High throughput multidimensional liquid chromatography approach for online protein removal and characterization of polysorbates and poloxamer in monoclonal antibody formulations.

The majority of commercially available monoclonal antibody (mAb) formulations are stabilized with one of three non-ionic surfactants: polysorbate 20 (PS20), polysorbate 80 (PS80), or poloxamer 188 (P188). All three surfactants are susceptible to degradation, which can result in functionality loss and subsequent protein aggregation or free fatty acid particle formation. Consequently, quantitative, and qualitative analysis of surfactants is an integral part of formulation development, stability, and batch release testing. Due to the heterogeneous nature of both polysorbates and poloxamer, online isolation of all the compounds from the protein and other excipients that may disturb the subsequent liquid chromatography with charged aerosol detection (LC-CAD) analysis poses a challenge. Herein, we present an analytical method employing LC-CAD, utilizing a combination of anion and cation exchange columns to completely remove proteins online before infusing the isolated surfactant onto a reversed-phase column. The method allows high throughput analysis of polysorbates within 8 minutes and poloxamer 188 within 12 minutes, providing a separation of the surfactant species of polysorbates (unesterified species, lower esters, and higher esters) and poloxamer 188 (early eluters and main species). Accuracy and precision assessed according to the International Council for harmonisation (ICH) guideline were 96 - 109 % and ≤1 % relative standard deviation respectively for all three surfactants in samples containing up to 110 mg/mL mAb. Subsequently, the method was effectively applied to quantify polysorbate 20 and polysorbate 80 in nine commercial drug products with mAb concentration of up to 180 mg/mL.

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.

Chocolate HILIC phases: development and characterization of novel saccharide-based stationary phases by applying non-enzymatic browning (Maillard reaction) on amino-modified silica surfaces.

Novel saccharide-based stationary phases were developed by applying non-enzymatic browning (Maillard Reaction) on aminopropyl silica material. During this process, the reducing sugars glucose, lactose, maltose, and cellobiose served as "ligand primers". The reaction cascade using cellobiose resulted in an efficient chromatographic material which further served as our model Chocolate HILIC column. (Chocolate refers to the fact that these phases are brownish.) In this way, an amine backbone was introduced to facilitate convenient manipulation of selectivity by additional attractive or repulsive ionic solute-ligand interactions in addition to the typical HILIC retention mechanism. In total, six different test sets and five different mobile phase compositions were investigated, allowing a comprehensive evaluation of the new polar column. It became evident that, besides the so-called HILIC retention mechanism based on partition phenomena, additional adsorption mechanisms, including ionic interactions, take place. Thus, the new column is another example of a HILIC-type column characterized by mixed-modal retention increments. The glucose-modified materials exhibited the relative highest overall hydrophobicity of all grafted Chocolate HILIC columns which enabled retention of lipophilic analytes with high water content mobile phases.

Enhanced methodology for porting ion chromatography retention data.

Porting is a powerful methodology to recalibrate an existing database of ion chromatography (IC) retention times by reflecting the changes of column behavior resulting from either batch-to-batch variability in the production of the column or the manufacture of new versions of a column. This approach has been employed to update extensive databases of retention data of inorganic and organic anions forming part of the "Virtual Column" software marketed by Thermo Fisher Scientific, which is the only available commercial optimization tool for IC separation. The current porting process is accomplished by performing three isocratic separations with two representative analyte ions in order to derive a porting equation which expresses the relationship between old and new data. Although the accuracy of retention prediction is generally enhanced on new columns, errors were observed on some columns. In this work, the porting methodology was modified in order to address this issue, where the porting equation is now derived by using six representative analyte ions (chloride, bromide, iodide, perchlorate, sulfate, and thiosulfate). Additionally, the updated porting methodology has been applied on three Thermo Fisher Scientific columns (AS20, AS19, and AS11HC). The proposed approach showed that the new porting methodology can provide more accurate and robust retention prediction on a wide range of columns, where average errors in retention times for ten test anions under three eluent conditions were less than 1.5%. Moreover, the retention prediction using this new approach provided an acceptable level of accuracy on a used column exhibiting changes in ion-exchange capacity.

