Masayuki Yabuta

Detection of Histidine Oxidation in a Monoclonal Immunoglobulin Gamma (IgG) 1 Antibody

Although oxidation of methionine and tryptophan are known as popular chemical modifications that occur in monoclonal antibody (mAb) molecules, oxidation of other amino acids in mAbs has not been reported to date. In this study, oxidation of the histidine residue in a human immunoglobulin gamma (IgG) 1 molecule was discovered for the first time by mass spectrometry. The oxidation of a specific histidine located at the CH2 domain of IgG1 occurred under light stress, but it was not observed under heat stress. With the forced degradation study using several reactive oxygen species, the singlet oxygen was attributed to a reactive source of the histidine oxidation. The reaction mechanism of the histidine oxidation was proposed on the basis of the mass spectrometric analysis of IgG1 oxidized in deuterium oxide and hydrogen heavy oxide.

An analysis of target preferences of Escherichia coli outer-membrane endoprotease OmpT for use in therapeutic peptide production: efficient cleavage of substrates with basic amino acids at the P4 and P6 positions.

The Escherichia coli outer‐membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids. The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2′. Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon‐like peptide‐1 (7–37) in 4 M urea. The P1–P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion. The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate. Conversely, cleavage efficiency increased on replacing Glu at P6. Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect. This was also shown to be true with synthetic decapeptide substrates in the absence of urea. The kcat/Km ratio increased with basic residues at P4 or P6, mainly due to a lower Km rather than an increase in kcat. On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure. OmpT released mature ANP from the E. coli‐expressed fusion protein. As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.

Genetic engineering of Escherichia coli for production of tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR). BH4 is a medicine used to treat atypical hyperphenylalaninemia. It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures. To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli (E. coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes. These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4. In order to increase BH4 productivity we made further improvements. First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced. Second, to augment the activity of GCHI, the folE gene from E. coli was replaced by the mtrA gene from Bacillus subtilis. These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth. The results suggest the possibility of commercial BH4 production by our method.

Substrate Specificity at the P1´ Site of Escherichia coli OmpT under Denaturing Conditions

Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues. The cleavage site of the substrate PRR was Arg140-Arg141 and the modified substrates PRX substituted all 19 natural amino acids at the P1´ site instead of Arg141. OmpT under denaturing conditions (in the presence of 4 M urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg140 except for the Arg140-Asp141, -Glu141, and -Pro141 pairs. In addition to Arg140 at the P1 site, similar results were obtained when Lys140 was substituted into the P1 site. In the absence of urea, an aspartic acid residue at the P1´ site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site.

Role of the ompT Mutation in Stimulated Decrease in Colony-Forming Ability Due to Intracellular Protein Aggregate Formation in Escherichia coli Strain BL21

Recently we found that the cells of Escherichia coli strain BL21 producing a fusion protein, GST-Sup35NM, show a much more rapid decrease in colony-forming ability in the stationary phase than control cells. In this study, it was found that an extract of the cells producing GST-Sup35NM forms fibrous protein polymers containing GST-Sup35NM. In the course of the study, we realized that strain BL21 carried the ompT mutation. We suspected that the deficiency in OmpT protease was responsible for the observed phenotype. To test this, we introduced the wild-type ompT gene into strain BL21, and found that the transformed cells recovered the wild-type phenotype. We concluded that OmpT protease, though known to localize on the cell surface, is involved in protein quality control within the cell.

Utilization of Escherichia coli Outer-Membrane Endoprotease OmpT Variants as Processing Enzymes for Production of Peptides from Designer Fusion Proteins

ABSTRACT Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp97 is proposed to interact with the P1′ amino acid of its substrates, OmpT variants with variations at Asp97 were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1′). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg↓motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.

Choosing the right protein A affinity chromatography media can remove aggregates efficiently

Protein A chromatography (PAC) is commonly used as an efficient capture step in monoclonal antibody (mAb) separation processes. Usually dynamic binding capacity is used for choosing the right PAC. However, if aggregates can be efficiently removed during elution, it can make the following polishing steps easier. In this study a method for choosing the right PAC media in terms of mAb aggregate removal is proposed. Linear pH gradient elution experiments of two different mAbs on various PAC columns are carried out, where the elution behavior of aggregates as well as the monomer is measured. Aggregates of one mAb are more strongly retained compared with the mAb monomer. Another mAb showed different elution behavior, where the aggregates are eluted as both the weakly and strongly retained peaks. In order to remove the two types of aggregates by stepwise elution two protocols are tested. The first protocol A consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. No detectable peaks are observed during the wash stage of protocol A whereas significant peaks are monitored during the arginine wash of protocol B. One of the PAC columns showed a smaller peak during the arginine wash. In addition, both aggregate removal and monomer yield are higher with protocol B compared with the other PAC columns. This method is found to be useful for choosing the right PAC column.

Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry

To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.

Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A detected by sodium dodecyl sulfate-capillary gel electrophoresis

&NA; This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal antibody‐A (mAb‐A), detected by sodium dodecyl sulfate‐capillary gel electrophoresis (SDS‐cGE). The fragment migrated close to the internal standard (10 kDa marker) of SDS‐cGE and increased about 0.5% under a 25 °C condition for 6 months. Generally, identification of fragments observed in SDS‐cGE is challenging to carry out due to the difficulty of collecting analytical amounts of fractionations from the capillary. In this study, in‐gel digestion peptide mapping and reversed phase liquid chromatography‐mass spectrometry (RPLC–MS) were employed to elucidate the structure of the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain HC1‐104. The fragment contained complementarity determining regions (CDRs), which are significant to antigen binding, and thus would affect the efficacy of mAb‐A. In addition, SDS‐cGE without the 10 kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment revealed that the fragment increases 0.2% per year in storage at 5 °C. The combination of the three analytical methodologies successfully identified the impurity peak detected by SDS‐cGE, providing information critical to assuring the quality and stability of the biotherapeutics. Graphical abstract Figure. No caption available. HighlightsA fragment of monoclonal antibody was detected close to the 10 kDa marker in SDS‐capillary gel electrophoresis and increased about 0.5% under 25 °C for 6 months.The fragment was identified and characterized by using in‐gel digestion peptide mapping, RPLC–MS, and a Gelfree 8100 fractionation system.Three analytical methods showed comparable results proving the fragment was a fraction of heavy chain HC1‐104.