Immunoglobulin Fusion Protein Research Articles

Expression of a soluble functional form of the integrin alpha4beta1 in mammalian cells.

The integrin alpha4beta1(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human alpha4 and beta1 subunits were fused to the genomic DNA encoding the human gamma1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote alpha4beta1 heterodimer formation. The soluble alpha4beta1-Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent Kd for VCAM-1 binding were similar for both the soluble and native forms of alpha4beta1. In addition, the integrin-Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble alpha4beta1 should be generally applicable to a range of integrins.

Characterization of TNFRSF19, a Novel Member of the Tumor Necrosis Factor Receptor Superfamily

By searching the expressed sequence tag database, a novel murine tumor necrosis factor receptor designated TNFRSF19 was identified. TNFRSF19 cDNA encodes a putative membrane protein of 348 amino acids with one incomplete and two complete cysteine-rich motifs within its extracellular region and a large cytoplasmic domain. TNFRSF19 mRNA can be detected in most murine tissues examined, particularly in brain, reproductive organs, and late developmental stages of murine embryo, but not in tissues of the immune system. The cell surface expression of the ligand of TNFRSF19 is highly restricted. Of 22 human and murine cell lines examined by FACS analysis, only Raji (B cell lymphoma cell line), GM847 (fibroblast cell line), 293 (embryonic kidney cell line), and K562 (chronic myeloid leukemia) were positive. TNFRSF19 did not bind newly cloned TNF ligands, including TWEAK (HGMW-approved symbol TNFSF12), VEGI/TL1 (HGMW-approved symbol TNFSF15), TL6/endokine (HGMW-approved symbol TNFSF18), APRIL (HGMW-approved symbol TNFSF13), OPGL (HGMW-approved symbol TNFSF11), LIGHT (HGMW-approved symbol TNFSF14), or BAFF/THANK (HGMW-approved symbol TNFSF13B) by enzyme-linked immunosorbent assay and FACS analyses. Overexpression of TNFRSF19 transduced neither apoptotic signaling nor signals leading to NF-κB induction. Taken together with the data that the TNFRSF19 extracellular domain–immunoglobulin fusion protein did not affect the allogeneic mixed lymphocyte reaction, our data indicate that TNFRSF19 is not involved in the modulation of immune responses.

Crucial role of the interleukin 1 receptor family member T1/ST2 in T helper cell type 2-mediated lung mucosal immune responses.

T1/ST2 is an orphan receptor of unknown function that is expressed on the surface of murine T helper cell type 2 (Th2), but not Th1 effector cells. In vitro blockade of T1/ST2 signaling with an immunoglobulin (Ig) fusion protein suppresses both differentiation to and activation of Th2, but not Th1 effector populations. In a nascent Th2-dominated response, anti-T1/ST2 monoclonal antibody (mAb) inhibited eosinophil infiltration, interleukin 5 secretion, and IgE production. To determine if these effects were mediated by a direct effect on Th2 cells, we next used a murine adoptive transfer model of Th1- and Th2-mediated lung mucosal immune responses. Administration of either T1/ST2 mAb or T1/ST2-Ig abrogated Th2 cytokine production in vivo and the induction of an eosinophilic inflammatory response, but failed to modify Th1-mediated inflammation. Taken together, our data demonstrate an important role of T1/ST2 in Th2-mediated inflammatory responses and suggest that T1/ST2 may prove to be a novel target for the selective suppression of Th2 immune responses.

