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Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dye-conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP - IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C 18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the two-dimensional analysis can be detected at low nanogram levels, comparable to results achieved in a one-dimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Hydrophobic interaction chromatography (HIC) and MS are leading techniques for the characterization of the critical quality attributes (CQA) of antibody-drug conjugates (ADCs). This includes the average drug-to-antibody ratio (DAR) and drug loading distribution. A workflow that effectively utilizes the synergy between chromatography and detection technologies has been developed and was assessed using cysteine-conjugated ADCs. The DAR of low, moderate and high drug-loaded ADC samples were calculated from the chromatographic peak areas using LC(HIC)/UV or the deconvoluted mass spectra using native LC(SEC)/MS. The results of DAR by both technologies produced comparable results. In addition, the 2D-LC/MS system has been evaluated in combination with HIC and reversed-phase chromatography for structural identification. Individual peaks from the 1st dimension of the HIC separation were isolated online and re-directed to the 2nd dimension reversed-phase column. ADC was detected as the sub-units by MS and the conjugation site was identified via a middle down approach.

In this study, we demonstrate the utility of ultra-performance liquid chromatography coupled to mass spectrometry (MS) and ion-mobility spectrometry (IMS) to characterize and compare reference and biosimilar monoclonal antibodies (mAbs) at an advanced level. Specifically, we focus on infliximab and compared the glycan profiles, higher order structures, and their host cell proteins (HCPs) of the reference and biosimilar products, which have the brand names Remicade® and Inflectra®, respectively. Overall, the biosimilar attributes mirrored those of the reference product to a very high degree. The glycan profiling analysis demonstrated a high degree of similarity, especially among the higher abundance glycans. Some differences were observed for the lower abundance glycans. Glycans terminated with N-glycolylneuraminic acid were generally observed to be at higher normalized abundance levels on the biosimilar mAb, while those possessing α-linked galactose pairs were more often expressed at higher levels on the reference molecule. Hydrogen deuterium exchange (HDX) analyses further confirmed the higher-order similarity of the 2 molecules. These results demonstrated only very slight differences between the 2 products, which, interestingly, seemed to be in the area where the N-linked glycans reside. The HCP analysis by a 2D-UPLC IMS-MS approach revealed that the same 2 HCPs were present in both mAb samples. Our ability to perform these types of analyses and acquire insightful data for biosimilarity assessment is based upon our highly sensitive UPLC MS and IMS methods.

The enormous dynamic range of proteinaceous species present in protein biotherapeutics poses a significant challenge for current mass spectrometry (MS)-based methods to detect low-abundance HCP impurities. Previously, an HCP assay based on two-dimensional chromatographic separation (high pH/low pH) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry and developed in the author's laboratory has been shown to achieve a detection limit of about 50 ppm (parts per milion) for the identification and quantification of HCPs present in monoclonal antibodies following Protein A purification.1 To improve the HCP detection limit we have explored the utility of several new analytical techniques for HCP analysis and thereby developed an improved liquid chromatography-mass spectrometry (LC-MS) methodology for enhanced detection of HCPs. The new method includes (1) the use of a new charge-surface-modified (CSH) C18 stationary phase to mitigate the challenges of column saturation, peak tailing, and distortion that are commonly observed in the HCP analysis; (2) the incorporation of traveling-wave ion mobility (TWIM) separation of coeluting peptide precursors, and (3) the improvement of fragmentation efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor fragmentation with their mobility drift time. As a result of these improvements, the detection limit of the new methodology was greatly improved, and HCPs present at a concentration as low as 1 ppm (1 ng HCP/mg mAb) were successfully identified and quantified. The newly developed method was applied to analyze two high-purity mAbs (NIST mAb and Infliximab) expressed in a murine cell line. For both samples, low-abundance HCPs (down to 1 ppm) were confidently identified, and the identities of the HCPs were further confirmed by targeted MS/MS experiments. In addition, the performance of the assay was evaluated by an interlaboratory study in which three independent laboratories performed the same HCP assay on the mAb sample. The reproducibility of this assay is also discussed.

