Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry

Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between 2 samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.     

Recognition of Porphyromonas gingivalis gingipain epitopes by natural IgM binding to malondialdehyde modified low-density lipoprotein

Objective

Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL.

Methods and Results

Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR^−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans.

Conclusion

Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis.     

The Cell Wall of the Arabidopsis Pollen Tube��Spatial Distribution, Recycling, and Network Formation of Polysaccharides

The pollen tube is a cellular protuberance formed by the pollen grain, or male gametophyte, in flowering plants. Its principal metabolic activity is the synthesis and assembly of cell wall material, which must be precisely coordinated to sustain the characteristic rapid growth rate and to ensure geometrically correct and efficient cellular morphogenesis. Unlike other model species, the cell wall of the Arabidopsis (Arabidopsis thaliana) pollen tube has not been described in detail. We used immunohistochemistry and quantitative image analysis to provide a detailed profile of the spatial distribution of the major cell wall polymers composing the Arabidopsis pollen tube cell wall. Comparison with predictions made by a mechanical model for pollen tube growth revealed the importance of pectin deesterification in determining the cell diameter. Scanning electron microscopy demonstrated that cellulose microfibrils are oriented in near longitudinal orientation in the Arabidopsis pollen tube cell wall, consistent with a linear arrangement of cellulose synthase CESA6 in the plasma membrane. The cellulose label was also found inside cytoplasmic vesicles and might originate from an early activation of cellulose synthases prior to their insertion into the plasma membrane or from recycling of short cellulose polymers by endocytosis. A series of strategic enzymatic treatments also suggests that pectins, cellulose, and callose are highly cross linked to each other.Upon contact with the stigma, the pollen grain swells through water uptake and develops a cellular protrusion, the pollen tube. During its growth in planta, the pollen tube invades the transmitting tissue of the pistil and finds its way to the ovary to deliver the male gametes for double fertilization to happen (Heslop-Harrison, 1987). Depending on the species, pollen tubes can grow extremely rapidly both in planta and in in vitro conditions. To fulfill its biological function, the pollen tube has to (1) adhere to and invade transmitting tissues (Hill and Lord, 1987; Lennon et al., 1998), (2) provide physical protection to the sperm cells, and (3) control its own shape and invasive behavior (Parre and Geitmann, 2005b; Geitmann and Steer, 2006). For all of these functions, the pollen tube cell wall plays an important regulatory and structural role. Although the pollen tube does not form a conventional secondary cell wall layer, its wall is assembled in two phases. The “primary layer” is mainly formed of pectins and other matrix components secreted at the apical end of the cell. The “secondary layer” is assembled by the deposition of callose in more distal regions of the cell (Heslop-Harrison, 1987). Depending on the species, cellulose microfibrils have been found to be associated either with the outer pectic or with the inner callosic layer. Unlike most other plant cells, cellulose is not very abundant representing only 10% of total neutral polysaccharides in Nicotiana alata pollen tubes, whereas callose accounts for more than 80% in this species (Schlüpmann et al., 1994).The biochemical composition of the pollen tube cell wall has been well characterized in many species such as Lilium longiflorum (Lancelle and Hepler, 1992; Jauh and Lord, 1996), tobacco (Nicotiana tabacum; Kroh and Knuiman, 1982; Geitmann et al., 1995; Ferguson et al., 1998; Derksen et al., 2011), Petunia hybrida (Derksen et al., 1999), Pinus sylvestris (Derksen et al., 1999), and Solanum chacoense (Parre and Geitmann, 2005a). But for Arabidopsis (Arabidopsis thaliana), the model for plant molecular biology studies (Arabidopsis Genome Initiative, 2000), there is a striking lack of quantitative information concerning the composition of the pollen tube cell wall as well as the spatial distribution of its components. This is all the more surprising because numerous mutants defective in enzymes involved in cell wall synthesis exhibit a pollen tube phenotype (for example, Jiang et al., 2005; Nishikawa et al., 2005; Wang et al., 2011). Two studies have characterized the Arabidopsis pollen germinating in vitro (Derksen et al., 2002) and in vivo (Lennon and Lord, 2000), but both are qualitative rather than quantitative. A biochemical study by Dardelle and coworkers investigated the cell wall sugar composition in a more quantitative way but does not provide any detailed spatial information (Dardelle et al., 2010; Lehner et al., 2010). This lack of information is not surprising given that until recently Arabidopsis pollen was known to be rather challenging to germinate reproducibly in vitro and more difficult to manipulate than the pollen of many other plant species (Bou Daher et al., 2009). With the publication of optimized methods for in vitro germination (Boavida and McCormick, 2007; Bou Daher et al., 2009), it has become much more feasible to germinate healthy-looking Arabidopsis pollen tubes in vitro in a highly reproducible way.The precisely controlled spatial distribution of biochemical components in the pollen tube cell wall is crucial for shape generation and maintenance of this perfectly cylindrical cell (Geitmann and Parre, 2004; Aouar et al., 2010; Fayant et al., 2010; Geitmann, 2010). The pollen tube, therefore, represents an ideal model system to study the link between intracellular signaling, biochemistry, cell mechanical properties, and morphogenesis in plant cells. Because of its typically fast growth rates, it responds quickly to any environmental triggers such as pharmacological, hormone, or enzymatic treatments. Adding Arabidopsis to the group of commonly studied pollen tube species is particularly timely, because one-third of the approximately 800 cell wall synthesis genes identified in this species are expressed in or are specific to its pollen (Pina et al., 2005). Therefore, the Arabidopsis pollen tube has become a valuable system for cell wall studies, especially with the increasing availability of cell wall mutant lines (Liepman et al., 2010).Here we describe the biochemical composition of the Arabidopsis pollen tube cell wall grown in in vitro conditions using immunocytochemical labeling coupled with epifluorescence and electron microscopic techniques. Rather than relying on imaging alone, we developed a quantitative strategy to assess the precise spatial distribution of cell wall components. This quantitative approach will provide an important tool and baseline dataset for the investigation of mutant phenotypes and for the interpretation of pharmacological studies. Furthermore, we used selective and strategically combined enzymatic digestions to determine the degree of connectivity between the individual types of cell wall polysaccharide networks.     

