Analysis of oligonucleotides using capillary zone electrophoresis and electrospray mass spectrometry.

This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry for the single-step desalting, and separation, as well as characterization of oligonucleotides in the framework of quality control after synthesis. Separation is performed using a 25 mM ammonium carbonate buffer supplemented with 0.2 mM trans-1,2-diaminocyclohexane-N, N, N', N' id (CDTA) (pH 9.7). During the electrophoretic process, sodium and potassium ions are removed from the polyanionic backbone of the oligonucleotides by exchange of these ions with ammonium ions or by chelation on CDTA, thus eliminating a sample preparation step. A sample stacking procedure used to concentrate the samples on the CZE capillary is described. After analysis, the obtained spectrum is deconvoluted to the zero charge spectrum to yield the molecular mass of the oligonucleotide. A misincorporation of one nucleotide can be detected by a difference in mass.

Rapid characterization of oligonucleotides by capillary liquid chromatography-nano electrospray quadrupole time-of-flight mass spectrometry.

A fast quality control method is developed allowing the desalting and characterization of oligonucleotides by capillary liquid chromatography and on-line nano-electrospray ionization quadrupole time-of-flight mass spectrometry using column switching. The influence of addition of ammonium acetate, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, formic acid or acetic acid to the sample, addition of ammonium acetate to the trapping solvent and variation of the trapping time on the further reduction of cation adduction was studied. Final conditions were the addition of 0.1 M ammonium acetate to the sample, the use of a trapping solvent consisting of 0.4 M aqueous 1,1,1,3,3,3-hexafluoro-2-propanol (HFLP) adjusted to pH 7.0 with triethylamine plus 10 mM ammonium acetate during 8 min and the elution of the oligonucleotides with 0.4 M HFIP in 50% methanol. The potential of the optimized procedure is demonstrated for different synthetic oligonucleotides.

Optimizing expression and purification from cell culture medium of trispecific recombinant antibody derivatives.

Antibody fragments offer the possibility to build multifunctional manifolds tailored to meet a large variety of needs. We optimized the production of a manifold consisting of one (bibody) or two (tribody) single-chain variable fragments coupled to the C-terminus of Fab chains. Different strong mammalian promoters were compared and the influence of expression media on production and recovery was investigated. Since the physical and chemical nature of these molecules largely depends on the nature of the antibody building blocks incorporated, a generally applicable process for the purification of recombinant antibody derivatives from serum containing mammalian cell culture medium was designed. To this end we compared protein L, hydroxyapatite, immobilized metal affinity chromatography, cation-exchange chromatography and hydrophobic charge induction chromatography.

New strategies in polypeptide and antibody synthesis: an overview.

The synthesis of radioligands can benefit considerably from optimized recombinant protein production, both on the aspect of economy of production and on the level of improving the targeting and pharmacokinetics of the ligand. This paper first describes a general production optimization strategy, and then elaborates on a protein design strategy tailored to targeting applications. Production in Escherichia coli will benefit from economy of goods and time as compared to other organisms. In order to increase the chance of finding a successful production system in this host, we have assembled a large number of expression strategies in a single, uniform expression system (FastScreen). The system allows rapid optimization of direct production of native proteins or via a fusion protein strategy with subsequent recovery of the desired protein. As an example of recombinant radioligand synthesis for improved targeting and clearing, a manifold of intermediate molecular size was synthesized by fusing one Fab and two single-chain variable fragments (scFv) antibody binding fragments into a trifunctional molecule (Tribody). Due to the use of the specific heterodimerization of the Fab chains, trispecific, bispecific, or trivalent antibody derived targeting reagents can easily be obtained. Recombinant production techniques also allow for specific incorporation of amino acids favoring a site specific labeling (labeling tags).

CD3 x CD28 cross-interacting bispecific antibodies improve tumor cell dependent T-cell activation.

