An Integrated Solution-Based Rapid Sample Preparation Procedure for the Analysis of N-Glycans From Therapeutic Monoclonal Antibodies.

Consistent glycosylation in therapeutic monoclonal antibodies is a major concern in the biopharmaceutical industry as it impacts the drug's safety and efficacy and manufacturing processes. Large numbers of samples are created for the analysis of glycans during various stages of recombinant proteins drug development. Profiling and quantifying protein N-glycosylation is important but extremely challenging due to its microheterogeneity and more importantly the limitations of existing time-consuming sample preparation methods.

Electroblotting from Polyacrylamide Gels.

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose.

Electroblotting from polyacrylamide gels.

This unit contains procedures for electrophoretically transferring proteins onto a variety of membranes including polyvinylidene difluoride (PVDF) and nitrocellulose, and derivatized membranes. The choice of membrane type for electrotransfer is dependent on the ultimate application for the blot membrane. An alternate protocol is provided for electroblotting in semidry systems.

Two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two-dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS-PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two-dimensional gels, and two-dimensional protein databases.

Two-dimensional gel electrophoresis.

While one-dimensional SDS-PAGE separates proteins on the basis of size, two-dimensional gel electrophoresis separates proteins first on the basis of isoelectric point, then on the basis of size. This method is capable of resolving 1000 to 2000 separate proteins when combined with sensitive detection methods. This unit describes methods for characterizing cell lysates by two-dimensional gel electrophoresis, including modifications for acidic and basic proteins, the use of immobilized pH gradients, and nonreducing/reducing electrophoretic separations.

Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?

Peptide mapping is a key analytical method for studying the primary structure of proteins. The sensitivity of the peptide map to even the smallest change in the covalent structure of the protein makes it a valuable 'finger-print' for identity testing and process monitoring. We recently conducted a full method validation study of an optimised reverse-phase high-performance liquid chromatography (RP-HPLC) tryptic map of a therapeutic anti-CD4 IgG1 monoclonal antibody.

An automated C-terminal sequencing method for analysis of proteins us ing an ABI 473A sequencer.

We developed a novel chemistry for C-terminal sequencing of proteins based on derivatization with acetylisothiocyanate to yield amino acid thiohydantoins (TH-AAs), and it was used for manual sequencing of biopharmaceutical products on a routine basis.This simple chemistry was automated using a ABI 473A N-terminal sequencer. All reagents (R1, trimethylsilylisothiocyanate; R3, alkaline thiocyanate for cleavage) and solvents required for sequencing were accommodated on the sequencer, which was modified to deliver liquid R2 (acetyl chloride) to the reaction vessel.

Protein sequence analysis using Hewlett-Packard biphasic sequencing cartridges in an applied biosystems 473A protein sequencer.

Protein sequence analysis using an adsorptive biphasic sequencing cartridge, a set of two coupled columns introduced by Hewlett-Packard for protein sequencing by Edman degradation, in an Applied Biosystems 473A protein sequencer has been demonstrated. Samples containing salts, detergents, excipients, etc. (e.

High-yield deblocking of amino termini of recombinant immunoglobulins with pyroglutamate aminopeptidase.

For larger proteins, efficient deblocking prior to Edman sequencing is especially important to obtain quality, extended sequencing data which is limited by the stepwise accumulation of background from the random acid hydrolysis of the protein. Therefore, any portion that remains blocked contributes to the undesirable background. We report an optimized procedure for the removal of pyroglutamate (pGlu) by pyroglutamate aminopeptidase (PGAP) and demonstrate its use for the quantitative deblocking of several humanized recombinant antibodies (rIgGs).

A pulmonary influenza virus infection in SCID mice can be cured by treatment with hemagglutinin-specific antibodies that display very low virus-neutralizing activity in vitro.

We have previously shown that a pulmonary influenza virus infection in SCID mice can be cured by treatment with monoclonal antibodies (MAbs) specific for the viral transmembrane protein hemagglutinin (HA) but not for matrix 2. Since both types of MAbs react with infected cells but only the former neutralizes the virus, it appeared that passive MAbs cured by neutralization of progeny virus rather than reaction with infected host cells. To prove this, we selected a set of four HA-specific MAbs, all of the immunoglobulin G2a isotype, which reacted well with native HA expressed on infected cells yet differed greatly (>10,000-fold) in virus neutralization (VN) activity in vitro, apparently because of differences in antibody avidity and accessibility of the respective determinants on the HA of mature virions.

Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membranes.

The most effective protein purification method of low picomole amounts for sequence analysis involves polyacrylamide gel electrophoresis followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. Since a critical factor in this procedure is the protein recovery at the blotting step, different types of PVDF membranes were systematically evaluated for their ability to bind proteins during electrotransfer. Differences in electroblotting recoveries occurred between types of PVDF membranes for some proteins.

High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels.

Optimal conditions of electroblotting that led to high protein recovery on polyvinylidene difluoride (PVDF) membranes were determined for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS concentrations in the gel and transfer buffer were found to be the most important factors affecting the amount of protein recovered on the PVDF membrane. The largest loss occurred during the first 10-30 min of transfer due to the relatively high initial SDS concentration in the gel.

Gamma-interferon-induced nerve growth factor receptors in colorectal carcinoma cell lines.

Gamma-interferon (gamma IFN) was found to induce expression of the 150,000 M(r) cell surface and the 35,000 M(r) chromatin receptors for nerve growth factor (NGF) in the SW1116 colorectal carcinoma cell line that does not express NGF receptors. In the SW707 colorectal carcinoma cell line that expresses a low level of NGF receptors, gamma IFN stimulated expression of the cell surface and the nuclear receptors. Induction of NGF receptors in SW1116 cells resulted in internalization and nuclear translocation of 125I-NGF.

[Therapeutic trial with fucidin in systemic scleroderma].

The signs and symptoms in systemic sclerosis were improved by systemic therapy with fucidine. In patients with untreated systemic sclerosis, asparaginic acid and glutaminic acid were increased in blood serum and asparagine and glutamine decreased. These compounds with fucidine therapy normalized.

Treatment of systemic scleroderma with fucidine with regard to some free amino acids contents before and after therapy.

In 7 patients with systemic scleroderma and acroscleroderma improvement was observed after the administration of fucidine. In the same time 4 amino acids contents, which had been abnormal prior to the therapy, normalized.