Cellular and Molecular Mechanisms Underlie the Anti-Tumor Activities Exerted by Walterinnesia aegyptia Venom Combined with Silica Nanoparticles against Multiple Myeloma Cancer Cell Types

Multiple myeloma (MM) is a clonal disease of plasma cells that remains incurable despite the advent of several novel therapeutics. In this study, we aimed to delineate the impact of snake venom extracted from Walterinnesia aegyptia (WEV) alone or in combination with silica nanoparticles (WEV+NP) on primary MM cells isolated from patients diagnosed with MM as well as on two MM cell lines, U266 and RPMI 8226. The IC₅₀ values of WEV and WEV+NP that significantly decreased MM cell viability without affecting the viability of normal peripheral mononuclear cells (PBMCs) were determined to be 25 ng/ml and 10 ng/ml, respectively. Although both WEV (25 ng/ml) and WEV+NP (10 ng/ml) decreased the CD54 surface expression without affecting the expression of CXCR4 (CXCL12 receptor) on MM cells, they significantly reduced the ability of CXC chemokine ligand 12 (CXCL12) to induce actin cytoskeleton rearrangement and the subsequent reduction in chemotaxis. It has been established that the binding of CXCL12 to its receptor CXCR4 activates multiple intracellular signal transduction pathways that regulate MM cell chemotaxis, adhesion, and proliferation. We found that WEV and WEV+NP clearly decreased the CXCL12/CXCR4-mediated activation of AKT, ERK, NFκB and Rho-A using western blot analysis; abrogated the CXCL12-mediated proliferation of MM cells using the CFSE assay; and induced apoptosis in MM cell as determined by PI/annexin V double staining followed by flow cytometry analysis. Monitoring the expression of B-cell CCL/Lymphoma 2 (Bcl-2) family members and their role in apoptosis induction after treatment with WEV or WEV+NP revealed that the combination of WEV with NP robustly decreased the expression of the anti-apoptotic effectors Bcl-2, Bcl_XL and Mcl-1; conversely increased the expression of the pro-apoptotic effectors Bak, Bax and Bim; and altered the mitochondrial membrane potential in MM cells. Taken together, our data reveal the biological effects of WEV and WEV+NP and the underlying mechanisms against myeloma cancer cells.     

Polyclonal antibodies against Staphylococcus aureus ATCC 12 600 catalase do not recognize any protein in cellular extracts from S. aureus subsp. anaerobius

Rabbits were immunized with electrophoretically pure catalase from Staphylococcus aureus ATCC 12 600. The antiserum was used to study whether S. aureus subsp. anaerobius was able to synthesize the apoprotein of catalase. Proteins were separated on polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes and were detected by immunoblotting. No protein reacting with the purified immunoglobulins against S. aureus ATCC 12,600 catalase could be detected in crude and partially purified cellular extracts from S. aureus subsp. anaerobius or its aerotolerant mutants.     

Raman spectroscopy can differentiate malignant tumors from normal breast tissue and detect early neoplastic changes in a mouse model

Raman spectroscopy shows potential in differentiating tumors from normal tissue. We used Raman spectroscopy with near-infrared light excitation to study normal breast tissue and tumors from 11 mice injected with a cancer cell line. Spectra were collected from 17 tumors, 18 samples of adjacent breast tissue and lymph nodes, and 17 tissue samples from the contralateral breast and its adjacent lymph nodes. Discriminant function analysis was used for classification with principal component analysis scores as input data. Tissues were examined by light microscopy following formalin fixation and hematoxylin and eosin staining. Discriminant function analysis and histology agreed on the diagnosis of all contralateral normal, tumor, and mastitis samples, except one tumor which was found to be more similar to normal tissue. Normal tissue adjacent to each tumor was examined as a separate data group called tumor bed. Scattered morphologically suspicious atypical cells not definite for tumor were present in the tumor bed samples. Classification of tumor bed tissue showed that some tumor bed tissues are diagnostically different from normal, tumor, and mastitis tissue. This may reflect malignant molecular alterations prior to morphologic changes, as expected in preneoplastic processes. Raman spectroscopy not only distinguishes tumor from normal breast tissue, but also detects early neoplastic changes prior to definite morphologic alteration.     

Utilization of a new biotinylation reagent in the development of a nondiscriminatory investigative approach for the study of cell surface proteins

In order to circumvent the various problems encountered during the study of membrane-bound proteins, we designed and synthesized a novel membrane-impermeable biotinylation reagent incorporating chemical properties compatible with this goal. We then developed a nondiscriminatory analytical procedure for such studies which overcomes possible selectivity, contamination and solubility problems. The necessary steps (labeling, limited in situ proteolysis, affinity purification) are all conducted in mild or near native conditions. This versatile method could provide an accurate picture of the cell surface proteome.     

The Neonatal and Adult Human Testis Defined at the Single-Cell Level

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