Physicochemical and Biological Examination of Two Glatiramer Acetate Products.

Herein we compared 40 mg/mL lots of the active ingredient, glatiramer acetate, manufactured by Mylan/Natco to the active ingredient, glatiramer acetate in Copaxone (Teva Pharmaceuticals, Ltd., Netanya Israel) using physicochemical (PCC) methods and biological assays. No differences were seen between the Mylan/Natco and Teva lots with some low resolution release PCC assays (amino acid analysis, molecular weight distribution, interaction with Coomassie Brilliant Blue G-250). Changes in polydispersity between Mylan/Natco and Copaxone lots were found using size exclusion chromatography and the high resolution PCC method, known as Viscotek, and suggestive of a disparity in the homogeneity of mixture, with a shift towards high molecular weight polypeptides. Using RPLC-2D MALLS, 5 out of 8 Mylan/Natco lots fell outside the Copaxone range, containing a high molecular weight and high hydrophobicity subpopulation of polypeptides not found in Copaxone lots. Cation exchange chromatography showed differences in the surface charge distribution between the Copaxone and Mylan/Natco lots. The Mylan/Natco lots were found to be within Copaxone specifications for the EAE model, monoclonal and polyclonal binding assays and the in vitro cytotoxicity assay, however higher IL-2 secretion was shown for three Mylan/Natco lots in a potency assay. These observations provide data to inform the ongoing scientific discussion about the comparability of glatiramer acetate in Copaxone and follow-on products.

Targeting Attenuated Interferon-α to Myeloma Cells with a CD38 Antibody Induces Potent Tumor Regression with Reduced Off-Target Activity.

Interferon-α (IFNα) has been prescribed to effectively treat multiple myeloma (MM) and other malignancies for decades. Its use has waned in recent years, however, due to significant toxicity and a narrow therapeutic index (TI). We sought to improve IFNα's TI by, first, attaching it to an anti-CD38 antibody, thereby directly targeting it to MM cells, and, second, by introducing an attenuating mutation into the IFNα portion of the fusion protein rendering it relatively inactive on normal, CD38 negative cells. This anti-CD38-IFNα(attenuated) immunocytokine, or CD38-Attenukine™, exhibits 10,000-fold increased specificity for CD38 positive cells in vitro compared to native IFNα and, significantly, is ~6,000-fold less toxic to normal bone marrow cells in vitro than native IFNα. Moreover, the attenuating mutation significantly decreases IFNα biomarker activity in cynomolgus macaques indicating that this approach may yield a better safety profile in humans than native IFNα or a non-attenuated IFNα immunocytokine. In human xenograft MM tumor models, anti-CD38-IFNα(attenuated) exerts potent anti-tumor activity in mice, inducing complete tumor regression in most cases. Furthermore, anti-CD38-IFNα(attenuated) is more efficacious than standard MM treatments (lenalidomide, bortezomib, dexamethasone) and exhibits strong synergy with lenalidomide and with bortezomib in xenograft models. Our findings suggest that tumor-targeted attenuated cytokines such as IFNα can promote robust tumor killing while minimizing systemic toxicity.

Functional protein microarrays.

Microarrays of immobilized functional proteins have the potential to increase dramatically the throughput of proteomic analysis. Micro-immunoassays, in which biological samples are exposed to arrays of immobilized antibodies, can be used for protein expression profiling. In addition, protein function can be elucidated by performing binding and enzymatic assays on arrays of biologically active proteins.

Challenges facing the development and use of protein chips to analyze the phosphoproteome.

Recent advances in analytical methods, particularly in the area of protein microarrays, have brought the field of proteomics to the forefront of biological science. Protein arrays have shown to be useful for the multiplexed analysis of several hundreds of proteins in parallel. While much of the effort has focused on developing methods to identify expressed proteins, the identification of post-translational modifications is equally important for comprehensive proteome characterization. Protein phosphorylation constitutes a major type of post-translational modification that mobilizes a high number of genes, is involved in many crucial cell functions and largely contributes to the complexity of the proteome. One of the major challenges to analyze phosphoproteins using arrays is the availability of specific antibodies. Thus far, this has hampered the development of highly complex phosphoprotein arrays. This review discusses some of the recent progress made in the development of techniques and reagents to quantitatively determine sites of protein phosphorylation.

Comparative immunogenicity assessment: a critical consideration for biosimilar development.

