SDS-capillary Electrophoresis Research Articles

Characterization of AT-02, a pan-amyloid-binding peptide fusion immunoglobulin with high binding potency, complement activation, and immune cell stimulation

Abstract Background Cardiac amyloidosis is an ominous manifestation of systemic amyloidosis, notably in AL and ATTR-associated types, resulting in a restrictive hypertrophic cardiomyopathy with electrical conduction and atrioventricular strain dysfunction (1). Amyloid is not naturally cleared by the immune system due to the presence of extracellular matrix components in the deposits (2). However, recruiting and stimulating the innate immune system, notably phagocytic macrophages, is a promising strategy to effect amyloid removal. To facilitate this, we have generated AT-02, a humanized IgG1 incorporating the pan-amyloid-binding peptide, p5R (3), at the light chain C-terminus. This reagent binds diverse types of amyloid via the peptide interactions and retains immunomodulatory capacity. Purpose The goal of these studies was to characterize the bioactivity of AT-02 that underlies its potential therapeutic activity. Methods AT-02 was produced from a CHO pool using a perfusion cell culture production process, purified by Protein A, anion exchange, and cation exchange chromatographies, and characterized by mass spectrometry and SDS-capillary electrophoresis. AT-02 was biotinylated and used for immunohistochemical detection of both AL and ATTR amyloidosis in formalin fixed tissues (Figure 1A). Binding to heparin (as a surrogate for amyloid-associated hypersulfated heparan sulfate proteoglycans) and synthetic AL fibrils was studied using both ELISA and surface plasmon resonance assays (Figure 1B). Activation of complement following binding to amyloid substrates was determined using a C5b9 liberation assay in the presence of human plasma. The effect of AT-02 on amyloid fibril growth was determined using a fluorescence fibril elongation assay. In vivo clearance of amyloid and cell activation was demonstrated using a murine model of AL amyloidoma. Results AT-02 bound human cardiac amyloid in tissue sections with high specificity and intensity. The potency for heparin and synthetic amyloid fibrils, as well as human cardiac amyloid extracts, was sub-nanomolar in the ELISA assay with kinetics consistent with a high affinity antibody. In the presence of plasma, complement activation of amyloid-bound AT-02 was significantly higher as compared to controls, and the reagent inhibited synthetic amyloid fibril growth by ∼40%. Treatment of human amyloid with AT-02 expedited phagocytosis by murine macrophages in vivo and enhanced the intensity of immune response to the implanted amyloid. Conclusion Clearance of cardiac amyloid is a significant clinical unmet need for patients with systemic amyloidosis. The humanized IgG1-peptide fusion, AT-02, potently binds cardiac amyloid and may serve as an opsonin and induce macrophage-mediated phagocytosis and clearance of cardiac amyloid deposits.

A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells

To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios. Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants

Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.

Identifying kinetically stable proteins with capillary electrophoresis

Unlike most proteins, which are in equilibrium with partially and globally unfolded conformations, kinetically stable proteins (KSPs) are trapped in their native conformations and are often resistant to harsh environment. Based on a previous correlation between kinetic stability (KS) and a protein's resistance to sodium dodecyl sulfate (SDS), we show here a simple method to identify KSPs by SDS-capillary electrophoresis (CE). Control non-KSPs were fully denatured by SDS and formed protein:SDS complexes that exhibited similar mobility in CE. In contrast, KSPs bound fewer SDS molecules, and showed a very different migration time and peak pattern in CE, thereby providing some insight about the structural heterogeneity of SDS:protein complexes and the relative KS of the various proteins.