August 11 Highlights

A Task Force of the North Atlantic Collaboration in MS (NATOMS) reviewed MRI as a diagnostic tool for MS in clinical practice based on current literature and expert opinion. Fazekas et al. (p. 448) present recommendations based on this review for indications, protocols, interpretations, and diagnostic implications of MRI. ♦The Lenercept Multiple Sclerosis Study Group and The University of British Columbia MS/MRI Analysis Group (p. 457) present the results of a randomized, placebo-controlled, multicenter study of lenercept (a recombinant tumor necrosis factor–alpha receptor p55 immunoglobulin fusion protein known to protect against experimental autoimmune encephalomyelitis). The study involved 168 patients, most with relapsing-remitting disease. The drug was poorly tolerated and, although there was no significant difference between groups with regard to MRI findings, the lenercept group had a worsening of clinical features. ♦Tubridy et al. (p. 466) report the results of their …

TNF neutralization in MS

<b><i>Objective:</i></b> A double-blind, placebo-controlled phase II study was conducted in 168 patients, most with relapsing-remitting MS, to evaluate whether lenercept would reduce new lesions on MRI. <b><i>Background:</i></b> Tumor necrosis factor (TNF) has been implicated in MS pathogenesis, has been identified in active MS lesions, is toxic to oligodendrocytes in vitro, and worsens the severity of experimental allergic encephalomyelitis (EAE) in animals. Lenercept, a recombinant TNF receptor p55 immunoglobulin fusion protein (sTNFR-IgG p55), protects against EAE. <b><i>Methods:</i></b> Patients received 10, 50, or 100 mg of lenercept or placebo IV every 4 weeks for up to 48 weeks. MRI scans and clinical evaluations were performed at screening, at baseline, and then every 4 weeks (immediately before dosing) through study week 24. <b><i>Results:</i></b> There were no significant differences between groups on any MRI study measure, but the number of lenercept-treated patients experiencing exacerbations was significantly increased compared with patients receiving placebo (<i>p</i> = 0.007) and their exacerbations occurred earlier (<i>p</i> = 0.006). Neurologic deficits tended to be more severe in the lenercept treatment groups, although this did not affect Expanded Disability Status Scale scores. Anti-lenercept antibodies were present in a substantial number of treated patients; serum lenercept trough concentrations were detectable in only a third. Adverse events that increased in frequency in treated patients included headache, nausea, abdominal pain, and hot flushes. <b><i>Conclusions:</i></b> Lenercept failed to be beneficial, but insight into the role of TNF in MS exacerbations was gained.

Relevance of individual CD5 extracellular domains on antibody recognition, glycosylation and co-mitogenic signalling.

CD5 is a type I glycoprotein which modulates T- and B-cell receptor-mediated signals and is expressed by thymocytes, mature T cells and a subset of mature B cells. The extracellular region of CD5 is composed of three scavenger receptor cysteine-rich domains (D1, D2, D3) for which only limited functional and structural data are available. Using cell transfectants expressing ectodomain-deficient CD5 molecules or CD5 immunoglobulin fusion proteins, we analysed individual CD5 domains with respect to monoclonal antibody binding specificity, glycosylation, and co-mitogenic signalling. Our results show the presence of N-linked oligosaccharides on D1 and D2, but not on D3. D1, the most amino-terminal domain, is predicted to be the most appropriately placed domain for an interaction with a ligand. This domain is recognised by a large panel of well characterised CD5 mAbs, reflecting its higher immunogenicity. In an attempt to develop mAbs with specificity for the more conserved membrane-proximal domains, we generated a unique mAb, named 83-C4, whose binding mapped to D3. Co-stimulatory studies revealed no significant differences between anti-D1 and anti-D3 mAbs. The high interspecies conservation of D3 implies a conserved role of this domain in CD5 function and the 83-C4 mAb promises to be a valuable tool in exploring this.

CD44 is not an adhesive receptor for osteopontin.

