Akiko Koga

C-terminal modification of monoclonal antibody drugs: Amidated species as a general product-related substance

Twelve therapeutic mAbs, comprising 10 IgG1s and 2 IgG4s, were analyzed by a peptide mapping technique to detect and quantify C-terminal modifications. C-terminal amidated structures were found in 8 out of the 12 mAbs. An in vitro study using a commercially available peptidylglycine alpha-amidating monooxygenase (PAM) revealed that both IgG1 and IgG4 can be substrates for PAM. This study showed that C-terminal amidation is a general C-terminal modification on the heavy chains of therapeutic mAbs and that C-terminal amidation of mAbs can be catalyzed by a certain PAM(s) in the Chinese hamster ovary (CHO) cells that are widely used for manufacturing therapeutic mAbs.

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals.

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.

VH/VL interface engineering to promote selective expression and inhibit conformational isomerization of thrombopoietin receptor agonist single-chain diabody

Thrombopoietin receptor agonist humanized VB22B single-chain diabody (hVB22B (scFv)(2)) was found to be expressed as a mixture of two conformational isomers, a single-chain diabody form and a bivalent scFv form, which had different V(H)/V(L) (variable region of the heavy chain/light chain) association patterns. The single-chain diabody form showed significantly higher biological activity than the bivalent scFv form and, when incubated at elevated temperatures, exhibited novel isomerization to the inactive bivalent scFv form. Therefore, therapeutic development of hVB22B (scFv)(2) would require separation of the purified single-chain diabody form from the mixture of the two conformational isomers and also inhibition of isomerization into an inactive bivalent scFv form during storage. Novel V(H)/V(L) interface engineering in hVB22 (scFv)(2), in which hydrogen bonding between H39 and L38 was substituted with electrostatic interaction to enhance the desired V(H)/V(L) association and inhibit the undesired V(H)/V(L) association, enabled selective expression of the desired conformational isomer without any reduction in biological activity or thermal stability. Moreover, V(H)/V(L) interface-engineered hVB22 (scFv)(2) was completely resistant to isomerization. Because the hydrogen bonding interaction between H39 and L38 and the surrounding residues are highly conserved in human antibody sequences, V(H)/V(L) interface engineering could be generally applied to various (scFv)(2) molecules for selective expression and inhibition of the isomerization of conformational isomers.

Thermodynamic and Fluorescence Analyses to Determine Mechanisms of IgG1 Stabilization and Destabilization by Arginine

ABSTRACTPurposeTo investigate mechanisms governing the stabilization and destabilization of immunoglobulin (IgG1) by arginine (Arg).MethodsThe effects of Arg on the aggregation/degradation, thermodynamic stability, hydrophobicity, and aromatic residues of IgG1 were respectively investigated by size-exclusion chromatography, differential scanning calorimetry, probe fluorescence, and intrinsic fluorescence.ResultsArg monohydrochloride (Arg–HCl) suppressed IgG1 aggregation at near-neutral pH, but facilitated aggregation and degradation at acidic pH or at high storage temperature. Equimolar mixtures of Arg and aspartic acid (Asp) or glutamic acid (Glu) suppressed aggregation without facilitating degradation even at high temperature. Arg–HCl decreased the thermodynamic stability of IgG1 by enthalpic loss, which was counteracted by using Asp or Glu as a counterion for Arg. The suppression of aggregation by Arg–HCl was well correlated with the decrease in hydrophobicity of IgG1. The intrinsic fluorescence of IgG1 was unaffected by Arg–HCl.ConclusionsSuppression of IgG1 aggregation can be attributed to the interaction between Arg and hydrophobic residues; on the other hand, facilitation of aggregation and degradation is presumably due to the interaction between Arg and some acidic residues, which could be competitively inhibited by simultaneously adding either Asp or Glu.

O-linked glucosylation of a therapeutic recombinant humanised monoclonal antibody produced in CHO cells.

