Intact Protein Mass Spectrometry of Cell Culture Harvest Guides Cell Line Development for Trispecific Antibodies.

IgG-like multispecific antibodies with asymmetric constructs have become widely used formats for therapeutic applications in recent years. Correct assembly of the subunits in this class of therapeutics is a critical quality attribute (CQA) with direct impact on biological activity. Therefore, early drug development efforts such as clone selection during cell line development must be guided by information on potential chain mispairing to enable timely decision making and risk mitigation. Here we describe a high-throughput analytical platform based on denaturing size-exclusion ultraperformance liquid chromatography (UPLC) coupled with intact protein mass spectrometry for profiling of mispairing and other product-related impurities, including half antibodies. This method can be performed directly on the clarified cell culture harvest fluid without the need for Protein A purification or other sample preparations and provides unbiased information on the product quality of the clones and the effect of growth conditions in a fast and cost-effective manner. Screening large numbers of clones expressing different trispecific antibody (tsAb) constructs revealed that although chain mispairing primarily depends on the antibody sequence and structure, it is also a characteristic of the clone. In addition, different growth conditions may affect the type and distribution of half antibodies and mispaired species impurities but not the quality ranking of the clones.

Clusterin glycopeptide variant characterization reveals significant site-specific glycan changes in the plasma of clear cell renal cell carcinoma.

Cancer-related alterations in protein glycosylation may serve as diagnostic or prognostic biomarkers or may be used for monitoring disease progression. Clusterin is a medium abundance, yet heavily glycosylated, glycoprotein that is upregulated in clear cell renal cell carcinoma (ccRCC) tumors. We recently reported that the N-glycan profile of clusterin is altered in the plasma of ccRCC patients. Here, we characterized the occupancy and the degree of heterogeneity of individual N-glycosylation sites of clusterin in the plasma of patients diagnosed with localized ccRCC, before and after curative nephrectomy (n = 40). To this end, we used tandem mass spectrometry of immunoaffinity-enriched plasma samples to analyze the individual glycosylation sites in clusterin. We determined the levels of targeted clusterin glycoforms containing either a biantennary digalactosylated disialylated (A2G2S2) glycan or a core fucosylated biantennary digalactosylated disialylated (FA2G2S2) glycan at N-glycosite N374. We showed that the presence of these two clusterin glycoforms differed significantly in the plasma of patients prior to and after curative nephrectomy for localized ccRCC. Removal of ccRCC led to a significant increase in the levels of both FA2G2S2 and A2G2S2 glycans in plasma clusterin. These changes were further confirmed by lectin blotting of plasma clusterin. It is envisioned that these identified glycan alterations may provide an additional level of therapeutic or biomarker sensitivity than levels currently achievable by monitoring expression differences alone.

Differential chemical derivatization integrated with chromatographic separation for analysis of isomeric sialylated N-glycans: a nano-hydrophilic interaction liquid chromatography-MS platform.

MS analysis of sialylated glycans is challenging due to their low ionization efficiency in positive ion mode as well as the possibility of in-source fragmentation. Chemical derivatization strategies have been developed to address this issue focused on removal of the labile acidic proton prior to MS analysis. Highly sialylated negatively charged glycans also exhibit high retention and unsatisfactory separation efficiency when analyzed by hydrophilic interaction liquid chromatography (HILIC) due to their high polarity. Here, we combined linkage specific derivatization of sialic acids by reaction with the condensation reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) in methanol with nanoscale liquid chromatographic separation prior to accurate mass Orbitrap MS analysis. Coupling DMT-MM charge neutralization of sialic acids with nano-HILIC-Orbitrap-MS not only allows for linkage specific characterization of sialylated glycans directly from the precursor mass but also improves the preceding HILIC separation by increasing the hydrophobicity and altering the selectivity of the oligosaccharide analytes. We focused on the trisialylated N-glycan fraction from haptoglobin and human plasma, enriched using weak anion exchange chromatography, as this trisialylated fraction has been linked with cancer associated changes in the serum N-glycome. The developed methodology was applied to investigate whether structural alterations in this oligosaccharide pool, enriched from the sera of pathological stage and sex matched patients bearing lung, breast, ovarian, pancreatic, or gastric cancer, demonstrate any degree of cancer specificity or whether changes in expression levels are purely cancer associated. The results of this pilot study indicate limited degrees of cancer specificity, particularly for pancreatic cancer, based on alterations in the relative abundance of specific trisialylated isomers.

Qualitative characterization of the rat liver mitochondrial lipidome using all ion fragmentation on an Exactive benchtop Orbitrap MS.

Untargeted lipidomics profiling by liquid chromatography-mass spectrometry (LC-MS) allows researchers to observe the occurrences of lipids in a biological sample without showing intentional bias to any specific class of lipids and allows retrospective reanalysis of data collected. Typically, and in the specific method described, a general extraction method followed by LC separation is used to achieve nonspecific class coverage of the lipidome prior to high-resolution accurate mass (HRAM) MS detection. Here we describe a workflow including the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. We also highlight how, in this method, all-ion fragmentation can be used to identify species of lower abundances, often missed by data-dependent fragmentation techniques. Here we describe the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition.

Multidimensional liquid chromatography platform for profiling alterations of clusterin N-glycosylation in the plasma of patients with renal cell carcinoma.

Identification of potential changes in the glycosylation of existing cancer biomarkers can result in a higher level of diagnostic sensitivity and specificity. Clusterin (Apolipoprotein J) has been implicated in renal cell carcinoma (RCC) and other types of malignancy as potential biomarker. In the present work, an automated multi-dimensional HPLC platform enabling high throughput affinity enrichment of clusterin from plasma samples was developed. Integrated with two dimensional gel electrophoresis, high purity clusterin in microgram quantities suitable for glycan characterization was isolated. The analytical platform was applied to study clusterin glycosylation in a small group of RCC patients before and after nephrectopy as a pilot study to evaluate the performance of the platform. A statistically significant decrease was observed in the levels of a bi-antennary digalactosyl disialylated (A2G2S(3)2) glycans while the levels of a core fucosylated bi-antennary digalactosyl disialylated glycan (FA2G2S(6)2) and a tri-antennary trigalactosyl disialylated glycan (A3G3S(6)2) were increased in the post-surgery plasma samples.

Technologies and strategies for glycoproteomics and glycomics and their application to clinical biomarker research.

Several approaches and technologies are currently available to study the glycosylated proteome (glycoproteomics) or the entire repertoire of glycans in a biological system (glycomics). The biological importance of glycosylation has driven the development of novel, sensitive separation and detection methods. New and improved methodologies, such as high throughput array systems and liquid chromatography-mass spectrometry for glycan profiling and sequencing, are emerging and are being applied in clinical research. A major thrust of glycoproteomics and glycomic clinical research is the application of these analytical tools to cancer research and is aimed at the discovery of glycan-based biomarkers for diagnosis of early stage human cancers, monitoring disease progression, measuring response to therapy, and detecting recurrence. The identification of cancer biomarkers requires a multidisciplinary approach and therefore this review discusses the strategies, technologies and methods currently used for N-glycoprotein/glycanbiomarker research.