Tackling Issues Observed during the Development of a Liquid Chromatography Method for Small Molecule Quantification in Antibody-Chelator Conjugate.

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.

Two-dimensional sequential selective comprehensive chiral×reversed-phase liquid chromatography of synthetic phosphorothioate oligonucleotide diastereomers.

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of Rp- and Sp-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.

Enantioseparation of chiral sulfonates by liquid chromatography and subcritical fluid chromatography.

Tert-butylcarbamoyl-quinine and -quinidine weak anion-exchange chiral stationary phases (Chiralpak® QN-AX and QD-AX) have been applied for the separation of sodium ß-ketosulfonates, such as sodium chalconesulfonates and derivatives thereof. The influence of type and amount of co- and counterions on retention and enantioresolution was investigated using polar organic mobile phases. Both columns exhibited remarkable enantiodiscrimination properties for the investigated test solutes, in which the quinidine-based column showed better enantioselectivity and slightly stronger retention for all analytes compared to the quinine-derived chiral stationary phase. With an optimized mobile phase (MeOH, 50 mM HOAc, 25 mM NH(3)), 12 of 13 chiral sulfonates could be baseline separated within 8 min using the quinidine-derivatized column. Furthermore, subcritical fluid chromatography (SubFC) mode with a CO(2)-based mobile phase using a buffered methanolic modifier was compared to HPLC. Generally, SubFC exhibited slightly inferior enantioselectivities and lower elution power but also provided unique baseline resolution for one compound.

Increments to chiral recognition facilitating enantiomer separations of chiral acids, bases, and ampholytes using Cinchona-based zwitterion exchanger chiral stationary phases.

The intramolecular distances of anion and cation exchanger sites of zwitterionic chiral stationary phases represent potential tuning sites for enantiomer selectivity. In this contribution, we investigate the influence of alkanesulfonic acid chain length and flexibility on enantiomer separations of chiral acids, bases, and amphoteric molecules for six Cinchona alkaloid-based chiral stationary phases in comparison with structurally related anion and cation exchangers. Employing polar-organic elution conditions, we observed an intramolecular counterion effect for acidic analytes which led to reduced retention times but did not impair enantiomer selectivities. Retention of amphoteric analytes is based on simultaneous double ion pairing of their charged functional groups with the acidic and basic sites of the zwitterionic selectors. A chiral center in the vicinity of the strong cation exchanger site is vital for chiral separations of bases. Sterically demanding side chains are beneficial for separations of free amino acids. Enantioseparations of free (un-derivatized) peptides were particularly successful in stationary phases with straight-chain alkanesulfonic acid sites, pointing to a beneficial influence of more flexible moieties. In addition, we observed pseudo-enantiomeric behavior of quinine and quinidine-derived chiral stationary phases facilitating reversal of elution orders for all analytes.

At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization.

Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding.

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

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.

Is hydrophobic interaction chromatography the most suitable technique to characterize site-specific antibody-drug conjugates?

Antibody drug conjugates (ADCs) belong to the fastest growing class of therapeutic agents for cancer therapy. In preclinical and clinical studies, there is a significant number of site-specific ADCs (also known as third generation ADCs), which are more homogeneous than their previous generations. These new ADC formats, in which the inter-chain disulphide bridges (hinge cysteines) are not reduced, also need to be deeply characterized. In particular, various quality attributes (QAs) have to be determined, such as free antibody level, average drug to antibody ratio (DAR) and drug distribution. In this contribution, a non-commercial site-specific conjugated ADC has been analyzed by RPLC. Our results demonstrated that RPLC has a huge potential to determine QAs and can replace the historically used HIC methods as RPLC provides better separation quality for such type of ADCs. Site-specific ADCs can be analyzed in RPLC at intact level without the need for sample preparation. A further advantage of RPLC is that it enables the direct coupling to MS and thus allows the fine identification of all eluting species.

Synergistic effects on enantioselectivity of zwitterionic chiral stationary phases for separations of chiral acids, bases, and amino acids by HPLC.

In an attempt to overcome the limited applicability scope of earlier proposed Cinchona alkaloid-based chiral weak anion exchangers (WAX) and recently reported aminosulfonic acid-based chiral strong cation exchangers (SCX), which are conceptionally restricted to oppositely charged solutes, their individual chiral selector (SO) subunits have been fused in a combinatorial synthesis approach into single, now zwitterionic, chiral SO motifs. The corresponding zwitterionic ion-exchange-type chiral stationary phases (CSPs) in fact combined the applicability spectra of the parent chiral ion exchangers allowing for enantioseparations of chiral acids and amine-type solutes in liquid chromatography using polar organic mode with largely rivaling separation factors as compared to the parent WAX and SCX CSPs. Furthermore, the application spectrum could be remarkably expanded to various zwitterionic analytes such as alpha- and beta-amino acids and peptides. A set of structurally related yet different CSPs consisting of either a quinine or quinidine alkaloid moiety as anion-exchange subunit and various chiral or achiral amino acids as cation-exchange subunits enabled us to derive structure-enantioselectivity relationships, which clearly provided strong unequivocal evidence for synergistic effects of the two oppositely charged ion-exchange subunits being involved in molecular recognition of zwitterionic analytes by zwitterionic SOs driven by double ionic coordination.