Osteopontin is a secreted glycoprotein with adhesive and migratory functions. Cellular interactions with osteopontin are mediated through integrin receptors which recognize the RGD domain. Recently, CD44, a non-integrin, multifunctional adhesion molecule was identified as an osteopontin receptor. CD44 is a ubiquitous surface molecule that exists as a number of different isoforms, generated by alternative splicing. To analyze which forms of CD44 mediate binding to osteopontin, we used the standard form of CD44 as CD44-human immunoglobulin fusion proteins and several splice variants in enzyme-linked immunosorbant assays. Multiple preparations of osteopontin were used including native osteopontin derived from smooth muscle cells, human urinary osteopontin, full-length recombinant osteopontin, and two recombinant osteopontin fragments expected to be formed following thrombin cleavage. Our data show that although the CD44-hlg fusion proteins could interact with hyaluronic acid as expected, there was no interaction between CD44H, CD44E, CD44v3,v8-v10, or CD44v3 with osteopontin. These studies suggest that CD44-osteopontin interactions may not be common in vivo and may be limited to a specific CD44 isoform(s), and/or a particular modified form of osteopontin.

Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis

Background & Aims: Membrane lymphotoxin (LT) α/β, a member of the tumor necrosis factor (TNF) family of immune regulatory molecules, is involved both in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in the adult. Defects observed in the mucosal immune system in animals with a genetically disrupted LTα/β pathway coupled with the expression of LTα/β in activated T cells motivated an examination of the importance of this pathway in experimental colitis. Methods: Soluble LTβ receptor (LTβR) immunoglobulin fusion protein was used to inhibit the LTα/β/light axis in two independent rodent models of colitis: CD45RB hi CD4 +–reconstituted SCID mice and bone marrow–transplanted tgϵ26 mice (BM → tgϵ26). Results: Treatment with LTβR immunoglobulin attenuated the development of both the clinical and histological manifestations of the disease in these two murine models of colitis. Given the success of TNF inhibitors in the treatment of human Crohn's disease, the effects of LTβR immunoglobulin have been compared with antibody to TNF in the BM → tgϵ26 model, and both treatments were equally efficacious. Conclusions: The LT pathway plays a role in the development of colitis as important as that of the TNF system and, therefore, represents a potential novel intervention point for the treatment of inflammatory bowel disease. GASTROENTEROLOGY 1998;115:1464-1475

Glycosylation provides both stimulatory and inhibitory effects on cell surface and soluble CD44 binding to hyaluronan.

Glycosylation has been implicated in the regulation of CD44-mediated cell binding of hyaluronan (HA). However, neither the relative contribution of N- and O-linked glycans nor the oligosaccharide structures that alter CD44 affinity for HA have been elucidated. To determine the effect of selective alteration of CD44 oligosaccharide composition on the affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant CD44-immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis. Using this cell line, a panel of recombinant glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surface CD44, the effect of any given glycosylation change on the ability of cell surface and soluble CD44 to bind HA could be compared. Four distinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal alpha2,3-linked sialic acid on N-linked oligosaccharides inhibited binding; (b) the first N-linked N-acetylglucosamine residue enhanced binding; (c) O-linked glycans on N-deglycosylated CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non-N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the intrinsic CD44 affinity for HA (Kd = 5 to >150 microM). The fourth augmented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44.

Discovery of a novel thrombopoietin mimic agonist peptide.

A random phage peptide library was constructed for the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing 1x10(9) different phages, was screened with a human immunoglobulin fusion protein containing the extracellular region of human thrombopoietin receptor. Several phages were isolated following four cycles of enrichment and amplification. These phages specifically bound to the fusion protein. One phage peptide acted as an agonist of the thrombopoietin receptor, since it stimulated the proliferation of thrombopoietin-dependent cells and the differentiation of mouse bone marrow cells to megakaryocytes. The amino acid sequence of this peptide is not present in the primary amino acid sequence of thrombopoietin. This discovery may lead to the design of a small-molecular mimic of thrombopoietin.

Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages.

