Efficient production of single-chain Fv antibody possessing rare codon linkers in fed-batch fermentation

Kumada, Yoichi,Sakan, Yoshinobu,Kajihara, Hideyuki,Kihara, Mana,Kikuchi, Yasufumi,Yamaji, Hideki,Seong, Gi Hun,Katoh, Shigeo Elsevier 2009 Journal of bioscience and bioengineering Vol.107 No.1

<P><B>Abstract</B></P><P>Single-chain Fv antibody (scFv) having 2 types of polypeptide linkers with or without rare codons, namely scFv (G<SUB>4</SUB>S)<SUB>3</SUB><SUP>R</SUP> and scFv No.10 (with rare codons) and scFv (G<SUB>4</SUB>S)<SUB>3</SUB> and scFv No.10<SUP>NR</SUP> (without rare codon), were expressed under controllable conditions in batch and fed-batch fermentation, in order to compare volumetric productivity and specific productivity levels of scFvs as a soluble form. In batch fermentation, volumetric productivity levels of scFv (G<SUB>4</SUB>S)<SUB>3</SUB><SUP>R</SUP> and scFv No.10, namely scFvs having the rare codon linkers were 3–5 times higher than those of scFvs that had linkers without the rare codon. In fed-batch fermentation controlled by an exponential feeding system, the cell concentrations of the transformants increased with similar specific growth rates (0.1 h<SUP>−1</SUP>), while the specific productivity levels of scFvs with the rare codon linkers were 1.6 times higher than those of scFvs without the rare codon linkers. These results indicate that the presence of several rare codons in the gene of a polypeptide linker increases soluble amount of scFvs. This might be caused by a temporary decrease in translation speed at the position of the polypeptide linker allowing time for the folding of the V<SUB>H</SUB> domain and avoiding unfavorable interactions between amino acid residues at the unfolded V<SUB>H</SUB> and V<SUB>L</SUB> domains. Higher specific productivity levels of both scFv No. 10 and scFv No. 10<SUP>NR</SUP> than those of scFv (G<SUB>4</SUB>S)<SUB>3</SUB><SUP>R</SUP> and (G<SUB>4</SUB>S)<SUB>3</SUB> might be caused by difference in stability of the polypeptide linkers on the basis of amino acid sequences. Thus, the rare codon linkers tested in this study will be considerably useful for large-scale production of soluble and active scFvs in fed-batch or continuous fermentations, in which high cell activity can be maintained.</P>

Hard Anodized Aluminum with Excellent Heat Cracking Resistance

Naohiro Kumada,Ayumi Hongo,Takamitsu Tsujimoto,Hajime Okumura,Kenji Hara 한국표면공학회 2023 한국표면공학회 학술발표회 초록집 Vol.2023 No.11-1

A Study on the High Performance Ceramic Heat Exchanger for Ultra High Temperatures

Masaya Kumada,Yuji Himeji 대한기계학회 1998 Heat transter conference Vol.6 No.1

Development of scFv-immobilized polystyrene support for sensitive immunodiagnosis

Yoichi KUMADA 한국생물공학회 2012 한국생물공학회 학술대회 Vol.2012 No.9

Treatment of hepatitis C with direct-acting antiviral agents: Japanese experience

( Fumitaka Suzuki ),( Hiromitsu Kumada ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.-