An appropriate assessment strategy with validated anti-drug antibody (ADA) assays is critical for comparative evaluation of immunogenicity between a proposed biosimilar and its reference product. The strategy should aim to identify potential differences in immune responses between these products. While an ADA assay employing the proposed biosimilar product as the detecting reagent has been generally recommended for such evaluation, a product-specific assay using the product of interest may be of use as it offers a capability of detecting antibodies against specific epitopes from the respective product. Regardless of assay strategy, the performance of the assay must be fully assessed and method needs to be validated to meet the comparative purpose of immunogenicity assessment.

Recent developments in protein microarray technology.

The sequencing of the human genome and the advent of DNA chips and sophisticated bioinformatics platforms have enabled molecular biologists to take a more global view of biological systems and to analyze naturally occurring genetic variation. Microarrays of antibodies can measure the concentrations of many proteins quickly and simultaneously. Microarrays of genomically encoded proteins allow scientists to screen entire genomes for proteins that interact with particular factors, catalyze particular reactions, or act as substrates for protein-modifying enzymes or as targets of autoimmune responses. The new protein microarray platforms will prove invaluable to basic biological research, and will dramatically accelerate the pace of discovery of drug targets and diagnostic biomarkers.

Improved method for pepsinolysis of mouse IgG(1) molecules to F(ab')(2) fragments.

Pepsinolysis of immunoglobulin (IgG) to yield F(ab')(2) fragments has been utilized for over 40 years, but the most common subclass of mouse immunoglobulin, IgG(1), is resistant to pepsin cleavage. We show here that this resistance is due to N-linked glycosylation. Deglycosylation by peptide: N-glycosidase F (PNGase F) improves pepsinolysis to generate F(ab')(2) fragments for mouse and rat monoclonal IgG(1) and, in some cases, IgG(2b). This effect was observed for antibodies derived from tissue cell culture or ascites fluid, as well as for polyclonal IgGs from mouse serum. As a result of this finding, the preparation of F(ab')(2) fragments from nearly all mouse IgG molecules should now be a robust procedure.

Protein expression profiling arrays: tools for the multiplexed high-throughput analysis of proteins.

The completion of the human genome sequence has led to a rapid increase in genetic information. The invention of DNA microarrays, which allow for the parallel measurement of thousands of genes on the level of mRNA, has enabled scientists to take a more global view of biological systems. Protein microarrays have a big potential to increase the throughput of proteomic research. Microarrays of antibodies can simultaneously measure the concentration of a multitude of target proteins in a very short period of time. The ability of protein microarrays to increase the quantity of data points in small biological samples on the protein level will have a major impact on basic biological research as well as on the discovery of new drug targets and diagnostic markers. This review highlights the current status of protein expression profiling arrays, their development, applications and limitations.

Recent applications of protein arrays in target identification and disease monitoring.

The focus in the field of protein arrays has shifted from technology development to applying the technology in biological research. This review will highlight several recently published examples of biologically relevant experiments using both planar and bead-based arrays. Examples of the use of antibody arrays, antigen arrays and protein activity arrays will be discussed.:

Antibody World Summit 2004-SRI Conference 19-21 July 2004, Philadelphia, PA, USA.

Overall, the sessions were well attended and a lot of questions were asked. Two-thirds of the speakers were from industry and the rest from academia. Of particular note, The academics presented cutting-edge science in the antibody discovery and development area. Business representatives from various therapeutic antibody companies discussed in detail various aspects and challenges of building a viable therapeutic antibody company.

Generation of bioreagents for protein chips.

Protein microarrays have the potential to dramatically increase the throughput of proteomic analysis. Protein expression profiling chips with distinct spots of immobilized protein capture agents will allow the simultaneous measurement of hundreds to thousands of proteins from one sample. In contrast to DNA chips, for which the capture probes are easily designed and synthesized, the development of content for protein biochips is a long and laborious process. Careful consideration must be given to the specificities desired, the format of the assay, and the requirements of the capture agents, as well as to process optimization to minimize development time and cost. Monoclonal antibodies have been the prime choice as protein capture agents for the majority of protein chips developed to date. New technologies for the production of protein capture agents are more amenable to automation than traditional monoclonal antibody production and therefore carry the promise for industrialization.

Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening.

Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

Chip-based analysis of protein-protein interactions by fluorescence detection and on-chip immunoprecipitation combined with microLC-MS/MS analysis.

A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by microLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein-protein interactions.

Optimizing antibody immobilization strategies for the construction of protein microarrays.

Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.