR) was challenged and confirmed during robustness testing, performed in-silico and experimentally by a Design of experiment (DoE) based approach. Finally, we validated the RPLC method for pivotal validation parameters (i.e. linearity, limit of quantification, and repeatability) and compared it for protein quantification against a well-established analytical methodology. The outcome of this study shows (i) a protocol for RPLC development using an AQbD principle for new method generation and (ii) a highly versatile RPLC method, suited for quick and straightforward recombinant protein titer measurement being applicable for the detection of a broad range of proteins.