Optimization of the anti-(human CD3) immunotoxin DT389-scFv(UCHT1) N-terminal sequence to yield a homogeneous protein.

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin-based anti-(human CD3) immunotoxin, DT389-scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1-389 of diphtheria toxin, scFv is single-chain variable-region antibody fragment and UCHT1is an anti-(human CD3) monoclonal antibody]. Biochemical characterization of this molecule by MS and N-terminal sequencing by Edman degradation revealed that the protein was heterogeneous at the N-terminus, containing species both with (60%) and without (40%) the initiator methionine residue. In an attempt to generate an N-terminally homogeneous molecule, a panel of seven N-terminal variants was designed, based on the published specificity of bacterial methionine aminopeptidase. Following bacterial expression, partial purification and separation on SDS/PAGE, these proteins were subjected to N-terminal sequencing by Edman degradation. Three of the mutants yielded a 100% homogeneous amino acid sequence. By contrast, the original DT389-scFv(UCHT1) protein and four variant proteins yielded two sequences with varying ratios corresponding to species with and without methionine. The N-terminal sequences of the three homogeneous clones were MLADD and MLDD, where the methionine was completely retained, and SADD, where the methionine was completely removed. One of the homogeneous mutants (SADD) was expressed, refolded and purified and found to be equipotent with the parent immunotoxin. Thus, using a rational mutagenesis approach, three N-terminally homogeneous variants of DT389-scFv(UCHT1) have been identified, at least one of which is functionally indistinguishable from the parent immunotoxin. This approach is generally applicable to biopharmaceutical production and immunotoxin development in particular.

Disease activated drugs: a new concept for the treatment of asthma.

Disease activated drugs (DAD) are pro-drugs of one active principle or combinations of two drugs, which have a proven efficacy for the treatment of the target disease. In opposition to pro-drugs, DAD are activated in inflamed but not normal tissues. Due to the disease specific activation, the amount of locally released drug(s) should be related directly to the severity of the inflammation. To test this concept in asthma a PDE4 inhibitor, an isoquinoline derivative, was chemically derivatized into pro-drugs or combined with corticosteroids. These new compounds were more readily cleaved into active PDE4 inhibitor, in bronchoalveolar lavage fluid (BALF) from Brown-Norway rats with lung inflammation than in BALF from rats without airway inflammation. The DAD concept (local selective release and improved therapeutic window) was validated in vivo using the inhibition of methacholine induced bronchoconstriction in guinea pigs with or without ozone induced lung inflammation. An example of DAD hydrolysis (isoquinoline-dexamethasone) was also examined in BALF from asthmatics and healthy volunteers.

Microbial conversion products of leptomycin B.

Leptomycin B (LMB), a secondary metabolite produced by Streptomyces sp. strain ATS 1287, with known antifungal and antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev and exhibits significant antiproliferative activity. Since LMB itself turned out to be distinctly cytotoxic, a bioconversion screening with a selected set of 29 bacterial and 72 fungal strains was performed in order to obtain metabolites of LMB with reduced antiproliferative effects. Several derivatives of LMB, more polar than the parent compound and produced in yields of > 5%, were detected. Liquid chromatography-mass spectroscopy analysis indicated the type of bioconversion. Fermentations (1-liter scale) of those strains with high rates of transformation were suitable for isolation and characterization of the most prominent metabolites. Thus, bioconversion of LMB with Aspergillus flavus ATCC 9170 and Emericella unguis ATCC 13431 served for isolation of the novel derivatives 26-hydroxy-LMB (30% was the concentration of the metabolite [with respect to LMB] used for bioconversion) and LMB-24-glutaminamide (90%), respectively. Streptomyces rimosus ATCC 28893 converted LMB into 4,11-dihydroxy-LMB (13%) and 2,3-dihydro-LMB (55%). Although the antiproliferative effects of the LMB metabolites could be reduced through microbial conversion, none of these metabolites inhibited the nuclear export of Rev better than LMB itself.

Microbial biotransformation products of cyclosporin A.

