Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique.

Fragmentation in protein-based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size-exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE-SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein-based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab-related low molecular weight fragments, a non-reducible thio-ether linked 75 kDa HL fragment was also identified.

The structural basis for binding of complement by immunoglobulin M.

An insight into the structural features of human IgM that are responsible for its capacity to bind the first component of complement (C) has been obtained by examining the ability of IgM subfragments to bind active C1 (C1). The smallest two fragments found to bind C1 were the major CNBr fragment of the Fc portion of IgM and the C(H)4 fragment of the carboxy-terminal domain. The smallest fragment which fixes C1 has a disaggregated mol wt of 6,800, consists of 60 residues, and contains no carbohydrate. Structural considerations and sequence overlaps suggest that the amino-terminal side of the C(H)4 domain (24 amino acid residues) might be responsible for fixing C1.

The K-chains of the immunoglobulin from a continuous culture of human lymphocytes (Daudi) have an unusual molecular size.

Membrane-associated immunoglobulin (M-Ig) was isolated from Daudi cells which had been radioiodinated using lactoperoxidase. This M-Ig was found to have a molecular size on SDS-PAGE of 330,000 daltons. The component protein chains (micro and K) have molecular sizes of 75,000 and 33,000 daltons, respectively. Further studies showed that [(3)H]galactose can be incorporated into K-chains, but that extra protein and not carbohydrate was responsible for the increased molecular size of this molecule.

Occurrence of surface IgM, IgD, and free light chains of human lymphocytes.

An analysis was made of the immunoglobulin surface markers of the cells of patients with chronic lymphatic leukemia (CLL) in view of previous evidence of their monoclonal B-cell character. The simultaneous presence of IgM and IgD on the surface of the majority of lymphocytes was demonstrated by both immunofluorescence and hemagglutination inhibition in most cases. However, cases were observed with surface IgM without IgD as well as cases with IgD without IgM. IgG and IgA were absent. Studies of the light chains indicated only a single class in a given case. In addition to bound light chains, free light chains were readily demonstrated in most cases through the use of antisera specific for "free chain" determinants. It thus appeared that there are three major types of surface Ig on CLL lymphocytes, IgM, IgD, and free light chains.

Phylogenetically associated residues within the VH3 subgroup of several mammalian species. Evidence for a "pauci-gene" basis for antibody diversity.

Immunoglobulin heavy chains from IgG pools of several mammalian species have been subjected to Edman degradation on an automated protein sequencer. The percentage of unblocked vs. blocked heavy chains was estimated from the yield of the invariant valine in the second position. Further analysis of these unblocked polypeptides unequivocally placed them in the V(HIII) subgroup on the basis of homology with known human heavy chain sequences. The mammals studied could be divided into three distinct categories on the basis of the distribution of the V(HIII) subgroup. In several species the V(HIII) subgroup could not be detected while, in others, virtually all of the heavy chains belonged to this subgroup. Several species had intermediate amounts with the level of the V(HIII) subgroup restricted to between 19 and 29% of the total pool. Within experimental error, all members of a given order had a similar V(HIII) subgroup distribution. Further amino acid sequence studies illustrated a high degree of structural homogeneity in the heavy chains of IgG isolated from pooled sera of a number of mammalian species. The very close amino acid sequence homologies of the amino terminal 24 residues of the various pools corroborated conclusions previously obtained using several myeloma proteins from some of these same species. In particular, certain phylogenetically associated residues were identifiable at characteristic positions in the pools in confirmation of their identification in the myeloma proteins. The simplest assumptions would suggest that these findings are more compatible with a pauci-gene than a multi-gene basis for the generation of antibody diversity.

Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice.