In Japan, an estimated 100-150 million people are persistently infected with hepatitis C virus (HCV). The present standard-of-care (SOC) therapy for patients infected with HCV of genotype 1b, the most prevalent genotype in Japan, is peginterferon (PEG-IFN) combined with ribavirin (RBV) for 48 weeks. However, sustained virological response (SVR), judged by the loss of detectable HCV RNA from serum 24 weeks after the completion of therapy, can be achieved in only 42-52% of the patients. To cope with this grim situation, a number of direct acting antivirals (DAAs) have been designed and developed, represented by NS3/4A protease inhibitors and NS5B polymerase or NS5A inhibitors. Among them, telaprevir (TVR) has shown promising results, when combined with PEG-IFN and RBV, in the phase 2 and 3 clinical trials, by improving SVR to ~70% in patients infected with HCV-1. Moreover, the triple therapy with TVR, PEG-IFN, and RBV for 12 weeks, followed by PEG-IFN and RBV for an additional 12 weeks (T12PR24) were reimbursed since November 2011 in Japan. In Japanese clinical studies (phase III, T12PR24), the SVR rates of naive, relapse and non-responder were 73% (92/126), 88% (96/109) and 35% (11/32). We investigate the predictive factors of SVR to a 12-week or 24-week regimen of triple therapy in 81 Japanese patients infected with HCV genotype 1. SVR was achieved by 45% and 67% in the 12- and 24-week regimens, respectively. Multivariate analysis identified rs8099917 near the IL28B gene (genotype TT) and substitution at aa 70 (Arg70) as significant determinants of SVR. Prediction of response to therapy based on a combination of these factors had high sensitivity, specificity, and positive and negative predictive values. The efficacy of triple therapy was high in the patients with genotype TT, who accomplished SVR (85%), irrespective of substitution of core aa 70. In the patients having genotype non-TT, those of Arg70 gained high SVR (57%), and SVR (17%) was the worst in patients who possessed both genotype non-TT and Gln70 (His70). Important adverse events were anemia and shin disorder (mainly rash, drug eruptions, and erythema). Next, dual oral therapy with the NS5A inhibitor (daclatasvir) and NS3 protease inhibitor (asunaprevir) in HCV genotype 1b-infected null responders or patients ineligible or intolerant to peginterferon/ribavirin were done (phase II) in Japan. This study (AI447-017) enrolled two populations infected with HCV genotype 1, including null responders and patients intolerant or medically ineligible to PEG-IFN and RBV. The overall SVR rate was 77%. A higher SVR rate was observed in the null responders (91%) compared to the ineligible and intolerant patients (64%). The overall response to therapy was not influenced by IL28B genotype. Potential association between virologic failure and pre-existing NS5A polymorphisms and low plasma concentrations of asunaprevir and daclatasvir requires further study.

MACROLAYER FORMATION AND MECHANISMS OF NUCLEATE BOILING , CRITICAL HEAT FLUX AND TRANSITION BOILING

Hiroto Sakashita,Toshiaki Kumada 대한기계학회 1996 KSME/JSME THERMAL and FLUID Engineering Conference Vol.1 No.1

Influence of Synthetic Conditions on Iron Oxide Nanoparticles

Wenli Pei,H. Kumada,H. Saito,S. Ishio 한국자기학회 2007 한국자기학회 학술연구발표회 논문개요집 Vol.- No.-

Treatment of hepatitis C with direct-acting antiviral agents: Japanese experience

( Fumitaka Suzuki ),( Hiromitsu Kumada ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.1

In Japan, an estimated 100-150 million people are persistently infected with hepatitis C virus (HCV). The present standard-of-care (SOC) therapy for patients infected with HCV of genotype 1b, the most prevalent genotype in Japan, is peginterferon (PEG-IFN) combined with ribavirin (RBV) for 48 weeks. However, sustained virological response (SVR), judged by the loss of detectable HCV RNA from serum 24 weeks after the completion of therapy, can be achieved in only 42?52% of the patients. To cope with this grim situation, a number of direct acting antivirals (DAAs) have been designed and developed, represented by NS3/4A protease inhibitors and NS5B polymerase or NS5A inhibitors. Among them, telaprevir (TVR) has shown promising results, when combined with PEG-IFN and RBV, in the phase 2 and 3 clinical trials, by improving SVR to ~70% in patients infected with HCV-1. Moreover, the triple therapy with TVR, PEG-IFN, and RBV for 12 weeks, followed by PEG-IFN and RBV for an additional 12 weeks (T12PR24) were reimbursed since November 2011 in Japan. In Japanese clinical studies (phase III, T12PR24), the SVR rates of naive, relapse and non-responder were 73% (92/126), 88% (96/109) and 35% (11/32). We investigate the predictive factors of SVR to a 12-week or 24-week regimen of triple therapy in 81 Japanese patients infected with HCV genotype 1. SVR was achieved by 45% and 67% in the 12- and 24 week regimens, respectively. Multivariate analysis identified rs8099917 near the IL28B gene (genotype TT) and substitution at aa 70 (Arg70) as significant determinants of SVR. Prediction of response to therapy based on a combination of these factors had high sensitivity, specificity, and positive and negative predictive values. The efficacy of triple therapy was high in the patients with genotype TT, who accomplished SVR (85%), irrespective of substitution of core aa 70. In the patients having genotype non-TT, those of Arg70 gained high SVR (57%), and SVR (17%) was the worst in patients who possessed both genotype non-TT and Gln70 (His70). Important adverse events were anemia and shin disorder (mainly rash, drug eruptions, and erythema). Next, dual oral therapy with the NS5A inhibitor (daclatasvir) and NS3 protease inhibitor (asunaprevir) in HCV genotype 1b-infected null responders or patients ineligible or intolerant to peginterferon/ribavirin were done (phase II) in Japan. This study (AI447-017) enrolled two populations infected with HCV genotype 1, including null responders and patients intolerant or medically ineligible to PEG-IFN and RBV. The overall SVR rate was 77%. A higher SVR rate was observed in the null responders (91%) compared to the ineligible and intolerant patients (64%). The overall response to therapy was not influenced by IL28B genotype. Potential association between virologic failure and pre-existing NS5A polymorphisms and low plasma concentrations of asunaprevir and daclatasvir requires further study.

