Earticle

세계 의약산업은 연구개발 생산성 저하, 블럭버스터 품목의 특허 만료, 각국의 의료비 절감 방안 등으로 성장률이 점차 둔화되고 있다. 그러나, 중국을 비롯한 인도, 브라질, 러시아, 터키 등 신흥 시장의 부상과 바이오 의약품 시장의 약진 등으로 새로운 사업 기회도 펼쳐지고 있다. 한국의 의약산업은 세계 12 위 권이며 규모로는 세계 시장의 1.4%에 불과하다. 특허를 통한 사업 기회 보호가 그어느 산업보다 뛰어난 의약산업은 신약이라는 연구개발 성과가 주요 성공요소로 작용하고 있다. 그러나 신약개발은 성공확률이 낮고, 개발 기간이 길며, 개발 비용이 높아서 한국은 국내 시장 위주의 제네릭 의약산업에 치중하고 있는 실정이다. 최근 바이오시밀러를 비롯한 바이오의약 산업이 새로운 사업기회로 부상하고 있다. 바이오의약품은 합성신약의 제네릭에 비해 가격, 설비 및 기술적인 측면에서 상대적으로 높은 진입장벽을 유지하고 있어 일부 다국적 제약사들도 꾸준한 관심을 보이고 있다. 한국 의약 산업의 글로벌 경쟁력 확보에는 바이오시밀러를 포함하는 바이오의약 산업이 그 대안이 될 수 있다. 합성의약품에 비해 상대적으로 성공확률이 높을 뿐만 아니라 생산설비 등의 진입장벽이 있기 때문이다. 따라서 한국 바이오의약 산업의 현주소를 조명하고 최근의 연구개발성과를 살펴 봄으로써 발전 전략을 모색한다.

Formation of non-native protein conformers is inherent to the process of de novo protein synthesis. Non-native protein conformations are recognized as being not correctly folded and retained by the cellular quality control of protein folding to become eventually degraded, mainly by the ubiquitin-proteasome system. Under various forms of cellular stress and due to mutations, a surplus of misfolded glycoproteins in the endoplasmic reticulum (ER) results in ER stress. Such ER stress is commonly associated with a large spectrum of human disorders caused by protein misfolding and results in the up-regulation of the unfolded protein response (UPR). Up-regulation of UPR has severe general consequences for cells and organs and eventually leads to inflammation and apoptotic cell death. Depending on the type of the protein affected, its misfolding can result in a loss-of-function or in a pathological-gain-of-function protein folding disorder (Roth, 2002; Roth et al., 2008). Attempts have been made to rescue the folding defect in various human protein folding disorders by synthetic chaperones. Different types of synthetic chaperones have been tested for therapeutic purposes: pharmacological chaperones such as ligand analogues and competitive enzyme inhibitors, and synthetic chaperones such as glycerol, DMSO and 4-phenylbutyrate. Two specific examples will be discussed to demonstrate the potential of synthetic chaperones: (i) use of 1-deoxygalactonojirimycin, a competitive inhibitor of alpha-galactosidase A, the protein affected in Fabry disease (Yam et al. 2005, 2006), and (ii) the use of 4-phenylbutyrate in myocilin-caused open-angle glaucoma (Yam et al. 2007 a-c).

SMB(Simulated Moving Bed), a continuous chromatography technique, has been used broadly in chemical industries, including for petrochemicals, fine chemicals, pharmaceuticals. Recently, SMB has found active applications in bioindustry, mainly for value-added proteins, such as enzymes, monoclonal antibodies, insulin. It is due to its higher productivity and less consumption of mobile fluid comparing to the conventional batch chromatography in addition to the general benefits of chromatography of operating at ambient temperature and pressure. SMB, consisting of closed loop of multi chromatography columns, two inlets for feed and eluent streams, and two outlets for streams of separated products, can generate separation of high efficiency by counter-current flowing of mobile and stationary phases with proper operation of valves which connect columns, inlets and outlets. The potency of SMB has been exploited for the separation of products of biological origin, including organic acids such as L-lactic acid which was produced by the saccharification of paper sludge, and amino acids such as phenylalanine and tryptophan. For the last decade, SMB has been employed for the recovery of sugar alcohols, the separation of fructo-oligosaccharides, and the purification of omega-3 fatty acids such as EPA and DHA with the supports from several industries such as the Wrigley and the CJ Corp. Though being originally designed for binary separation, SMB has been modified for wider applications, such as 2-zone SMB/chromatography hybrid system for the center-cut separation, one-column SMB analogue retrofitting conventional batch chromatography, solvent gradient SMB, and thermally-assisted SMB. SMB operation of size exclusion chromatography was successfully adapted to the continuous refolding of lysozyme, recording first in the world. Currently, rare sugar separation of D-psicose and L-ribose and elimination of endotoxins from antibody products are under study using SMB.