Four monoclonal antibodies against carcinoembryonic antigen (CEA) have been selected from 32 hybrids that produce antibodies against this antigen, by the criteria of high affinity for CEA and low cross-reactivity with granulocyte glycoprotein(s). The specificity of tumor localization in vivo of the four MAb, and their F(ab')2 and Fab fragments was compared in nude mice bearing grafts of a serially transplanted, CEA-producing, human colon carcinoma. The distribution of radiolabeled MAb and their fragments after intravenous injection was analyzed by direct measurement of radioactivity in tumor and normal organs, as well as by whole-body scanning and by autoradiography of tumor sections. Paired labeling experiments, in which 131I-labeled antibody or fragments and 125I-labeled control IgG are injected simultaneously, were undertaken to determine the relative tumor uptakes of each labeled protein. The tumor antibody uptake divided by that of control IgG defines the specificity index of localization. Tumor antibody uptakes (as compared with the whole mouse), ranging between 7 and 15, and specificity indices ranging between 3.4 and 6.8, were obtained with the four intact MAb at day 4-5 after injection. With F(ab')2 fragments of the four MAb, at day 3, the tumor antibody uptakes ranged between 12 and 24 and the specificity indices between 5.3 and 8.2. With the Fab fragments prepared from the two most promising MAb, the antibody uptakes reached values of 34 and 82 at day 2-3 and the specificity indices were as high as 12 and 19. The scanning results paralleled those obtained by direct measurement of radioactivity. With intact MAb, tumor grafts of 0.5-1 g gave very contrasted positive scans 3 d after injection. Using MAb fragments, tumors of smaller size were detectable earlier. The best results were obtained with Fab fragments of MAb 35, which gave clear detections of tumors weighing only 0.1 g as early as 48 h after injection. Autoradiographs of tumor sections from mice injected with 125I-labeled MAb demonstrated that the radioactivity was localized in the tumor tissues and not in the stromal connective tissue of mouse origin. The highest radioactivity concentration was localized in areas known to contain CEA such as the pseudolumen of glands and the apical side of carcinoma cells. The penetration of radioactivity in the central part of tumor nodules and the pseudolumen appeared to be increased with the use of MAb fragments.

Heterogeneity of the BALB/c antiphosphorylcholine antibody response at the precursor cell level.

Immune responsiveness to phosphorylcholine (PC) in BALB/c mice has been characterized by combining (a) usuage of highly sensitive radioimmunoassays for quantitation of antibody, heavy-chain class, and idiotype on a weight basis; (b) isolation of PC-specific B cells in fragment cultures; and (c) stimulation in a carrier-primed environment with the PC hapten coupled to carrier through a tripeptide spacer in order to maximize carrier recognition. The specificity and accuracy of the radioimmunoassays have veen verified by specific inhibition, lack of nonspecific binding, and excellent concordance of values for monoclonal antibody concentration obtained independently for Fab and idiotype content. The latter evidence also serves as strong confirmation of the monoclonality of in vitro monofocal responses as well as the preservation of the idiotype on antibodies of differing immunoglobulin classes. The results indicate that while B cells expressing the TEPC 15 idiotype predominate, other idiotypes may be represented by 2-50% of PC-specific precursors, and monoclonal antibodies even of the TEPC 15 idiotype are produced in both the IgM and IgG1 immunoglobulin classes. These findings are confirmed by the analysis of serum antibodies produced in carrier-primed mice immunized with hapten coupled through a tripeptide spacer, thus re-emphasizint the enhancement of primary responsiveness, particularly IgG1 production, by maximizing carrier recognition. The finding of idiotype diversity in the PC response, as well as diversity of expression in terms of quantity and immunoglobulin class of antibody synthesized by the clonal progeny of B cells within the TEPC 15 clonotype, emphasize the heterogeneity of the B-cell population both in terms of specificity repertoire and the physiological state of cells even within a single clonotype.

The synthesis, assembly, and secretion of gamma globulin by mouse myeloma cells. VI. Assembly of IgM proteins.

The study of the synthesis, assembly, and secretion of IgM by seven murine myeloma tumors has revealed that free mu chain can be detected intracellularly after release from the ribosome. It combines with light chains to form microL. The major intracellular protein in six of the seven tumors was the 8S subunit. One tumor contained considerable amounts of 19S material intracellularly. Those tumors that did not contain 19S IgM intracellulariy appeared to assemble the subunits outside the cell.