Safety and Efficacy of Elbasvir/Grazoprevir in Asian Participants with Hepatitis C Virus Genotypes 1 and 4 Infection

( Wei Lai ),( Hiromitsu Kumada ),( Ponni Perumalswami ),( Tawesak Tanwandee ),( Wendy Cheng ),( Jeong Heo ),( Pin Nan Cheng ),( Peggy Hwang ),( Sheng Mei Mu ),( Xu Min Zhao ),( Michael Robertson ),( B 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: Clinical experience with direct-acting antiviral treatments for hepatitis C virus (HCV) infection is emerging in the Asia-Pacific region. We conducted an integrated analysis of the safety and efficacy of elbasvir (EBR)/grazoprevir (GZR) in self-identified Asian participants who were enrolled in 11 EBR/GZR phase 2/3 studies. Methods: All participants received EBR/GZR 50 mg/100 mg alone for 12 weeks or in combination with ribavirin (RBV) for 16 weeks. The primary endpoint of all studies was sustained virologic response (HCV RNA < 15 IU/mL) 12 weeks after end of therapy (SVR12). Results: A total of 780 Asian participants with HCV GT1 or 4 infection were included (GT1b, n=715; GT1non-b, n=63; GT4, n=2). Most participants were enrolled from Japan (n=366, 46.9%), mainland China (n=146, 18.7%), Taiwan (n=109, 14.0%) and South Korea (n=90, 11.5%). Overall, 12.4% of participants had cirrhosis, and 20.4% were treatment-experienced. SVR12 was achieved by 756/780 (96.9%, 95% CI 95.5-98.0) of all Asian participants, including 748/772 (96.9%, 95% CI 95.4- 98.0) who received EBR/GZR for 12 weeks and 8/8 (100%, 95% CI 63.1-100.0) who received EBR/GZR + RBV for 16 weeks. The frequency of safety events among Asian participants was: any adverse event (AE), 58.1% (453/780), drug-related AEs, 23.6% (184/780), serious AEs, 2.6% (20/780), and discontinuation due to an AE, 0.9% (7/780). Fifteen participants (1.9%) had elevated ALT/AST levels that met the criteria for an event of clinical interest (ALT/AST >3× baseline and >100 U/L), 3 of whom discontinued treatment. The efficacy and safety profile of EBR/GZR was comparable to that observed among non-Asians. Conclusions: The combination of EBR/GZR was safe and highly effective in this large population of Asian participants with primarily HCV GT1b infection. Late transaminase elevations were reported in approximately 2% of participants, which is consistent with the safety profile of EBR/GZR in non-Asians.

A STUDY ON THE RELATIONSHIP BETWEEN THE THEMAL CONDUCTIVITY OF POLYURETHANE COMPOSITES AND THE ORIENTATION OF DISPERSED MICA FLAKES

Yukihiro Hamada,Masaya Kumada,Ryo Yasuda,Yo Aoyama,Fumiaki Takahashi,Kenji Yamauchi 대한기계학회 1996 KSME/JSME THERMAL and FLUID Engineering Conference Vol.2 No.1