Use of SDS to Extract Sorghum and Maize Proteins for Free Zone Capillary Electrophoresis (FZCE) Analysis

ABSTRACTTwo different extraction methods for extracting sorghum (Sorghum bicolor L. Moench.) storage proteins for free zone capillary electrophoresis (FZCE) analysis were compared. A traditional solvent based on 60% t‐butanol was compared with a pH 10 borate buffer containing the anionic detergent SDS followed by precipitation of nonkafirins using 60% t‐butanol. FZCE analysis of both types of extracts showed identical patterns, despite the fact that the SDS should have given all proteins equal charge‐to‐mass ratios. This methodology was also successfully applied to maize proteins. The use of t‐butanol to precipitate nonkafirins, combined with electrophoresis at low pH, is thought to have removed the SDS from the storage proteins. The SDS extraction procedure produced more stable extracts for FZCE analysis. These extracts could also be used directly for SDS capillary electrophoresis (SDS‐CE) separations. Kafirins from 15 genotypes were extracted with this procedure and analyzed by FZCE and SDS‐CE. Resolution of the kafirins by FZCE was much higher than the SDS‐CE, demonstrating that the kafirin proteins possessed a high level of charge density variability within a relatively small molecular size distribution. Two distinct groups of α‐kafirins could be seen in the FZCE electropherograms.

Study of the LMW glutenin composition of some old Hungarian wheat cultivars using capillary electrophoresis

The LMW glutenin composition of nine old Hungarian wheats (several lines per variety), and one Canadian wheat was studied using one-step, one-dimensional SDS-PAGE (Gupta and MacRitchie, 1991), after which a method was developed for the determination of wheat LMW glutenin patterns using SDS capillary electrophoresis (CE). In both cases the samples were prepared as described by Gupta and MacRitchie (1991), with some modifications in the case of CE. Both methods were used to analyse the homogeneity of the varieties, and the electropherograms obtained with the two methods were compared. It was found that the new method based on CE was suitable for the demonstration of differences and similarities between the wheat LMW glutenins. The method can be used to reveal differences not only between varieties (e.g. between the Hungarian variety Bankuti 1205 and the Canadian variety Marquis), but also between the genotypes making up a variety population (e.g. the sublines of Bankuti 1205). The results indicate that CE is a satisfactory alternative to the traditional PAGE method for the determination of the LMW glutenin subunits of wheat varieties.

Enzymatic hydrolysis, grease permeation, and water barrier properties of zein isolate coated paper.

An inexpensive zein-lipid mixture was isolated from yellow dent, dry-milled corn. Grease permeation through zein isolate applied to brown Kraft paper was found to be independent of loading levels at zein isolate levels above 30 mg/16 in.(2). The data shows that water vapor transmission rates depended on the amount of coating applied. Triacylglycerols were the most abundant lipid in milled corn but were absent in the zein isolate (perhaps due to hydrolysis by lipases). Zein from the paper was hydrolyzed enzymatically and the hydrolysis monitored by SDS-capillary electrophoresis. At an E:S ratio of 1:100 no further increase in the hydrolysate peak occurred after 10 and 30 min for alpha-chymotrypsin and pancreatin 8 x; however, zein and lipid were still present 1 h after hydrolysis by pancreatin 1 x.

Characterization of an intracellular oligopeptidase from Lactobacillus paracasei

An intracellular oligopeptidase from Lactobacillus paracasei Lc-01 has been purified to homogeneity by Fast Flow Q Sepharose, hydroxyapatite, and Mono Q chromatography. The molecular mass of the enzyme was determined to be 140 kDa by gel filtration and approximately 30 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis. The pI of the enzyme was at pH 4.5. The enzyme expressed maximum activity at pH 8.0 and 40 degrees C. Oligopeptidase activity on bradykinin was inhibited strongly by 1,10-phenantroline and EDTA and partly by p-chloromercuribenzoic acid but not by phosphoramidon or phenylmethylsulfonyl fluoride. Marked inhibition by beta-casein fragment 58 to 72 was demonstrated. The enzyme showed neither general aminopeptidase nor caseinolytic activity, and it degraded only oligopeptides between 8 and 13 amino acids. The enzyme readily hydrolyzed the Phe-Ser and Pro-Phe bonds of bradykinin; the Phe-His bond of angiotensin I; the Pro-Gln, Gln-Phe, and Phe-Gly bonds of substance P; and the Pro-Tyr bond of neurotensin. Weak activity toward the Ala-Tyr and Pro-Ser bonds of alpha(s1)-casein fragment 157 to 164, was observed. The N-terminal amino acid sequence of the oligopeptidase showed a high degree of homology to the lactacin B inducer from Lactobacillus acidophilus.