Immunoglobulin and glucose-6-phosphate dehydrogenase as markers of cellular origin in Burkitt lymphoma.

Two independent marker systems, G-6-PD isoenzymes and cell membrane-associated IgM, were used to trace the cellular origin of Burkitt lymphoma. Application of the G-6-PD system is dependent upon the fact that, in accordance with inactivity of one X chromosome in each somatic cell, females heterozygous for the usual B gene (Gd(B)) at the X-linked G-6-PD locus and the variant allele Gd(A) (or Gd(A-)) have two types of cells. Gd(B) is active in one cell population, which consequently produces B type enzyme; in the other population Gd(A) is active, producing the variant A enzyme. Therefore, tumors with a clonal origin in a Gd(B)/Gd(A) heterozygote should exhibit only one enzyme type (B or A) whereas those with multicellular origin may show both A and B enzymes. Utilization of the immunoglobulin system is based upon the supposition that in lymphoid neoplasms with clonal origin either all or none of the tumor cells should have surface-associated IgM and kappa-reactivities. 33 of 34 relatively homogeneous (with respect to content of neoplastic cells) individual Burkitt tumors from 19 G-6-PD heterozygotes had single enzyme phenotypes. Similarly, of 95 tumors tested, 92 consisted essentially of IgM(+) or (-) cells. Two neoplasms could not be definitely classified and one tumor had two cell populations. These data suggest a clonal origin for most Burkitt tumors, but the one neoplasm with a double G-6-PD phenotype (A/B) and the one tumor that had two populations of cells with respect to surface IgM, could have originated from multiple cells. G-6-PD was determined in each of two tumors from seven heterozygotes and in all cases both tumors had the same single enzyme phenotype. Surface-associated IgM was tested in four tumors from one patient, three from another, and in two neoplasms from 11 patients. With one exception, all tumors from the same patient were concordant with respect to IgM. These findings suggest that the entire disease has a clonal origin, i.e., it emerges at one focus and then spreads to other parts of the body. Cells from 36 recurrent neoplasms were typed for G-6-PD (in heterozygotes) and/or IgM. In one previously reported patient, initial and recurrent tumors were discordant for G-6-PD. Two other patients had IgM phenotypes in recurrences that were discordant with those found in their initial tumors. Phenotypes from three of nine relapses which occurred after 5 mo were discordant for G-6-PD or IgM but no discordance was detected among 27 earlier recurrences. Thus, some "late" recurrences may be due to emergence of "new" maligant cell lines whereas most early relapses are due to reemergence of the original malignant clones. The probable unicellular origin of Burkitt lymphoma and the findings in tumor recurrences are discussed in terms of the disease's putative viral etiology.

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis.

Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.

Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies.

In the companion paper (Sztul, E. S., K. E. Howell, and G. E. Palade, J. Cell Biol., 100:1248-1254), we have shown that pulse labeling of hepatic proteins with [35S]cysteine can be obtained in vivo in intact rats. Soluble label clears the plasma in approximately 5 min, and incorporated label reaches peak values in the liver approximately 20 min after injection. In the present study, we show that the 105,000-mol-wt protein (105K), kinetically the earliest intracellular form of secretory component (SC), is the predominant form found, between 5 and 20 min postinjection, in homogeneous rough microsomal fractions. The second kinetically defined form, i.e., 116K, is the predominant species present in relatively homogeneous, light Golgi fractions in which it appears at approximately 15 min, and peaks at approximately 25 min, postinjection. The third kinetically defined form, 120K, is found 30 min after injection as the major SC species (albeit still accompanied by its immediate precursor, 116K), in a sinusoidal plasmalemmal fraction isolated by immunoadsorption to anti-SC-coated Sepharose beads. These findings lead to the following conclusions: (a) SC is synthesized on polysomes attached to the rough endoplasmic reticulum (ER) membrane; (b) it is partially translocated across the ER membrane and core glycosylated co-translationally to give a 105K peptide; (c) 105K moves from the ER to the Golgi complex where it is terminally glycosylated to give the 116K form; (d) the latter moves to the sinusoidal plasmalemma where it appears together with the final mature form, 120K. Kinetic evidence indicates that the vesicular carriers involved in the transport of SC from the Golgi complex to the sinusoidal plasmalemma, and from the latter to the biliary front of the hepatocytes, are present in a Golgi heavy fraction and a crude carrier vesicle fraction from which they remain to be isolated, purified, and characterized.

