Rapid method for monitoring galactosylation levels during recombinant antibody production by electrospray mass spectrometry with selective-ion monitoring.

This report describes a simple and rapid method to determine the relative amounts of glycoforms differing in terminal galactose on a recombinant antibody produced in Chinese hamster ovary (CHO) cells. The method uses a single quadrupole mass spectrometer coupled to an HPLC system to quantify the glycoform amounts found on a recombinant antibody that binds to the human CD20 antigen. Samples from the recombinant antibody process are reduced and injected directly into the HPLC system where the heavy and light chain antibody fragments, as well as host-cell protein contaminants, are separated chromatographically. Mass-selective detection is performed in the selected-ion monitoring (SIM) mode to monitor the most abundant (38+) ions corresponding to the glycoforms found on the heavy chain of the recombinant antibody. Results obtained using the assay demonstrate good sensitivity, linearity and reproducibility. Comparison to a method using capillary electrophoresis (CE) of the labeled free oligosaccharides demonstrates similar quantitation of the glycoforms in the recombinant antibody. The LC-MS method provides a simple and rapid means for accurately quantifying antibody glycoforms directly from cell culture and other process samples.

Identification of amino acids that modulate mannose phosphorylation of mouse DNase I, a secretory glycoprotein.

We have reported that bovine DNase I, a secretory glycoprotein, acquires mannose 6-phosphate residues on 12.6% of its Asn-linked oligosaccharides when expressed in COS-1 cells and that the extent of phosphorylation increases to 79.2% when lysines are placed at positions 27 and 74 of the mature protein (Nishikawa, A., Gregory, W. , Frenz, J., Cacia, J., and Kornfeld, S. (1997) J. Biol. Chem. 272, 19408-19412). We now demonstrate that murine DNase I, which contains Lys27 and Lys74, is phosphorylated only 20.9% when expressed in the same COS-1 cell system. This difference is mostly due to the absence of three residues present in bovine DNase I (Tyr54, Lys124, and Ser190) along with the presence of a valine at position 23 that is absent in the bovine species. We show that Val23 inhibits phosphorylation at the Asn18 glycosylation site, whereas Tyr54, Lys124, and Ser190 enhance phosphorylation at the Asn106 glycosylation site. Tyr54 and Ser190 are widely separated from each other and from Asn106 on the surface of DNase I, indicating that residues present over a broad area influence the interaction with UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, which is responsible for the formation of mannose 6-phosphate residues on lysosomal enzymes.

Effect of electric field on liquid chromatographic separation of peptide digests. Combining capillary separation techniques.

A system is described which allows operation of a range of capillary based liquid phase separations including capillary electrophoresis, isocratic and gradient capillary electrochromatography, isocratic and gradient capillary liquid chromatography and electrically assisted gradient capillary liquid chromatography. The system was coupled to electrospray ionization mass spectrometry in the electrically assisted capillary liquid chromatography mode to investigate the effect of applied voltage on the selectivity in peptide mapping separations. Analyses were performed on tryptic digests of recombinant human growth hormone and tissue plasminogen activator. The results show a small but useful effect on selectivity that can be used to fine tune specific separations.

Human DNase I contains mannose 6-phosphate and binds the cation-independent mannose 6-phosphate receptor.

DNase I isolated from human urine (hDNase) or expressed in Chinese hamster ovary (CHO) cells contains mannose-phosphorylated oligosaccharides. hDNase binds to a column of immobilized cation-independent mannose 6-phosphate receptor, with the strongest binding exhibited by the protein bearing diphosphorylated oligosaccharides. The binding is inhibited by 5 mM mannose 6-phosphate, and can be prevented by prior treatment of hDNase with alkaline phosphatase. Phosphorylated high-mannose oligosaccharides were observed at both sites of glycosylation in hDNase by high-performance liquid chromatography-mass spectrometry of a tryptic digest. These results indicate that hDNase, though not an acid hydrolase, may enter the lysosomal trafficking pathway, and may have evolved from a lysosomal enzyme.

The phosphorylation of bovine DNase I Asn-linked oligosaccharides is dependent on specific lysine and arginine residues.