신약개발의 변화가 2000년대 들어오면서 일어나고 있다. 2000년도 10대 약물중 9개가 저분자화합물 신약으로 최근까지도 신약하면 합성화합물에 의한 것으로 인식이 되어왔다. 그러나 눈부신 분자생물 학 및 바이오테크놀로지의 발전으로 바이오 의약 특히 재조합 단백질 및 항체의약이 눈부시게 발전하였다. 2008년 10대 약물의 절반이 이들 바이오의약품이며, 더욱 중요한 것은 미래 2014년에는 10개중 7개가 바이오 의약품이며, 상위 1-6위를 바이오의약품 특히 항체가 포진하게 된다는 예측이다. 이러한 변화에 따라 많은 글로벌 제약사들은 바이오 신약개발을 위한 투자를 대대적으로 시작하였으며, 이 들 바이오 신약들이 미래 제약산업의 성장동력으로 기여하게 될것이다. 본 세미나에서는 VEGF 에 대한 완전인간항체 발굴 및 최근TWO-IN-ONE 항체에 대한 소개를 할것이다.

생명(연)은 “2016년 세계적 바이오 기술혁신을 선도하는 전문연구원”이라는 비전을 설정하고 기술 경쟁력 확보 및 기술사업화 성과창출을 연구소의 최우선 과제로 선정하고 2011년까지 원천특허 2개, 누적 기술료수입 200억원 등의 기술사업화 성과창출 목표를 설정하였다. 2003년 성과확산팀을 신설, 2005년부터 성과확산실로 전담조직을 확대 발전시켰으며, 변리사채용, BT분야 연구 및 연구원 창업 경영 노하우가 풍부한 박사급 인력을 전담부서장으로 등용하는 등 기술사업화 전담조직 및 인적역량을 대폭 강화하였다. 맞춤형 특허․기술사업화 교육을 시행하고, R&D 중복투자, 특허분쟁 예방을 위하여 출연(연) 최초로 특허맵(Patent Map)을 도입․운영하고 있다. 2009년부터는 “강한 특허 만들기 운동”을 전사적으로 추진하고 있으며, 특허 출원 시부터 원내 전담 변리사의 Lab밀착 컨설팅, BT 전담 특허사무소 8社 선정 및 연구자 1인 1변리사 갖기운동으로 전담변리사 활용을 극대화하였으며, 매주 목요일 특허상담의 날을 지정․운영하고 있다. 기술사업화 전략에 힘입어 최근 양적․질적으로 우수한 기술사업화 성과를 창출하고 있다. 특허출원의 경우 2006년193건, 2007년 249건, 2008년 266건으로 매년 30% 이상 획기적으로 증가하고 있으며, 특허등록의 경우 매년 150여건의 특허를 국내외에 등록하고 있다. 또한 기술이전․기술료의 경우 최근 3년 동안 약 290%가 증가하는 초고성장세에 있으며, 2009년도에는 400억원의 기술이전계약을 성과를 이룰 것으로 예상되고 있다.

고부가가치 의약용 단백질 수요의 급격한 증가로 인하여 효율적인 발현시스템 개발 필요성이 더욱 증대되고 있다. 메탄올자화 효모 한세눌라 폴리모파는 다양한 발현 도구 활용과 고농도 배양이 가능하기 때문에 유망한 재조합 단백질 발현숙주로 주목 받고 있다. 실제 한세눌라 폴리모파를 이용하여 생산된 B형 간염백신, 히루딘, 인슐린 등이 성공적으로 시장에 진입함으로써 이미 바이오시밀러 생산시스템으로서 경제성이 입증되었다. 본 연구팀은 지난 십 수년간 한세눌라 폴리모파를 보다 효율적인 의약용 단백질 생산 시스템으로 개발하기 위한 세포 재설계 연구를 수행해 오고 있다. 강력한 발현 프로모터나 다중병렬 도입벡터 등 기반 분자생물학 도구 개발은 물론 한세눌라 폴리모파를 이용하여 의약용 단백질을 보다 고품질로 생산하기 위한 플랫폼 기술개발에 주력해 오고 있다. 일례로 각종 단백질 분해효소 유 전자가 제거된 한세눌라 폴리모파 균주를 개발하여 단백질분해 효소에 의한 부적절한 절단없이 고효율로 재조합 단백질을 분비 생산하는 기술을 개발하였다. 또한 의료용 단백질의 60% 이상을 차지하는 당단백질을 생산하기 위하여 효모 특이적인 당사슬 생합성경로 유전자를 제거하거나 당사슬수식효소 유전자를 도입하여 인체에 적합한 당사슬이 부가된 단백질을 생산하는 숙주를 개발하였다. 한편 최근 독자적으로 해독한 한세눌라 폴리모파의 유전체 정보를 바탕으로 전사체, 단백질체 등 각종 기능유전체 연구방법을 도입하여 재조합 단백질 발현, 수식 및 관련 신호전달 네트워크 등을 세포 수준에서 분석하고 리모델링 하기 위한 연구를 수행하고 있다.