Biogenesis of the polymeric IgA receptor in rat hepatocytes. I. Kinetic studies of its intracellular forms.

The polymeric IgA receptor (or secretory component [SC]) is a major biliary secretory protein in the rat. It was identified as an 80,000-mol-wt (80 K) glycoprotein by coprecipitation (with IgA) by anti-IgA antibodies (Sztul, E. S., K. E. Howell, and G. E. Palade, 1983, J. Cell Biol., 97:1582-1591) and was used as antigen to raise anti-SC antibodies in rabbits. Pulse labeling with [35S]cysteine in vivo, followed by the immunoprecipitation of solubilized total microsomal fractions with anti-SC sera, made possible the identification of three intracellular forms of SC (all apparently membrane proteins) and the definition of their kinetic and structural interrelations. At 5 min postinjection of [35S]cysteine, a major band of Mr 105,000 was maximally labeled. This peptide lost radioactivity concomitantly with the appearance of a radioactive doublet of Mr 116,000 and 120,000 at 15-30 min postinjection. Loss of radioactivity from 116K paralleled increased labeling of the 120K peptide which appears to be the mature form of the receptor. The 105K form was sensitive to endoglycosidase H which converted it to a 96K peptide. The 116K and 120K forms were resistant to endoglycosidase H but sensitive to endoglycosidase F which converts them to 96K and 100K forms, respectively. Taken together, these findings support the following conclusions: (a) All rat hepatic SC forms are the products of a single gene; (b) all SC forms are N-glycosylated; (c) the 116K form is the result of the terminal glycosylation of the 105K form; and (d) the 120K peptide is probably produced by modifications at other sites than its complex oligosaccharide chains.

Complete amino acid sequence of the Mu heavy chain of a human IgM immunoglobulin.

The amino acid sequence of the micro, chain of a human IgM immunoglobulin, including the location of all disulfide bridges and oligosaccharides, has been determined. The homology of the constant regions of immunoglobulin micro, gamma, alpha, and epsilon heavy chains reveals evolutionary relationships and suggests that two genes code for each heavy chain.

Mouse immunoglobulin kappa chain MPC 11: extra amino-terminal residues.

The kappa chain from the immunoglobulin of myeloma tumor MPC 11 has 12 extra residues at its amino terminus, the first six of which are identical to the residues at positions 1 to 6 of typical mouse kappa chains and at positionss 13 to 18 of MPC 11 itself. Two of the peptide bonds within this extra 12-residue segment are cleaved under very mild conditions.

Partial amino acid sequence of the precursor of immunoglobulin light chain programmed by messenger RNA in vitro.

The five proteins programmed in a cell-free system by a mouse kappa light chain messenger RNA were labeled with [3H]leucine and subjected to amino acid sequence analyses. In all five proteins, 20 amino acid residues precede the amino terminus of the mature protein, indicating that there is one major point for the initiation of messenger RNA translation. The abundance (30 percent) of leucine residues in the extra piece (leucine at positions 6, 7, 8, 11, 12, and 13) indicates that this moiety is hydrophobic. Furthermore, it seems that the precursor may have an additional extra piece at the carboxyl terminus.

Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV).

BACKGROUND: Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. RESULTS: In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent. CONCLUSION: For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

A double epitope tag for quantification of recombinant protein using fluorescence resonance energy transfer.