In order to mimic the human metabolic pathway of cyclosporin A (CyA) a total of 28 bacterial and 72 fungal strains was screened for their ability to transform CyA. Among 3 bacteria and 11 fungi, which produced the main human metabolite OL-17 [eta HyMeBmt1]CyA, Actinoplanes sp. (ATCC 53771) achieved the best transformation rate (5.4%). Furthermore, the two N-demethylated minor products [Leu4]CyA (3.2%) and [Leu9]CyA (4.7%) were isolated, both known as minor natural metabolites and the first one also as a human biotransformation product. Microbial conversion of CyA using the actinomycete Sebekia benihana (NRRL 11111) yielded [gamma HyMeLeu4]CyA (35%), [gamma HyLeu4]CyA (4.5%) and [gamma HyMeLeu4, gamma HyMeLeu6]CyA (8.6%). The structures of these derivatives correspond with those of the human metabolic pathway. The related compounds [Nva2]CyA (CyG) and [D-MeSer3]CyA were similarly converted to the corresponding 4-gamma-hydroxylated analogues. None of the biotransformation products showed a better immunosuppressive effect than CyA, although in various cases the cyclophilin binding affinity was comparable to that of CyA.

Taxoid side-chain structure determination by electrospray ionization tandem mass spectrometry.

A method to elucidate the side-chain structure of novel taxoids was developed through use of electrospray ionization tandem mass spectrometry. The intact side-chain fragments produced by source fragmentation in the atmosphere-to-vacuum interface were further dissociated in the collision cell of a triple quadrupole mass spectrometer. The distinctive collisionally induced dissociation mass spectra were remarkably informative and gave detailed information on the side-chain functionalities. This technique proved valuable in the characterization of new taxoids produced by yew-tree cell cultures.

Analysis of taxol and related diterpenoids from cell cultures by liquid chromatography-electrospray mass spectrometry.

Authentic taxanes (taxol, 10-deacetyltaxol, cephalomannine, 10-deacetylcephalomannine, baccatin III) and extracts from cell cultures derived from various yew tree species have been analyzed by microbore high-performance liquid chromatography (HPLC)-electrospray mass spectrometry (ESMS). All gave excellent positive-ion ES spectra with dominant protonated molecules at low nozzle-to-skimmer bias value (45 V). By increasing the voltage value to 85 V, fragmentation increased and structurally informative spectra were obtained. The fragments found were both of the C-13 side-chain and of the taxane ring, so their analysis gave important information about the taxane structure and any chemical modifications at different positions of the molecule. When tandem MS was used (argon gas, 25 eV collision energy), fragments similar to those obtained from collision-induced dissociation in the source were detected. The cell culture extracts were analyzed by microbore HPLC-ESMS and excellent spectra were obtained on 5-10 ng of separated compounds; even greater selectivity and sensitivity were obtained through use of selected-ion monitoring (SIM). With SIM, 100 pg of all taxanes could readily be detected. In the HPLC-ESMS mode, only 10% of the eluent was mass-analyzed, so 90% would be available for recovery through fraction collecting.

Expediting rare variant hemoglobin characterization by combined HPLC/electrospray mass spectrometry.

Microscale analysis of a variant hemoglobin (Hb) has been achieved by combination of high performance liquid chromatography (HPLC) and electrospray mass spectrometry (ESMS) and the method should be almost universally applicable. We have eliminated preparative scale HPLC of globin chains and semi-preparative HPLC of proteolytic digests which had been used prior to mass spectrometry. Use of microbore HPLC columns reduced the time required for analysis substantially and solvent usage by 100x. Molecular masses of intact globins and masses and sequence information of tryptic peptides could be obtained without collecting and separately analyzing chromatographic fractions. As an example of the use of these methods, we report the characterization of an unknown hemoglobinopathy case that was finally authenticated as Hb P-Galveston [beta 117(G19)His-->Arg], using the following sequence of analyses: 1) ESMS of complete hemolysate, 2) analytical HPLC of globin chains, 3) combined microbore HPLC/ESMS of globin chains to determine their molecular masses, 4) cysteine derivatization and tryptic digestion of mixture of all globins, followed by microbore separation of the peptides, molecular mass determination, and generation of fragmentation patterns allowing confirmation of amino acid sequences. This four-part strategy should allow characterization of almost all variant Hbs. Exceptions would be mutations in regions of globin chains which give rise to small (< four residues) tryptic peptides, either normal or produced by addition of new tryptic sites and mutations that introduce only minute difference in molecular weight (MW) of tryptic peptides. Since only 10% of each separated peptides is mass analyzed, 90% is available for collection and further structural identification (e.g. by tandem MS or Edman sequencing) if the identity is still in doubt.