The secretory glycoprotein DNase I acquires mannose 6-phosphate moieties on its Asn-linked oligosaccharides, indicating that it is a substrate for UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase) (Cacia, J., Quan, C., and Frenz, J. (1995) Glycobiology 4, 99). Phosphotransferase recognizes a conformation-dependent protein determinant that is present in lysosomal hydrolases, but absent in most secretory glycoproteins. To identify the amino acid residues of DNase I that are required for interaction with phosphotransferase, wild-type and mutant forms of bovine DNase I were expressed in COS-1 cells and the extent of oligosaccharide phosphorylation determined. Phosphorylation of DNase I oligosaccharides decreased from 12.6% to 2.3% when Lys-50, Lys-124, and Arg-27 were mutated to alanines, indicating that these residues are required for the basal level of phosphorylation. Mutation of lysines at other positions did not impair phosphorylation, demonstrating the selectivity of this process. When Arg-27 was replaced with a lysine, phosphorylation increased to 54%, showing that phosphotransferase prefers lysine residues to arginines. Mutation of Asn-74 to a lysine also increased phosphorylation to 50.3%, and the double mutant (R27K/N74K) was phosphorylated 79%, equivalent to the values obtained with lysosomal hydrolases. Interestingly, Lys-27 and Lys-74 caused selective phosphorylation of the neighboring Asn-linked oligosaccharide. Finally, mutation of Lys-117 to an alanine stimulated phosphorylation, demonstrating that some residues may be negative regulators of this process. We conclude that selected lysine and arginine residues on the surface of DNase I constitute the major elements of the phosphotransferase recognition domain present on this secretory glycoprotein.

Cognitive deficits in patients with daytime sleepiness.

A chief complaint of subjects with daytime sleepiness is the disturbance of cognitive functions like concentration, learning and memory. Since sleepiness interferes with the regulation of vigilance, one may assume that a disturbance of this basic dynamic variable causes deficiencies in information processing which in turn reduce the capacity for learning and memory. In two studies the time course of vigilance was measured by means of the critical flicker fusion (CFF) test in patients with narcolepsy or with an obstructive sleep apnea syndrome (OSAS). The CFF test was applied at 15 min intervals. The total test duration was ten hours in the study with narcoleptic patients and three hours in the study with OSAS patients. The mean level of performance was similar in healthy subjects and those with narcolepsy, while the latter displayed a three- to four-fold increase in temporal variability. Such an increase in variability of performance was not seen in subjects with OSAS. These data suggest that clinically distinguishable groups of patients with daytime sleepiness differ also in the pattern of performance impairment.

Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: identification and effect on binding affinity.

This report describes the effect on antigen binding of an isomerized aspartate residue located in the complementarity-determining regions (CDRs) of a recombinant monoclonal antibody. The antibody, which binds human IgE, contains two Asp-Gly sequences within its CDRs, but only one site was found to be labile to isomerization. Isolation and characterization of antibody fragments differing in the labile sequence were facilitated by using a technique involving hydrophobic interaction chromatography (HIC) that separates aspartyl, isoaspartyl, and cyclic imide variants to the residue located in CDR-L1. The variants were isolated for structural characterization and for determination of their relative antigen binding affinities. Mutants were constructed with altered residues to obviate the effects of isomerization and were evaluated for their ability to bind to IgE. Inspection of published crystal structures of CDRs of antibodies indicated that hydrogen binding of the Asp side chain of the unreactive residue may be the constraint that prevents isomerization. The strategy outlined here may prove to be of general utility in the biochemical and immunochemical characterization of recombinant antibodies.

Symposium: Cognitive processes and sleep disturbances: The disturbance of cognitive processes in narcolepsy.

Neuropsychological methods have been applied by different authors to investigate cognitive processes such as attention, information processing, memory and psychomotor performance in narcoleptic patients. A review of the results strongly suggests that cognitive processes in narcoleptic patients are not impaired on a functional but only on a temporal level. Providing that short and challenging tasks were used, the performance of narcoleptic patient did not differ significantly from that of healthy subjects. Performance was impaired mainly when a low and monotonous information input had to be processed, a situation which is typical for tests of vigilance. This was supported by this study measuring critical flicker fusion (CFF) at 15-min intervals for 10 hours in 10 narcoleptic patients and matched healthy controls. While peak performance did not differ between groups, narcoleptic patients were unable to perform at a steady level through the day. Tiredness and episodes of sleepiness seem to be the main reason for cognitive impairments in narcolepsy.

Protein sorting by high performance liquid chromatography. 2. Separation of isophosphorylates of recombinant human DNase I on a polyethylenimine column.

Anion exchange HPLC with a polyethylenimine (PEI) column separates recombinant human deoxyribonuclease I (rhDNase) glycoforms according to the extent and positions of phosphorylation of mannose residues in N-linked oligosaccharides. The separation provides a selectivity unavailable by anion exchange HPLC with other columns or by isoelectric focusing gel electrophoresis and can be used to quantify the phosphate content of preparations of rhDNase. Tryptic mapping of fractions collected from the column and treated with alkaline phosphatase was used to identify the sites of phosphorylation. Unnatural forms of rhDNase, bearing oligosaccharide structures at only one of the two sites of glycosylation, were prepared by cleaving the phosphate-containing high mannose and hybrid structures from the purified isophosphorylates fractionated on the PEI column. The separation of rhDNase isophosphorylates on the PEI column mimics the relative affinities for the mannose 6-phosphate receptor that traffics acid hydrolases to lysosomes and provides a useful example of protein sorting by biomimetic interaction chromatography.