An unpredictable modification of a therapeutic recombinant humanised monoclonal antibody (rh-mAbX) produced using CHO cells was found. LC/MS analysis of rh-mAbX indicated the presence of heterogeneity in the light chain with a corresponding mass shift of 162 Da compared to the theoretical mass. To characterise the heterogeneity, that is, the attached moiety, several analyses were performed. Peptide mapping of rh-mAbX indicated that the attached moiety was located in the amino acid sequence from Leu20 to Lys45, which is a part of the variable region of the light chain. The peptide was efficiently purified in two-steps by RP-HPLC by utilising two different types of RP columns. N-terminal sequencing and LC/MS/MS analysis of the peptide suggested that Ser29 of the light chain was the modification site, and that the attached moiety was a single O-linked hexose. HPAEC-PAD analysis following β-elimination indicated the presence of an O-linked glucose in the modified peptide. Monosaccharide composition analysis after acid hydrolysis supported this result. The content of antibodies containing this species was determined to be approximately 10% by Lys-C peptide mapping detected at 280 nm. Thus, this study demonstrated the formation of a unique O-linked glucosylation posttranslational modification in a recombinant humanised monoclonal antibody produced in CHO cells.

Small-scale screening method for low-viscosity antibody solutions using small-angle X-ray scattering

Graphical abstract Figure. No caption available. Abstract In this study, we investigated the concentration range in which self‐association starts to form in humanized IgG monoclonal antibody (mAb) solutions. Furthermore, on the basis of the results, we developed a practical method of screening for low‐viscosity antibody solutions by using small‐angle X‐ray scattering (SAXS) measurements utilizing small quantities of samples. With lower‐viscosity mAb3, self‐association was not detected in the range of 1–80 mg/mL. With higher‐viscosity mAb1, on the other hand, self‐association was detected in the range of 10–20 mg/mL and was clearly enhanced by a decrease in temperature. The viscosities of mAb solutions at 160, 180, and 200 mg/mL at 25 °C quantitatively correlated very well with the particle size parameters obtained by SAXS measurements of mAb solutions at 15 mg/mL at 5 °C. The quantity of mAb sample required for the SAXS measurements was only 0.15 mg, which is about one‐hundredth of that required for actual viscosity measurements at a high concentration, and such quantities could be available even at an early stage of development. In conclusion, the SAXS analysis method proposed in this study is a valuable tool for the development of concentrated mAb therapeutics with high manufacturability and high usability for subcutaneous injection. Abbreviations: Dmaxapp: apparent maximum dimension; Rgapp: apparent radius of gyration; EMS: electromagnetically spinning; IFT: indirect Fourier transformation; p(r): pair–distance distribution function; q.s.: quantity sufficient; SAXS: small‐angle X‐ray scattering.

Establishment of powder dustiness evaluation method by dustmeter with small amount of pharmaceutical ingredients

The ratio of high potent materials in the new chemical entities has recently increased in the pharmaceutical industry. Generally, most of them are highly hazardous, but there is little toxicity information about the active pharmaceutical ingredients in the early development period. Even if their handling amount is quite small, the dustiness of high potent powder generated in the manufacturing process has an important impact on worker health; thus, it is important to understand the powder dustiness. The purpose of this study was to establish a method to evaluate the powder dustiness by the consumption of small amount of samples. The optimized measurement conditions for a commercially available dustmeter were confirmed using lactose monohydrate and naproxen sodium. The optimized test conditions were determined: the dustmeter mode, the flow rate, the drum rotation speed, the total measurement time, and sample loaded weight were type I mode, 4 L/min, 10 rpm, 1 min and 1-10 g , respectively. The setup conditions of the dustmeter are considerably valuable to pharmaceutical industries, especially, at the early development stage and especially for expensive materials, because the amount of air-borne dust can be evaluated with accuracy by the consumption of small amount of samples.