Determination of disulfide bridges in natural and recombinant insect defensin A.

The primary-structure comparison of natural insect defensin A from Phormia terranovae and recombinant insect defensin A from Saccharomyces cerevisiae has been accomplished using a combination of Edman degradation and liquid secondary ion mass spectrometry. The natural and recombinant proteins have the same primary structure with identical disulfide-bond designations (formula; see text) as determined from the peptides obtained after thermolysin digestion. The combined use of Edman degradation and mass spectometry allowed the disulfide-bridge structure to be determined with a total of only 40 micrograms (9.9 nmol) natural peptide. Mass spectrometry provides a rapid means of disulfide-bridge verification, requiring not more than 20 micrograms recombinant insect defensin A, which is compatible with use in batch analysis.

Cuttlefish sperm protamines. 2. Mass spectrometry of protamines and related peptides.

The sequence of very basic proteins such as protamines (more than 50% arginines) and related peptides has been determined using mass spectrometry in conjunction with Edman degradation. The capabilities of three mass spectrometric (MS) techniques [fast-atom-bombardment (FAB), 252Cf plasma desorption (252CFPD) and electrospray (ES)] have been evaluated on stallion protamine 1, cuttlefish protamine, and the corresponding cleavage peptides. In contrast to FAB-MS and 252Cf PD-MS, ES-MS made possible an easy determination of the molecular mass of the intact protamines (approximately 8 kDa). With ES-MS about 0.2 nmol was sufficient to yield a mass measurement with an accuracy of 0.05%. On peptides smaller than 3500 Da, both FAB-MS and 252Cf PD-MS allowed mass measurements with an accuracy of 0.1%. 252Cf PD-MS appeared more sensitive than FAB-MS by about a factor of 10. FAB-MS is nevertheless particularly interesting since in most cases it produced spectra with intense A-type fragmentation ions which provided reliable primary structure information.

Application of electrospray mass spectrometry to the characterization of recombinant proteins up to 44 kDa.

Mass measurement by electrospray mass spectrometry (ESMS) is used as a rapid preliminary verification of the identity of various recombinant proteins ranging from 7 to 44 kDa with an accuracy of 0.01-0.03%. ESMS not only improves the speed but also the reliability of the protein structure determination when used in conjunction with other methods of protein analysis. Modifications of these large molecules, for example the loss of C-terminal amino acids, N-terminal acetylation, 2-mercaptoethanol addition to a cysteine, and trace formation of a covalent dimer (3%), are easily detected individually or in mixtures by mass measurement using ESMS; feats which would be very difficult to achieve using classical biochemical methods. As little as 1% of several structurally related protein contaminants have been identified in a 15 kDa recombinant protein preparation.

Mass spectrometry analyses of recombinant hirudins (7 kDa).

The use of liquid secondary ion mass spectrometry (LSIMS) in the characterization of related recombinant 7-kDa peptides illustrates the adequacy of average mass measurement by scanning at low resolution. The difficulty in using the high-resolution technique in the case of poor LSIMS sensitive peptides is discussed, as well as the fact that it does not give, for these molecular weights, any real advantage. The average (or chemical) molecular weights of three recombinant hirudin molecules, hirudin variant 2 (rHV2, 6892.4 Da), hirudin variant 2-Lys47 (rHV2-Lys47, 6906.5 Da), and hirudin variant 2-Arg47 (rHV2-Arg47, 6934.5 Da), less than or equal to 10 micrograms each, have been measured with an accuracy less than or equal to 0.3 Da in the narrow-scan mode and less than or equal to 0.5 Da (from the protonated molecular ion) in the wide-scan mode within 10-15 min; this allows easy distinction of the three 65 amino acid proteins, which differ by a single amino acid. These three molecules could also be distinguished from one another in a mixture. Mass spectrometry and limited sequence characterization of several minor, similarly isolated peptides identified them to be N-terminal additions and/or C-terminal deletions of rHV2-Lys47. LSIMS analysis is consistent with there being no covalent dimer of rHV2-Lys47 as a narrow scan of the 7-kDa molecular ion cluster at high resolution shows it not to be a doubly charged ion.