Ultrasensitive plasmid mapping by high performance capillary electrophoresis.

This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely related plasmids with three different restriction enzymes. The plasmids express full length and truncated forms of a growth factor receptor oncogene product and were digested with HpaII, HaeIII and RsaI. The resulting oligonucleotide fragments were under 2000 base pairs in length, a size well suited to separation by HPCE with linear polyacrylamide as a sieving matrix. Plasmid mapping is an essential tool in biotechnology both for the design of an expression system and for monitoring the stability of the expression system during fermentation. HPCE can yield much higher resolution of oligonucleotides than attainable in conventional agarose gel electrophoretic procedures for plasmid mapping. In the examples described here, the HpaII digests provided the surest identification of individual plasmids in the HPCE analysis and could discriminate among all four plasmids. In conventional slab electrophoresis, however, the RsaI digests provided the best discrimination, although two of the plasmids in this system yielded essentially identical electrophoretic patterns. Hence the optimal restriction enzyme for plasmid mapping applications with HPCE may differ from that selected on the basis of conventional slab gel analysis, and the former technique can provide higher discrimination among related plasmids. The advantages of the HPCE format with respect to speed, low sample consumption and resolution are described.

Protein sorting by high-performance liquid chromatography. I. Biomimetic interaction chromatography of recombinant human deoxyribonuclease I on polyionic stationary phases.

Chromatographic separations can be tailored to exploit specific interactions between a stationary phase ligand and a protein structural feature of interest. Variations in this feature then form the basis for sorting a mixture of closely related proteins into defined subpopulations. This report describes the sorting of variants of recombinant human deoxyribonuclease I (rhDNase) that differ in the occurrence of deamidation at a single residue. rhDNase, an enzyme that non-specifically hydrolyzes DNA, is glycosylated and exhibits considerable charge heterogeneity owing to the sialylation and phosphorylation of its N-linked oligosaccharides. This heterogeneity obscures the relatively subtle differences between deamidated and intact rhDNase, preventing separation on this basis in conventional ion-exchange HPLC. Published structural information on bovine DNase reveals that the analogous labile asparagine residue is involved in DNA binding, so stationary phases containing polyanionic ligands mimicking nucleic acids were employed to separate the deamidation variants of rhDNase. Electrostatically immobilized DNA, a "tentacle" cation exchanger (TCX) and immobilized heparin columns all resolved the deamidated and intact forms of rhDNase when operated at pH 4.5. The ligands of the TCX and heparin columns are sufficiently long, flexible and polyanionic to interact with rhDNase in a manner similar to DNA and to sort rhDNase variants on the basis of the charge difference of a single residue involved in that interaction. A non-hydrolyzable double-stranded oligonucleotide analogue of DNA was also synthesized and immobilized to an HPLC support. This column, operated at pH 6, where rhDNase is active, resolved the two isomeric products of deamidation of rhDNase, i.e., variants of the enzyme containing either aspartate or isoaspartate in lieu of asparagine at the deamidation site in rhDNase. This is the first reported separation of intact variants of a glycoprotein differing on the basis of these isomeric products of deamidation through the common cyclic imide mechanism.

Characterization of a tryptic digest by high-performance displacement chromatography and mass spectrometry.

High-performance displacement chromatography (HPDC) provides a means of increasing the capacity of a chromatographic column, while maintaining the resolution afforded by high-performance liquid chromatographic (HPLC) instruments. The high capacity and high resolution of HPDC can be exploited in tryptic mapping to facilitate the characterization of a protein preparation. In this manner, minor constituents of the mixture, which may be difficult to isolate by conventional chromatographic methods, can be obtained in sufficient amounts to permit chemical characterization by established techniques. The isolation by HPDC of peptides obtained by digestion of recombinant human growth hormone (rhGH) and the subsequent characterization of the peptides are described. The identification of certain of these peptides revealed information on the specificity of trypsin for the substrate, rhGH, and for autolysis. Fractions from the HPDC tryptic map were collected and analyzed by electrospray ionization mass spectrometry (ESI-MS) either directly or following further separation by gradient elution HPLC. Fragment ions observed in the ESI mass spectra facilitated identification of peptides obtained by HPDC tryptic mapping.

High performance capillary electrophoresis.

The application of recombinant-DNA methods for the production of therapeutic proteins has, over the past decade, driven the development of new technology for the analysis and characterization of biological molecules. High performance capillary electrophoresis (HPCE) has generated enormous interest among biochemists, analytical chemists and chromatographers, and is emerging as an extremely high-resolution separation technique, that may rival high performance liquid chromatography (HPLC) in its efficiency and breadth of application.

