Margit Jeschke

Determination of the Origin of Charge Heterogeneity in a Murine Monoclonal Antibody

AbstractPurpose. The aim of this study was to elucidate the molecular basis of charge heterogeneity found in a purified monoclonal IgG1 antibody, MMA383. Methods. Cation exchange chromatography (CEX) and isoelectric focusing (IEF) were used to monitor charge heterogeneity. CEX in conjunction with carboxypeptidase B digests of the antibody was used to determine the contribution of C-terminal lysines to MMA383 charge heterogeneity. Potential chemical degradation sites were identified by peptide mapping of individual chains, with peptide identification by mass spectrometry (MALDI-TOF MS). Peptide sequencing was used to determine specific deamidation sites. Binding constants of predominant isoforms were compared by surface plasmon resonance (SPR). Results. Extensive charge heterogeneity of purified MMA383 was detected by CEX and IEF. Removal of C-terminal lysines simplified the IEF pattern to nine predominant isoforms. Quantitation of isoaspartate in each of the isoforms indicated deamidation of MMA383 as a major cause of charge heterogeneity. CEX of the individual isoform chains suggested the presence of one deamidation site on each of the heavy and light chains. The two sites of deamidation were identified using peptide mapping, sequencing and mass spectrometry. SPR results showed no significant difference in the binding parameters among the isoforms. Conclusions. C-terminal lysine microheterogeneity and deamidation of Asn141 in the heavy chain and Asn161 in the light chain are the major causes of MMA383 charge heterogeneity. Identification of the two deamidation sites will allow replacement of these amino acids in order to create a product less susceptible to degradation.

Discrimination Between Silicone Oil Droplets and Protein Aggregates in Biopharmaceuticals: A Novel Multiparametric Image Filter for Sub-visible Particles in Microflow Imaging Analysis

ABSTRACTPurposeAccurate monitoring of the sub-visible particle load in protein biopharmaceuticals is increasingly important to drug development. Manufacturers are expected to characterize and control sub-visible protein particles in their products due to their potential immunogenicity. Light obscuration, the most commonly used analytical tool to count microscopic particles, does not allow discrimination between potentially harmful protein aggregates and harmless pharmaceutical components, e.g. silicone oil, commonly present in drug products. Microscopic image analysis in flow-microscopy techniques allows not only counting, but also classification of sub-visible particles based on morphology. We present a novel approach to define software filters for analysis of particle morphology in flow-microscopic images enhancing the capabilities of flow-microscopy.MethodsImage morphology analysis was applied to analyze flow-microscopy data from experimental test sets of protein aggregates and silicone oil suspensions.ResultsA combination of four image morphology parameters was found to provide a reliable basis for automatic distinction between silicone oil droplets and protein aggregates in protein biopharmaceuticals resulting in low misclassification errors.ConclusionsA novel, custom-made software filter for discrimination between proteinaceous particles and silicone oil droplets in flow-microscopy imaging analysis was successfully developed.

BIOTECHNOLOGICAL AND GENE THERAPEUTIC STRATEGIES IN CANCER TREATMENT

New anti-cancer agents are being developed which incorporate cancer-cell-specific recognition functions and are thus able to distinguish between normal and tumor cells. Recognition is dependent on the enhanced expression of antigenic determinants on the surface of tumor cells. The ErbB-2 receptor (ErbB-2R) is overproduced in a high percentage of adenocarcinomas arising in the breast, ovary, lung and stomach, when compared to normal cells. The tumor-enriched expression and extracellular accessibility make this receptor a suitable target for directed tumor therapy. A gene expressing the single-chain antibody molecule (scFv), specific for the extracellular domain of the ErbB-2R, was constructed by joining cDNAs encoding the light- and heavy-chain variable domains of the monoclonal antibody (mAb) FRP5. This scFv-encoding gene has been used as a targeting domain for two effectors: (i) A recombinant immunotoxin-encoding gene was constructed by adding sequences encoding a modified Pseudomonas aeroginosa exotoxin A (ETA) to the scFv-encoding DNA. (ii) Cytotoxic T-lymphocytes (CTL) with specificity for ErbB-2R-producing tumor cells were generated by retroviral transfer of a chimeric gene which encodes the scFv(FRP5), a hinge region and the zeta-chain of the T-cell receptor (TCR) complex. The bacterially produced recombinant immunotoxin scFv(FRP5)-ETA binds specifically to the ErbB-2R and displays both in vitro and in vivo cytotoxic effects selective for tumor cells producing high levels of the ErbB-2R. Target cells expressing the ErbB-2R gene were lysed in vitro with high specificity by the scFv::hinge::zeta-expressing T-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoroaluminate Induces Activation and Association of Src and Pyk2 Tyrosine Kinases in Osteoblastic MC3T3-E1 Cells