Cysteine-conjugated antibody-drug conjugates (ADCs) are manufactured using controlled partial reduction and conjugation chemistry with drug payloads that typically occur in intervals of 0, 2, 4, 6, and 8. Control of heterogeneity is of particular importance to the quality of ADC product because drug loading and distribution can affect the safety and efficacy of the ADC. Liquid chromatography ultra-violet (LC-UV)-based methods can be used to acquire the drug distribution profiles of cysteine-conjugated ADCs when analyzed using hydrophobic interaction chromatography (HIC). However, alternative analysis techniques are often required for structural identification when conjugated drugs do not possess discrete ultra-violet absorbance properties for precise assessment of the drug-to-antibody ratio (DAR). In this study, multidimensional chromatography was used as an efficient method for combining non-compatible techniques, such as HIC, with analysis by mass spectrometry (LC/LC/QTOF-MS) for rapid on-line structural elucidation of species observed in HIC distribution profiles of cysteine-conjugated ADCs. The methodology was tested using an IgG1 mAb modified by cysteine conjugation with a non-toxic drug mimic. Structural elucidation of peaks observed in the HIC analysis (1(st) dimension) were successfully identified based on their unique sub-unit masses via mass spectrometry techniques once dissociation occurred under denaturing reversed phase conditions (2(nd) dimension). Upon identification, the DAR values were determined to be 2.83, 4.44, and 5.97 for 3 drug load levels (low-, medium-, and high-loaded ADC batches), respectively, based on relative abundance from the LC-UV data. This work demonstrates that multidimensional chromatography coupled with MS, provides an efficient approach for on-line biotherapeutic characterization to ensure ADC product quality.

A generic method for the identification and quantification of host-cell proteins (HCPs) in protein biopharmaceuticals is described. Therapeutic proteins and HCPs were converted to complex peptide mixtures following tryptic digestion. Comprehensive peptide separations were performed using online two-dimensional capillary liquid chromatography-(LC) involving high-pH reversed phase (RP)/low-pH RP separations. We applied this method to the analysis of HCP impurities in monoclonal antibody (mAb) preparations.

Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a "discovery" assay, the latter as a "monitoring" assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique ("Hi3" method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.

Influenza vaccination is recognized as the most effective method for reducing morbidity and mortality due to seasonal influenza. To improve vaccine supply and to increase flexibility in vaccine manufacturing, cell culture-based vaccine production has emerged to overcome limitations of egg-based production. The switch of production system and the need for annual re-evaluation of vaccines for the effectiveness due to frequent viral antigenic changes call for methods for complete characterization of the hemagglutinin (HA) antigens and the final vaccine products. This study describes advanced liquid chromatography-mass spectrometry (LC-MS) methods for simultaneous identification of HA proteins and process-related impurities in a trivalent influenza candidate vaccine, comprised of purified recombinant HA (rHA) antigens produced in an insect cell-baculovirus expression vector system (BEVS). N-linked glycosylation sites and glycoforms of the three rHA proteins (corresponding to influenza A subtypes H1N1 and H3N2 and B virus, respectively) were profiled by peptide mapping using reversed-phase (RP) LC-MS(E) (data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode). The detected site-specific glycoforms were further confirmed and quantified by hydrophilic interaction LC (HILIC)-multiple reaction monitoring (MRM) assays. LC-MS(E) was used to characterize the vaccine candidate, providing both protein identities and site-specific information of glycosylation and degradations on each rHA protein. HILIC-MRM methodology was used for rapid confirming and quantifying site-specific glycoforms and potential degradations on each rHA protein. These methods can contribute to the monitoring of vaccine quality especially as it pertains to product comparability studies to evaluate the impact of production process changes.