High-performance displacement chromatography-mass spectrometry of tryptic peptides of recombinant human growth hormone.

The combination of high-performance displacement chromatography with continuous flow fast atom bombardment (FAB)-mass spectrometry (MS) offers a means of overcoming the sample capacity limitations imposed by the low flow-rates tolerated in microbore systems employed for directly coupled liquid chromatography-MS. Displacement chromatography is performed at high concentrations with the same equipment and columns as typically used in chromatography at low concentrations. By using this mode of chromatography with a solution of cetyltrimethylammonium bromide as the displacer, the capacity of a reversed-phase column can be increased 50- to 100-fold for separation of a tryptic digest of biosynthetic human growth hormone. Despite the high load, the use of displacement chromatography allowed high-resolution separation of the complex mixture of eighteen major components. On-line analysis by continuous flow FAB-MS yielded high-quality spectra of these peptides and demonstrated that sharp, single-component bands can be obtained in this separation. Along with the major fragments, the chromatogram showed other peptides originating from protein variants in the sample, from non-specific cleavage in the enzymatic digest or from autolysis of trypsin. On-line analysis also allowed selective ion monitoring of the column effluent for individual peptides and confirmed the high efficiency and resolution obtained by preparative displacement separations on HPLC columns and equipment.

The influence of two behavioral regimens on the distribution of sleep and wakefulness in narcoleptic patients.

Thirty-two hours (night-day-night) of polygraphic recordings were performed on 14 patients with a diagnosis of narcolepsy-cataplexy. Half of the patients stayed in bed during the day, whereas the other half were seated at a table. Patients were free to nap whenever they wanted to. Patients under continuous bedrest slept 2-3 times more during the day than patients who were sitting at the table. Rapid-eye-movement (REM) sleep and slow-wave sleep (SWS, stages 3 and 4) were nearly absent during daytime sleep in the table group, but not in the bed group. The differential behavioral regimes during the day resulted in different amounts of SWS in the consecutive night sleep. Although SWS increased from the first to the second night in the table group, it decreased in the bed group. This result suggests that the presumably homeostatic regulation of SWS is intact in narcoleptic patients.

Characterization of human growth hormone by capillary electrophoresis.

Production of proteins by recombinant DNA technology for use as pharmaceuticals requires the use of the most powerful tools of analytical protein chemistry in order to confirm purity and identity of the product and reliability of the process. Capillary electrophoresis is an emerging technology that shows high sensitivity and selectivity and may have promise in this application. The technique combines the instrumental control and quantification features of high-performance liquid chromatography with the separating power of electrophoresis, and thereby has attracted broad interest. In this report, human growth hormone expressed in bacteria has been analyzed by both free zone electrophoresis and isoelectric focusing in a coated capillary to demonstrate the separation of the native molecule from its deamidated variant. A capillary zone electrophoretic tryptic map has also been developed and characterized. This map complements the widely employed reversed-phase high-performance liquid chromatography tryptic mapping systems that are important in protein characterization. Certain drawbacks to capillary zone electrophoresis compared to other analytical methods are noted, including relatively poor reproducibility and low sample tolerance. For applications as demonstrated here, however, the speed, separating power and sensitivity of the technique compensate for these shortcomings.

Hydrocarbon recovery and biocompatibility of solvents for extraction from cultures of Botryococcus braunii.

Various water-immiscible solvents were tested for biocompatibility and hydrocarbon recovery under different contact conditions with the hydrocarbon-rich microalga Botryococcus braunii. Eighteen solvents were first selected from a database of 1500 compounds (compiled for solvent selection for ethanol recovery from Saccharomyces cerevisiae fermentation). Nine of these candidate solvents were shown to be biocompatible with B. braunii following short contact times. This biocompatibility tends to be associated with high molecular weights and high boiling points but strongly depends on solvent chemical structure. A low polarity is essential to biocompatibility and calculated octanol-water partition coefficients, or capacity factors determined by reversed-phase high-performance liquid chromatography (HPLC), are suitable predictors of biocompatibility with B. braunii. High recoveries of hydrocarbons directly from the algal culture require relatively polar solvents and are, therefore, inimical with maintenance of cell viability. The inaccessibility of weakly polar solvents to the cell surface appears to protect the algae but also prevents substantial recovery of the hydrocarbons stored in B. braunii outer walls. In order to achieve a high recovery, contact with the solvent must be carried out on algae concentrated by filtration. Then, a large fraction of B. braunii hydrocarbons can be recovered, after a short contact time, without impairing cell viability. Under these conditions, the pertinent solvent property is affinity for the nonpolar hydrocarbons, and the highest recovery yield, approximately 70% after contact for 30 min, is achieved with hexane.