Human monocytes undergo spontaneous apoptosis upon culture in vitro; removal of serum from the media dramatically increases the rate of this process. Monocyte apoptosis can be significantly abrogated by the addition of growth factors or proinflammatory mediators. We have evaluated the role of the endogenous Fas-Fas ligand (FasL) interaction in the induction of this spontaneous apoptosis and found that a Fas-immunoglobulin (Ig) fusion protein, an antagonistic anti-Fas monoclonal antibody and a rabbit anti-FasL antibody all greatly reduced the onset of apoptosis. The results indicate that spontaneous death of monocytes is mediated via an autocrine or paracrine pathway. Treatment of the cells with growth factors or cytokines that prevented spontaneous apoptosis had no major effects on the expression of Fas or FasL. Additionally, monocyte-derived macrophages were found to express both Fas and FasL but did not undergo spontaneous apoptosis and were not sensitive to stimulation by an agonistic anti-Fas IgM. These results indicate that protective mechanisms in these cells exist at a site downstream of the receptor-ligand interaction.

Preclinical Safety Assessment of the Recombinant TNF Receptor–Immunoglobulin Fusion Protein

The tumor necrosis factor (TNF) is now recognized as one of the most pleiotropic mediators of host defense, immune regulation, and inflammatory response. Due to its broad spectrum of effects, TNF has been implicated as a key mediator in the pathogenesis of acute and chronic inflammatory conditions. The inhibition of bioactive TNF could therefore provide a substantial therapeutic benefit. One approach to a potent TNF antagonist is to use recombinant protein technology in the design of a molecule in which the heavy-chain sequences of an immunoglobulin are fused with the ligand-binding region of the TNF receptor. This paper describes some aspects of the preclinical safety evaluation of the recombinant human TNF receptor–immunoglobulin fusion protein (TENEFUSE). Specific emphasis was placed on transgenic and gene-deleted mice models as central sources of information in the safety evaluation of this TNF antagonist.

Molecular Cloning, Mapping to Human Chromosome 1 q21-q23, and Cell Binding Characteristics of Spα, a New Member of the Scavenger Receptor Cysteine-rich (SRCR) Family of Proteins

CD5 and CD6, two type I cell surface antigens predominantly expressed by T cells and a subset of B cells, have been shown to function as accessory molecules capable of modulating T cell activation. Here we report the cloning of a cDNA encoding Spalpha, a secreted protein that is highly homologous to CD5 and CD6. Spalpha has the same domain organization as the extracellular region of CD5 and CD6 and is composed of three SRCR (scavenger receptor cysteine rich) domains. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid panel analysis indicated that the gene encoding Spalpha is located on the long arm of human chromosome 1 at q21-q23 within contig WC1.17. RNA transcripts encoding Spalpha were found in human bone marrow, spleen, lymph node, thymus, and fetal liver but not in non-lymphoid tissues. Cell binding studies with an Spalpha immunoglobulin (Spalpha-mIg) fusion protein indicated that Spalpha is capable of binding to peripheral monocytes but not to T or B cells. Spalpha-mIg was also found to bind to the monocyte precursor cell lines K-562 and weakly to THP-1 but not to U937. Spalpha-mIg also bound to the B cell line Raji and weakly to the T cell line HUT-78. These findings indicate that Spalpha, a novel secreted protein produced in lymphoid tissues, may regulate monocyte activation, function, and/or survival.

Identification of the Tumor Antigen 90K Domains Recognized by Monoclonal Antibodies SP2 and L3 and Preparation and Characterization of Novel Anti-90K Monoclonal Antibodies

The tumor antigen 90K (Mac-2BP, L3 antigen), which has been shown to have T cell costimulatory activity, is a ∼90 kDa secreted protein found in high levels in plasma, saliva, breast milk and other human fluids. The 90K antigen can be divided into three domains: an amino terminal scavenger receptor cysteine-rich (SRCR)-like domain (D1), followed by a heavily glycosylated mucin-like domain (D2) and a ∼27 kDa carboxy-terminal domain (D3). In this study we report on the construction of six different 90K immunoglobulin (Ig) fusion proteins containing different 90K domain combinations. Initially these fusion proteins were used to identify which 90K domain contains the epitopes recognized by the anti-90K monoclonal antibodies (mAb) SP2 and L3. Both of these mAbs were found to recognize 90K-D2. A new panel of anti-90K mAb was then generated by immunizing mice with ascites derived 90K protein. The 90K domain specific fusion proteins were then used to identify novel anti-90K mAbs which recognize the amino terminal SRCR domain and the carboxy terminal ∼27 kDa domain of 90K. Two novel anti-90K SRCR (D1) and one anti-90 27 kDa domain (D3) mAbs were obtained. These 90K-Ig fusion proteins, as well as the novel and existing anti-90K mAbs, provide a set of tools which will allow further dissection of the structure and function of this immune modulatory protein.