Extending the limits of size exclusion chromatography: Simultaneous separation of free payloads and related species from antibody drug conjugates and their aggregates.

Size exclusion chromatography (SEC) is commonly performed in isocratic conditions to separate partially excluded molecules from the pores of the stationary phase, based on their difference in hydrodynamic volume. In this work, a baseline resolution was obtained between the monomeric antibody drug conjugate (ADC) and high molecular weight species (HMWS). Besides HMWS, small free payloads, linkers and linker-payloads of ADCs, which would not be discriminated solely based on their size (MW < 1.5 kDa), were also separated on the same SEC column by applying sequentially an acetonitrile gradient after the elution of the largest species. Such an approach allowed a simultaneous i) measurement of the HMWS amount under native conditions, and ii) quantitation of the free payloads, within one generic SEC run. For this purpose, a state-of-the-art 150 × 4.6 mm SEC column packed with 2.0 µm particles and 250 Špore size, was selected to achieve fast separations of the species within 10 min. A second dimension (RPLC) was also developed to further extend the possibility offered by this experimental setup. The SECxRPLC multiple heart cutting mode was operated by using a modern 2D-LC instrument containing twelve 120 µL sampling loops. Repeatabilities (0.01% < RSD < 3.68%) and recoveries (between 82% and 107%) were found to be suitable with both approaches (SEC and SECxRPLC), whereas the LOQs remain similar. Finally, the SEC method was applied for the screening of ADC crude reaction mixtures, whereas the SEC x RPLC method facilitated separating some additional impurities. The streamlined methodology will further support the development and characterization of ADC products.

Potential of chiral anion-exchangers operated in various subcritical fluid chromatography modes for resolution of chiral acids.

Anion-exchange-type chiral stationary phases (CSPs) derived from quinine or quinidine were applied in subcritical fluid chromatography (SFC) for the direct separation of chiral acidic compounds. Employing subcritical (sc) mobile phase modes (CO2 + methanol as co-solvent and acids and bases as additives) first the influence of type and amount of acidic and basic additives on separation performance was investigated. Secondly, water was tested as a neutral additive and the influence of temperature variation on enantioselectivity was studied. Thirdly, we could chromatographically confirm that the often verbalized "inherent acidity" of sc CO2 + methanol is manifested by the in situ formation of methylcarbonic acids in the sc mobile phase and thus functioning as acidic additive. Accordingly the dissociated methylcarbonic acid, acting as a counterion, enables an anion exchange mechanism between the cationic CSP and the corresponding acidic analyte. In the absence of a dissociable acid in the mobile phase such an ion exchange mode would not work following a stoichiometric displacement model. This finding is further corroborated by the use of ammonia in methanol as co-solvent thus generating in situ the ammonium salt of methylcarbonic acid. In summary, we report on ion-exchange mediated chromatographic separations in SFC modes by merely using (i) sc CO2 and MeOH, (ii) sc CO2 and ammonia in MeOH, and (iii) sc CO2 and MeOH plus acids and bases as additives. Comparisons to HPLC mode have been undertaken to evaluate merits and limitations. This mode exhibits high potential for preparative chromatography of chiral acids combining pronounced enantioselectivity with high column loadability and avoiding possibly troublesome mobile phase additives, as the in situ formed methylcarbonic acid disintegrates to CO2 and methanol upon pressure release.

Mechanistic investigations of cinchona alkaloid-based zwitterionic chiral stationary phases.

Novel zwitterionic cinchona alkaloid-based chiral selectors (SOs) were synthesized and immobilized on silica gel. The corresponding brush-type chiral stationary phases (CSPs) were characterized as zwitterionic ion-exchange-type materials and exhibited remarkable enantioselectivity for their zwitterionic target analytes, viz. underivatized amino acids and aminosulfonic acids. We rationally designed structural modifications on the strong cation exchange (SCX) subunit of the zwitterionic SO and investigated the influence on chiral recognition power for amphoteric solutes. SOs with chiral isopropyl- or cyclohexyl-moieties in vicinity to the SCX site showed broadest application range by baseline resolving 39 out of 53 test compounds, including α-, ß-, and γ-amino acids with different substitution patterns. Furthermore, we introduced two pseudoenantiomeric zwitterionic CSPs which combined the unique features of providing comparable enantioselectivities but reversed enantiomer elution orders. By application of slightly acidic polar organic mobile phases as preferred elution mode, we found that certain amounts of aprotic acetonitrile in protic methanol substantially increased enantioselectivity and resolution of amino acids in a structure-dependent manner.