Stoichiometry of Subunits and Heme Content of Hemoglobin from the Earthworm Lumbricus terrestris

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major O2-binding chains, a, b, c (forming a disulfide-linked trimer), and d ("monomer"). Additional structural chains, "linkers," are required for the assembly of the approximately 200-polypeptide molecule. The proportion of linker chains had been reported to be one-third of the total mass on the basis of densitometry of Coomassie Blue-stained SDS-gels. Reverse-phase high performance liquid chromatography (HPLC), however, gave 16.3% linkers on the basis of both 220-nm absorbance and amino acid analysis (Ownby, D. W., Zhu, H., Schneider, K., Beavis, R. C., Chait, B. C., and Riggs, A. F. (1993) J. Biol. Chem. 268, 13539-13547). The subunit proportions have now been redetermined by SDS capillary electrophoresis as a test of the HPLC results. The electrophoresis, monitored at 214 nm, avoided the use of Coomassie Blue and provided results identical with those obtained by HPLC. Capillary electrophoresis monitored at both 214 and 415 nm was used to show that linker chains do not bind heme. Heme content has been found to be 2.9% by determination of hemin, amino acid analysis and dry weight. Measurement of the rate of hemin loss from oxidized L. terrestris Hb shows that high rates of loss can account for values of heme content significantly below 2.9% (or 0.26% iron).

Examination of capillary zone electrophoresis, capillary isoelectric focusing and sodium dodecyl sulfate capillary electrophoresis for the analysis of recombinant tissue plasminogen activator

The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of θ-amino acid buffers (pH≈5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an ϵ-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 μm I.D. capillary or narrow bore capillaries without a polymer solution (25 μm I.D.) were employed. rtPA was resolved into at least eight species within a p I range of 6.4–9.2 using Ampholine 3.5–10. Migration time precision for the major peaks ranged from 0.2% for CZE to ≤2–3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.

Characterization of enterotoxigenic Bacteroides fragilis by a toxin-specific enzyme-linked immunosorbent assay.

Within the past decade, certain strains of Bacteroides fragilis have been associated with diarrhea in humans and cytotoxic activity on certain colon carcinoma cell lines. An enzyme-linked immunosorbent assay (ELISA) for detecting the enterotoxin of B. fragilis in cultures and stools was developed by using high-titer monospecific goat and rabbit antitoxins in an indirect format. The lower limit of detection for purified toxin was approximately 0.05 micrograms/ml; the linear range was from 0.05 to 10 microgram/ml. Using the ELISA to screen cultures of toxigenic and nontoxigenic strains of B. fragilis, we observed 100% correlation with 16 known toxigenic strains which had various cytotoxic activities on HT-29 cells. In addition, we found 6 of 62 previously untested strains also to be positive in both assays. Stability studies revealed that although the cytotoxic activities of crude and purified toxin preparations incubated at elevated temperatures were rapidly lost, the ELISA responses were not significantly reduced. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis showed that the purified toxin autodigested to several stable peptides. Studies on partially purified membranes from the toxigenic strains revealed the presence of several membrane-associated components which were noncytotoxic but strongly immunoreactive in the ELISA. Preliminary studies with spiked feces indicated that the ELISA may be useful for screening not only cultures for the enterotoxigenic B. fragilis but also stool specimens. Ongoing studies are focusing on determining the nature of the toxin's apparent proteolytic capabilities and investigating the feasibility of using the ELISA on stool specimens from healthy and diarrheic humans.