Fluoride is known to increase bone mass in vivo, probably through stimulation of osteoblast proliferation; however, the mechanisms of fluoroaluminate action in osteoblasts have not yet been elucidated. We have previously shown that in osteoblastic MC3T3-E1 cells, fluoroaluminate stimulates G protein-mediated protein tyrosine phosphorylation (Šuša, M., Standke, G. J. R., Jeschke, M., and Rohner, D. (1997) Biochem. Biophys. Res. Commun. 235, 680–684). Although the Ser/Thr kinases Erk1, Erk2, and p70S6K were activated in response to fluoroaluminate, the identity of fluoroaluminate-activated tyrosine kinase(s) remained elusive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate induces a 110-kDa tyrosine-phosphorylated protein that we identify as Pyk2, a cytoplasmic tyrosine kinase related to Fak (focal adhesion kinase). The tyrosine phosphorylation of Pyk2 increased in a dose- and time-dependent manner. The autophosphorylation activity of Pyk2 increased 3-fold and reached its maximum within 10 min of fluoroaluminate treatment. Fluoroaluminate also induced activation of Src and the association of Pyk2 with Src. The phosphorylation of Src-associated Pyk2 increased >20-fold in in vitro kinase assays, suggesting that Pyk2 is phosphorylated by Src. Although MC3T3-E1 cells express much more Fak than Pyk2, Src preferentially associated with Pyk2. In vitro, Pyk2 bound to the Src SH2 domain, suggesting that this interaction mediates the Src-Pyk2 association in cells. These data indicate that osteoblastic cells express Pyk2, which is tyrosine-phosphorylated and activated in response to G protein activation by fluoroaluminate, and that the mechanism of Pyk2 activation most likely involves Src. Thus, Src and Pyk2 are tyrosine kinases involved in G protein-mediated tyrosine phosphorylation in osteoblastic cells and may be important for the osteogenic action of fluoroaluminate.

Expression of Src Family Kinases and Their Putative Substrates in the Human Preosteoclastic Cell Line FLG 29.1

Several lines of evidence suggest that the c‐Src tyrosine kinase has a specific role in bone‐resorbing osteoclasts. To investigate this further, we examined the expression of c‐Src, its kinase family members, and their putative substrates in the human leukemia cell line FLG 29.1. Western blot analysis with specific antibodies against Src family members showed expression of Src, Fyn, and Lyn, lower levels of Yes and Hck, and the absence of Lck tyrosine kinase. During a 3‐day treatment with phorbol 12‐myristate, 13‐acetate (PMA), which induces differentiation of FLG 29.1 cells toward an osteoclast‐like phenotype, the levels of Src and Fyn increased and the levels of Lyn decreased. In a similar leukemia cell line, HL‐60, Src protein was not constitutively expressed and not induced by PMA treatment, which leads to monocytic differentiation. PMA treatment of FLG 29.1 cells induced a strong increase in the expression of p120 Cbl and Pyk2 kinase, which are putative Src substrates. Pyk2 phosphorylation increased upon adherence of FLG 29.1 cells to fibronectin and to ST2 stromal cells. The expression of other Src substrates and interacting proteins, such as p120 Cas, p130 Cas, vinculin, Fak kinase, and the p85 phosphatidylinositol 3‐kinase subunit either did not change or slightly increased during PMA treatment. The elevated total protein tyrosine phosphorylation in PMA‐treated FLG 29.1 cells was abolished by herbimycin A, a Src inhibitor. These data are consistent with the proposed role of Src in the osteoclastic function and support the use of FLG 29.1 cells as a model to study Src substrates in the cells of the osteoclastic lineage.

Size Fractionation of Microscopic Protein Aggregates Using a Preparative Fluorescence-Activated Cell Sorter

Protein aggregation, which takes place both in vivo and in vitro, is an important degradative pathway for all proteins. Protein aggregates have distinct physicochemical and biological properties that are important to study and characterize from the perspective of both fundamental and applied sciences. The size of protein aggregates varies across a huge range, spanning several orders of magnitude. Currently, protein aggregates larger than hundreds of nanometers in diameter are impossible to physically fractionate. Here, we present a new method to fractionate microscopic proteinaceous particles using preparative fluorescence-activated cell sorting technology.