Diastereoselective discrimination of lysine-alanine-alanine peptides by zwitterionic cinchona alkaloid-based chiral selectors using electrospray ionization mass spectrometry.

Electrospray ionization-mass spectrometry (ESI-MS) was used to investigate stereoselective interactions between seven zwitterionic alkylsulfonate-modified cinchona alkaloid chiral selectors and biologically relevant lysine-alanine-alanine tripeptide and alanine-alanine dipeptide selectands in modified methanolic solutions. Ion intensities from full scan mass spectra were used to assess degrees of association, the ratios of which were used to calculate selectivities for different selector-selectand pairs. The results support prior work on similar systems using HPLC, in that binding is mediated in these systems primarily through the quinuclidine amine on the selector and the C-terminal carboxylate of the peptide. N(α)- and N(α), N(ɛ)-acetylated forms of the tripeptide were used to study the relative contribution to binding imparted by the presence of multiple basic amines on the tripeptide with the selectors; this was not previously investigated by HPLC. The ability of the sulfonate group on the selector to reach and preferentially interact with the N(ɛ)-amine on the side chain of lysine was revealed. Overall, in acidic methanol conditions (0.5% acetic acid), degrees of association ranged from 1.5% to 17%, and selectivities ranged from non-selective to a 5.5:1 preference for binding one peptide stereoisomer over another with a given chiral selector. In sodium acetate (100 µM)-modified methanol solutions, significant changes in degrees of association (ranging from 4% to 25%) and selectivities (ranging from non-selective to 4.2:1 preference) were observed. These mass spectrometry experiments help to clarify the chiral recognition mechanism for these selectors and suggest that retention and selectivity could be further modulated in HPLC experiments through the utilization of alkali salt-containing mobile phases.

Tailoring of integrin ligands: probing the charge capability of the metal ion-dependent adhesion site.

Intervention in integrin-mediated cell adhesion and integrin signaling pathways is an ongoing area of research in medicinal chemistry and drug development. One key element in integrin-ligand interaction is the coordination of the bivalent cation at the metal ion-dependent adhesion site (MIDAS) by a carboxylic acid function, a consistent feature of all integrin ligands. With the exception of the recently discovered hydroxamic acids, all bioisosteric attempts to replace the carboxylic acid of integrin ligands failed. We report that phosphinates as well as monomethyl phosphonates represent excellent isosters, when introduced into integrin antagonists for the platelet integrin αIIbß3. The novel inhibitors exhibit in vitro and ex vivo activities in the low nanomolar range. Steric and charge requirements of the MIDAS region were unraveled, thus paving the way for an in silico prediction of ligand activity and in turn the rational design of the next generation of integrin antagonists.

Enantiomer separation and indirect chromatographic absolute configuration prediction of chiral pirinixic acid derivatives: Limitations of polysaccharide-type chiral stationary phases in comparison to chiral anion-exchangers.

Chiral alpha-arylthiocarboxylic acids with different substitution patterns, representing new pirinixic acid derivatives with dual PPARalpha/gamma agonistic activities, have been separated into enantiomers on tert-butylcarbamoylquinine and quinidine based chiral anion-exchangers and amylose tris(3,5-dimethylphenylcarbamate) coated silica on analytical and preparative scale. Absolute configurations of individual enantiomers were assigned chromatographically via elution orders on the chiral anion-exchangers and were confirmed by stereoselective syntheses via Ewans auxiliaries that have lead to enantiomeric products with known absolute configurations. The results of both methods were in full agreement. Moreover, the receptor stereoselectivity in PPARalpha transactivation activities was consistent within the test set of structurally related compounds. Limited correlation (between elution order and substitution) was observed within the set of alpha-arylthiocarboxylic acids on the amylose tris(3,5-dimethylphenylcarbamate) based chiral stationary phase (CSP), in particular the elution order changed with remote substitution. This clearly demonstrates the risks of chromatographic absolute configuration assignments by prediction from one structural analog to another one, especially with CSPs such as polysaccharide CSPs that are recognized for their broad applicability due to multiple binding and chiral recognition modes. It is therefore of utmost importance that such chromatographic absolute configuration predictions by extrapolation to structural analogs are combined with orthogonal methods for verification of the results.