Bispecific antibodies (Bs-Abs) containing an anti-CD3 and an anti-TAA specificity can recruit T cells to the tumor for cancer immunotherapy. To be effective, efficient activation at the tumor site is a prerequisite. This can be achieved by triggering both the T-cell receptor and the co-stimulatory molecule CD28. We engineered two recombinant cross-interacting Bs-Abs (CriBs-Abs) by incorporating a peptide tag and its cognate single-chain variable fragment (scFv), respectively, into a pair of (tumor x CD3) and (tumor x CD28) binding Bs-Abs. A 30-fold lower concentration of the activating CriBs-Ab as compared to non interacting Bs-Ab was sufficient for strong T-cell activation in the presence of tumor cells. One thousand-fold higher concentrations of both CriBs-Abs were required for marginal T-cell activation (70-fold below maximal response) in the absence of tumor cells. An optimized stoichiometry (1 : 1000) of activating versus co-stimulating CriBs-Ab thus allowed low doses of activating CriBs-Ab to induce tumor-cell dependent T-cell activation when used in combination with high concentrations of the pre-targeted co-stimulating CriBs-Ab in vitro. This indicates a large window of operation in which only tumor cell dependent T-cell activation is induced and systemic tumor cell independent T-cell activation is avoided, while ensuring optimal activation with a low concentration of the activating CriBs-Ab, which has the highest potential to induce toxic effects in vivo.

Analysis of nucleic acid constituents by on-line capillary electrophoresis-mass spectrometry.

This review is focused on the capillary electrophoresis-mass spectrometric (CE-MS) analysis of nucleic acid constituents in the broadest sense, going from nucleotides and adducted nucleotides over nucleoside analogues to oligonucleotides. These nucleic acid constituents play an important role in a variety of biochemical processes. Hence, their isolation, identification, and quantification will undoubtedly help reveal the process of life and disease mechanisms, such as carcinogenesis, and can also be useful for antitumor and antiviral drug research to provide valuable information about mechanism of action, pharmacokinetics, pharmacodynamics, toxicity, therapeutic drug level monitoring, and quality control related to this substance class. Fundamental investigations into their structure, the search for modifications, the occurrence and biochemical impact of structural variation amongst others, are therefore of great value. In view of the related bioanalytical procedures, the coupling of CE to MS has emerged as a powerful tool for the analysis of the complex mixtures of nucleic acid constituents: CE confers rapid analysis and efficient resolution, while MS provides high selectivity and sensitivity with structural characterization of minute amounts of compound. After an introduction about the biochemical and analytical perspectives on the nucleic acid constituents, the different modes of CE used in this field of research as well as the relevant CE-MS interfaces and the difficulties associated with quantitative CE-MS are briefly discussed. A large section is finally devoted to field-oriented applications.

Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis-electrospray ionization-quadrupole time of flight-mass spectrometry.

A capillary zone electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometric method was developed for the characterization of oligonucleotides after synthesis, using model compounds. The major difficulty is the adduction of metal cations to the polyanionic backbone of the oligonucleotide sample, resulting in complex spectra and decreased sensitivity. Several approaches were investigated to circumvent this problem. Separation was performed in an ammonium carbonate buffer. During separation, the interfering metal ions were exchanged for ammonium ions, which are less tightly bound to the oligonucleotide when ionized. The influence of the addition of piperidine and imidazole or trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) to the running buffer for further reduction of cation adduction was investigated. Addition of CDTA to the buffer system resulted in a deconvoluted spectrum with very little adducts. On-line sample stacking proved vital to preconcentrate the samples. The pH and the concentration of the ammonium carbonate buffer as well as the electrophoresis voltage were optimized to achieve the best signal response for the oligonucleotides and a maximum reduction of the cation adducts as well as a short analysis time. Finally, the sheath liquid composition was examined for further improvement of the signal. The developed method was used to analyze different oligonucleotides (5000-9200 Da) in light of its use as a final quality control method for oligonucleotides in terms of purity and sequence homogeneity of the synthesized products. In all cases, very little adducts were observed in the deconvoluted spectra, and the relative errors of the measured molecular masses ranged from 3 to 35 ppm.

Analysis of benzo[a]pyrene diol epoxide-DNA adducts by capillary zone electrophoresis- electrospray ionization-mass spectrometry in conjunction with sample stacking.

Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.