Genetically Transferred Central and Peripheral Immune Tolerance via Retroviral-Mediated Expression of Immunogenic Epitopes in Hematopoietic Progenitors or Peripheral B Lymphocytes

Based on the hypothesis that IgGs are potent tolerogens and that immature lymphohematopoietic antigen-presenting cells (APC), and even mature peripheral B cells, may be effective APC for tolerance induction, we designed an immunoglobulin fusion protein retroviral expression vector to test the role of B cells in a novel gene therapy strategy for the transfer of immune tolerance. An immunodominant epitope (residues 12-26 of the lambda repressor cI protein) was fused in frame to an IgG heavy chain in a retroviral vector, which was used to infect either bone marrow cells or activated peripheral B lymphocytes. These cells were transferred into syngeneic recipients, who were subsequently challenged with the 12-26 peptide in adjuvant. Bone marrow (BM) chimeras generated with retrovirally transduced bone marrow were shown to be profoundly unresponsive to the 12-26 peptide at both the humoral and cellular levels, but were competent to respond to an unrelated protein (lysozyme or PPD). Importantly, we also show that immunocompetent adult recipients infused with transduced mature, activated B lymphocytes, are rendered unresponsive by this treatment. Surprisingly, lymphoid-deficient BM progenitors from syngeneic SCID donors could also be transduced to produce tolerogenic APC. Our data suggest that activated B cells are sufficient to be effective tolerogenic APC in immunocompetent adult mice, but that nonlymphoid cells may also induce tolerance in reconstituted hosts. This approach for gene-transferred tolerogenesis has the potential to be maintained indefinitely, and it requires only knowledge of cDNA sequences of target antigens.

The Ly-1.1 and Ly-1.2 epitopes of murine CD5 map to the membrane distal scavenger receptor cysteine-rich domain.

CD5 is a member of a superfamily of proteins which contain one or more extracellular domains homologous to the type I macrophage Scavenger Receptor cysteine-rich (SRCR) domain. The extracellular region of CD5 is composed of three SRCR domains (D1, D2, D3). Murine CD5 (mCD5) is polymorphic (Ly-1.1 and Ly-1.2 alleles), however, the only murine CD5 gene characterized to date encodes the Ly-1.2 allele (mCD5.2). Likewise, the domain specificity of many of the available anti-mCD5 mAb recognizing either Ly-1.1 or Ly-1.2 or both has not been examined. Herein we describe the isolation and characterization of cDNA encoding the Ly1.1 allele (mCD5.1) and map the location and molecular nature of the mCD5 allelic variation. We also determined which SRCR domain of mCD5 is recognized by a panel of anti-mCD5 mAb. The mCD5.1 protein differs from mCD5.2 in only three amino acids, all of which map to the most amino terminal SRCR domain (D1) of mCD5. An additional seven silent substitutions were observed in the nucleotide sequence encoding mCD5 D1, D2 and transmembrane domains. Immunoglobulin (Ig) fusion proteins consisting of various combinations of mCD5.1 or mCD5.2 SRCR domains were produced and used to determine that allele specific mAb bound to D1, confirming sequence data. MAb against monomorphic determinants on mCD5 bound to each mCD5D11g.