Current chromatographic and mass spectrometric techniques have limitations for analyzing heparin and heparin oligomers due to their high polarity, structural diversity, and sulfate lability. A rapid method for the analysis of heparin oligosaccharides was developed using ion-pair reversed-phase ultraperformance liquid chromatography coupled with electrospray quadruple time-of-flight mass spectrometry (IPRP-UPLC ESI Q-TOF MS). The method utilizes an optimized buffer system containing a linear pentylamine and a unique additive, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), to achieve highly efficient separation together with enhanced mass response of heparin oligosaccharides. Analyses of a heparin oligosaccharide test mixture, dp6 through dp22, reveal that the chromatographic conditions enable baseline resolution of isomeric heparin oligosaccharides (dp6) and produce intact molecular ions with no sulfate losses during mass spectrometric analysis. In addition, the described conditions are amenable to the detection of heparin oligosaccharides in positive ion mode, yield stronger positive ion signals for corresponding oligosaccharides compared to the negative ion mode, and allow identification of structural isomers by an MS/MS approach. Because sensitive detection of oligosaccharides is also achieved with ultraviolet (UV) detection, the method utilizes a dual detection scheme (UV and MS in series) along with IPRP UPLC to simultaneously obtain quantification (UV) and characterization (MS) data for heparin oligosaccharides. The broad potential of this new method is further demonstrated for the analysis of a low-molecular-weight heparin (LMWH) preparation from porcine heparin. This approach will be of particular utility for profiling the molecular entities of heparin materials, as well as for structural variability comparison for samples from various sources.

RATIONALE: Electrospray ionization mass spectrometry (ESI-MS)-based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation. Adducts directly impact the sensitivity of MSbased analyses as the available charge is distributed across the parent peak and adduct(s). The current study systematically evaluated common liquid chromatography (LC) components in LC/ESI-MS configurations used in oligonucleotide analysis to identify metal adduct contributions from LC instrumentation. METHODS: A UPLC liquid chromatography system was configured with a single quadrupole MS detector (ACQUITY QDa, Waters Corp.) to monitor adduct formation in oligonucleotide separations. An ion-pairing mobile phase comprised of 15 mM triethylamine and 400 mM hexafluoro-2-propanol was used in conjunction with an oligonucleotide separation column (Waters OST BEH C18, 2.1 mm ? 50 mm) for all separations. A 10-min method was used to provide statistical figures of merit and evaluate adduct formation over time. RESULTS: Trace alkali metal salts in the mobile phase and reagents were determined to be the main source of metal salt adducts in LC/ESI-MS-based configurations. Non-specific adsorption sites located throughout the fluidic path contribute to adduct formation in oligonucleotide analyses. Ion-pairing mobile phases prepared at neutral or slightly basic pH result in up to a 57% loss of spectral abundance to adduct formation in the current study. CONCLUSIONS: Implementation of a short low pH reconditioning step was observed to effectively displace trace metal salts non-specifically adsorbed to surfaces in the fluidic path and was able to maintain an average MS spectral abundance ≥94% with a high degree of repeatability (relative standard deviation (R.S.D.) 0.8%) over an extended time study. The proposed method offers the ability to rapidly regenerate adsorption sites with minimal impact on productivity while retaining assay sensitivity afforded by MS detection with reduced adduct formation. [ABSTRACT FROM AUTHOR]

An improved method for peptide de novo sequencing by MALDI mass spectrometry is presented. The method couples a charge derivatization reaction with C-terminal digestion to modify tryptic peptides. The charge derivatization attaches a fixed charge group onto the N-termini of peptides, and the enzymatic digestion after the derivatization step removes C-terminal basic amino acid residues such as arginine and lysine The fragmentation of the modified peptide(s) under low-energy CID conditions (MALDI Q-TOF mass spectrometer) yields a simplified yet complete ion series of the peptide sequence. The validity of the method is demonstrated by the results from several model protein digests, where peptide sequences were correctly deduced either manually or through an automated sequencing program. [ABSTRACT FROM AUTHOR]

A pair of isotopically coded light and heavy reagents, tris-(2 ,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide esters (1 and 2) were synthesized and used to derivatize low molecular weight (< 500 Da) molecules containing primary or secondary amine functional groups for MALDI-TOF MS analysis. The light and heavy reagents added a 573 and 600 Da positively charged tag to each analyte, respectively. In the presence of 10 times excess of tag reagents, the coupling reactions reached near completion within 10 mm. The derivatization greatly facilitated MALDI analysis of small molecules and significantly improved the sensitivity of analysis, allowing a limit of detection in the low femtomole range. Additionally, the reaction mixtures were directly analyzed by MALDI without sample cleanup. The quantification of small molecules by MALDI-TOF MS was successfully achieved by analysis of isotopically coded light and heavy derivatives. MALDI-TOF quantitative analysis of a mixture of antibiotics yielded calibration curves in the concentration range from 0.3 to 30 pmol/μL with r² values greater than 0.9995. [ABSTRACT FROM AUTHOR]