Performance comparison of partial least squares-related variable selection methods for quantitative structure retention relationships modelling of retention times in reversed-phase liquid chromatography.

The relative performance of six multivariate data analysis methods derived from or combined with partial least squares (PLS) has been compared in the context of quantitative structure-retention relationships (QSRR). These methods include, GA (genetic algorithm)-PLS, Monte Carlo uninformative variable elimination (MC-UVE), competitive adaptive reweighted sampling (CARS), iteratively retaining informative variables (IRIV), variable iterative space shrinkage approach (VISSA) and PLS with automated backward selection of predictors (autoPLS). A set of 825 molecular descriptors was computed for 86 suspected sports doping compounds and used for predicting their gradient retention times in reversed-phase liquid chromatography (RPLC). The correlation between molecular descriptors selected by each technique and the retention time was established using the PLS method. All models derived from a selected subset of descriptors outperformed the reference PLS model derived from all descriptors, with very small demands of computational time and effort. A performance comparison indicated great diversity of these methods in selecting the most relevant molecular descriptors, ranging from 28 for CARS to 263 for MC-UVE. While VISSA provided the lowest degree of over-fitting for the training set, CARS demonstrated the best compromise between the prediction accuracy and the number of selected descriptors, with the prediction error of as low as 46s for the external test set. Only ten descriptors were found to be common for all models, with the characteristics of these descriptors being representative of the retention mechanism in RPLC.

Comparative characterization of hydrophilic interaction liquid chromatography columns by linear solvation energy relationships.

22 commercially available and home-made stationary phases with different surface modifications were compared under hydrophilic interaction liquid chromatographic (HILIC) conditions. The column set comprised neutral, basic, acidic, zwitterionic and mixed surface modifications. Retention data of 68 differently structured test solutes were acquired to generate retention models based on a linear solvation energy relationship (LSER) approach. A recently modified solvation parameter model with two additional molecular descriptors was evaluated in terms of its universal applicability when electrostatic forces are enabled in addition to predominant partition phenomena. The suggested method could not be confirmed to be a standardized way to characterize HILIC systems when different operating conditions are applied. However, the significant contribution of the recently introduced charge descriptors (D⁻ and D⁺) on explaining the interactions within HILIC systems was confirmed. The solvation parameter model was found to be a useful tool in the course of column development, to affirm or dismiss the preceding educated guess on how certain immobilized ligands will behave. Acidic modified surfaces (stationary phases) exhibit a very small hydrogen bond acceptor property and are less versatile when it comes to an even distribution of solutes along the retention window. Furthermore, the results indicate that basic and neutral columns are more preferable for HILIC applications and might explain why only a limited variety of strong acidic modified HILIC columns, although found in literature, are available commercially.

Additional investigations into the retention mechanism of hydrophilic interaction liquid chromatography by linear solvation energy relationships.

In analogy to our previous publication, the hydrophilic interaction liquid chromatography mechanism was examined in terms of hydrogen bonding, coulombic interactions and phase ratio using linear solvation energy relationships. At first, 23 commercially available and in-house synthesized chromatographic supports are discussed in order to obtain system constants at pH 5.0 with ammonium acetate as buffer salt. Subsequently we compared these outcomes with our former results obtained at pH 3.0 with ammonium formate as buffer additive. Goodness of fit in terms of the adjusted multiple correlation coefficient was found to be reduced under the new conditions. No universal model which simultaneously comprised acidic, basic and neutral analytes could be performed. A significant enhancement of the HILIC systems hydrogen bond basicity was found when changing the pH and buffer counter ions. Even though packing materials showed similar selectivity profiles during the collection of the experimental retention data, different forces were found to account for the overall retention (e.g. Shiseido PC HILIC and Nucleodur HILIC). This indicates that HILIC type selectivity is rather based on a sum of additive or multiplicative phenomena.