The expression of recombinant proteins is a well-accepted technology, but their detection and purification often require time-consuming and complicated processes. This paper describes the development of a novel double epitope tag (GEPGDDGPSGAEGPPGPQG) for rapid and accurate quantification of recombinant protein by a homogeneous immunoassay based on fluorescence resonance energy transfer. In our double epitope tagging system, recombinant proteins can be simply measured on a microtiter plate by addition of a pair of fluorophore-labeled monoclonal antibodies (their epitopes; GEPGDDGPS and GPPGPQG). The sensitivity of the immunoassay with an incubation time of only 5 min is almost equal to that of labor-intensive Western blotting. In addition, culture media and extracts of host cells generally used for protein expression have little effect on this immunoassay. To investigate the utility of our proposed tag for protein production, several different proteins containing this tag were practically expressed and purified. The data presented demonstrate that the double epitope tag is a reliable tool that can alleviate the laborious and troublesome processes of protein production.

Purification of antibody fragments for the reduction of charge variants using cation exchange chromatography.

Recently, antibody fragments have been studied as therapeutic agents because they lack Fc effector function while having affinity similar to their original monoclonal antibody and can be produced using E. coli. Antibody fragments can be purified using affinity chromatography in the capture step, although they need a polishing step because of product-related impurities, mainly charge variants. Unlike monoclonal antibodies, few studies exist regarding the separation of charge variants in antibody variants. In this study, an efficient separation of charge variant method was assessed using a cation exchange chromatography resin with salt and a pH gradient. The SP ImpRes resin and pH gradient exhibited the most effective separation potency using combinations of resin and the separation method. The antibody fragment that did not undergo the charge variant separation process exhibited a difference in the tertiary structure of the protein and in vivo pharmacokinetics. However, the antibody fragment was similar to the reference protein when the charge variant separation process was performed. These results are expected to support efficient charge variant separation of antibody fragments and to be applied to the industrial production of therapeutic antibody fragments.

The relationship between L-chain synthesis and gamma-globulin production.

The simultaneous administration of labeled human gammaG globulin and L-chains to subjects has allowed examination of the metabolic relationship between a component part and the whole immunoglobulin molecule. Studies were carried out in a series of control subjects and in a group of patients where substandard production of gamma-globulin was anticipated, i.e., patients with the nephrotic syndrome on chronic Imuran therapy and patients with uremia. Full expression of the plasma decay curve was obtained for both substances, that for L-chain requiring only 4-5 days except in uremic subjects and that for gamma-globulin requiring up to 30-40 days. Urinary excretion of inorganic iodide was also quantitated for 20-30 days. Equilibrium of the extravascular, vascular, and urinary radioactivity from the labeled gammaG globulin was usually not seen during this time interval suggesting more than one site of catabolism of the protein. Excess L-chain poduction was about 45% of the total L-chain production and probably in no instance could account for the low gamma-globulin production seen in certain patients with renal disease.

Immunoproliferative small intestinal disease associated with Campylobacter jejuni.

BACKGROUND: Immunoproliferative small intestinal disease (also known as alpha chain disease) is a form of lymphoma that arises in small intestinal mucosa-associated lymphoid tissue (MALT) and is associated with the expression of a monotypic truncated immunoglobulin alpha heavy chain without an associated light chain. Early-stage disease responds to antibiotics, suggesting a bacterial origin. We attempted to identify a causative agent. METHODS: We performed polymerase chain reaction (PCR), DNA sequencing, fluorescence in situ hybridization, and immunohistochemical studies on intestinal-biopsy specimens from a series of patients with immunoproliferative small intestinal disease. RESULTS: Analysis of frozen intestinal tissue obtained from an index patient with immunoproliferative small intestinal disease who had a dramatic response to antibiotics revealed the presence of Campylobacter jejuni. A follow-up retrospective analysis of archival intestinal-biopsy specimens disclosed campylobacter species in four of six additional patients with immunoproliferative small intestinal disease. CONCLUSIONS: These results indicate that campylobacter and immunoproliferative small intestinal disease are associated and that C. jejuni should be added to the growing list of human pathogens responsible for immunoproliferative states.