Structure–Function Studies on Synthetic Peptides Derived from the 109–118 Lectin Domain of Selectins

Previously, we identified several peptides corresponding to amino acid sequences within the lectin domains of selectins that inhibit neutrophil (PMN) adhesion to P-selectin. Here we focused on one of the active regions, 109-118, which contains residues that have been identified as critical for E-selectin binding to the sialyl Lexis X (sLex) counter-receptor. Analogues were synthesized and examined for their inhibitory effect on PMN binding to P-selectin and E-selectin immunoglobulin fusion proteins (P-IgG, E-IgG) and also on P-IgG and E-IgG binding to sLex coated surfaces. Peptide sequences which inhibited PMN binding to the fusion proteins were not necessarily those that inhibited fusion protein binding to sLex. In addition, various amino acid substitutions could be tolerated at the 111 and 113 positions without altering inhibitory activity. Modeling suggests that structural conformations of peptide analogues could explain the differences in biological activity of peptide analogues compared to mutants of the native protein.

The Amino-terminal Immunoglobulin-like Domain of Activated Leukocyte Cell Adhesion Molecule Binds Specifically to the Membrane-proximal Scavenger Receptor Cysteine-rich Domain of CD6 with a 1:1 Stoichiometry

Activated leukocyte cell adhesion molecule (ALCAM) was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain.

Human dendritic cells activate T lymphocytes via a CD40: CD40 ligand-dependent pathway.

The CD40:CD40 ligand (CD40L) interaction provides T lymphocyte-mediated help for B lymphocyte and monocyte function but has also been shown to serve as a co-stimulus for T lymphocyte activation. In this report, we studied the regulation of CD40 expression and its functional relevance for the human dendritic cell (DC) stimulation of T lymphocytes. Only a small subpopulation of directly isolated blood DC expressed CD40. However, CD40 was rapidly up-regulated by culture, and its expression was further enhanced by interleukin (IL)-1 alpha, IL-1 beta, IL-3, tumor necrosis factor-alpha and granulocyte/macrophage-colony-stimulating factor. Expression of CD40L on DC was not detected. The proliferation of T lymphocytes in an allogeneic mixed leukocyte reaction, stimulated by blood DC or epidermal Langerhans cells, was significantly reduced in the presence of the CD40 immunoglobulin (CD40Ig) fusion protein or CD40L monoclonal antibodies. Cross-linking of CD40 on directly isolated DC with mouse CD40L trimer (mCD40LT) markedly augmented CD80 and CD86 up-regulation. Nevertheless, the same cross-linking mCD40LT inhibited DC stimulated T lymphocyte proliferation. When CD40Ig was added simultaneously with CTLA-4Ig, only minimal and variable additional inhibition of DC-stimulated allogeneic T lymphocyte proliferation and IL-2 secretion was observed, compared to each fusion protein alone. These results suggest that both CD80/CD86-dependent and -independent components of DC-T lymphocyte CD40:CD40L co-stimulation exist and further emphasize that the majority of blood DC have to differentiate or be activated to express co-stimulatory molecules.

Liquid chromatographic and capillary electrophoretic examination of intact and degraded fusion protein CTLA4Ig and kinetics of conformational transition

Methods have been developed for the CE and HPLC analysis of CTLA4Ig, an immunoglobulin fusion protein. Two different LC approaches, size-exclusion (SEC) and “mixed-mode” ion-exchange (ABx), were developed along with a CE method that uses a micellar electrokinetic chromatographic buffer consisting of borate ions, sodium dodecyl sulfate and acetonitrile. These assays measure the presence of several CTLA4Ig-related species, and the observed changes resulting from multiple modes of degradation. In an attempt to identify possible degradation products, collections were taken from the ABx and SEC liquid chromatographic systems and further analyzed by matrix-assisted laser desorption time-of-flight (MALDI TOF) mass spectrometry. Multiple species were detected covering a wide molecular mass range. In addition, the CE method was used to study conformational kinetics between two forms of CTLA4Ig and to estimate the activation energy of the conformer-conformer transition. Pseudo-first-order reaction kinetics were demonstrated for CTLA4Ig samples stressed with papain, H 2O 2, and sodium dodecyl sulfate/heat.