The technology for the production of recombinant proteins has been recognized as an essential field not only for the biopharmaceuticals but also for the functional genomics. In vast quantities of genomic information, numerous uncovered proteins with values for human drugs and industrial enzymes are waiting for their characterizations and proper applications. Unfortunately, however, about 20-30% of tested proteins could be produced under the current expression systems. Thus, post-genomic biology requires novel expression strategies and improved throughput for the breakthrough of the current limitations. A GRAS yeast Saccharomyces cerevisiae has an excellent protein secretion pathway and post-translational modification function necessary for higher eukaryotic proteins. During the last two decades, S. cerevisiae has been frequently chosen for the secretory production of numerous proteins. But the yield of target proteins was generally low and unpredictable. In fact, there has been limited number of vectors available with secretion signals from several known proteins, such as mating factor alpha, invertase, and acid phosphatase etc. Such could not completely support the secretion of numerous target proteins having different physicochemical properties. For the improvement of the yeast secretion systems, more signals from different types of secretory protein and screening methods of an optimal signal from the pool of secretion signals for each target protein, are required. There are about 600 yeast ORFs among 6,000 encoding secretory proteins which pass the ER and having different secretion signals. To utilize them all for the secretion of numerous target proteins, genome-wide TFP (translational fusion partner) library containing different signal sequences have been constructed. Simple insertion of target gene into TFP library vectors and a positive selection on media could find an optimal fusion partner in a high throughput manner. The technology could rapidly screen a TFP capable of inducing hyper-secretion of target proteins, especially proteins that are difficult to produce using conventional recombinant production ethods. The platform technology will be useful for the production of massive recombinant proteins of bio-industry and basic research in the post-genome era. Several industrial applications of recombinant proteins produced up to grams per liter, especially for bioenergy field will be presented.

What takes priority in technology commercialization is to select the best technology that has high commercialization potential. The total number of patents that Korea Research Institute of Bioscience & Biotechnology(KRIBB) has filed is 957, and among these large number of patents, it is nearly impossible to find the superior technology that qualifies both commercialization potential and transfer condition. Markpro has developed online marketing software called eTTMS(easy Technology Transfer Management System). It is a program that searches technology demanding companies and automatically matches available technology in response to their state of need. Because eTTMS focuses on the perspective of the demanding companies or client, it is also called as "Client-dependant technology matching", and is a way to find a technology that has best commercialization potential. It first searches for demanding companies, classifies them into passive and active categories, analyzes and suggests business model of the company, completes the portfolio of related technologies from KRIBB, then offers these total proposal to the selected demanding company. Another way to find a technology is called "Technologydependant matching". This process increases accuracy when finding the most appropriate technology out of large number of related technology. With the software that automatically evaluate the technology, technology transaction experts can analyze and evaluate the market feasibility, and commercialization potential. Both Client-dependant technology matching and technology-dependant matching methods assist experts to find the technology that has the highest commercialization potential, and it also helps experts to come up with the performances even faster. Moreover, by continually managing the technology and demanding companies, it could promote technology commercialization and technology exchange from industry, academia, and institute.

Currently, a bulk amount of raw glycerol is formed as the main by-product during biodiesel production, which is as high as 10% (w/w) of biodiesel generated. The surplus of raw glycerol has not only greatly disturbed the market of traditional glycerol in the preparation and price, but also brought a significant environmental problem since it cannot be discharged direct into the environment without any treatment. As a result of these issues, a large research effort has been devoted to developing methods to refine glycerol as a low-cost feedstock into industrially valuable materials such as fuels, building blocks, and bioactive substances. 1,3-Propanediol (1,3-PD) is a valuable chemical that is used mainly for the synthesis of polymethylene terephalates by polymerization with terephthalates. Use of this polymer in the manufacturing of textile fiber, film, plastic, etc. is rapidly expanding. Klebsiella pneumoniae is a typical microbial strain capable of producing 1,3-PD, in which the metabolic pathway responsible for the production of 1,3-PD has been well studied. The microbial production of 1,3-PD by K. pneumoniae involves the formation of various by-products. 2,3-Butanediol, one of the main by-products, may serve as an obstacle for obtaining a high purity of 1,3-PD in downstream processes because of its similar boiling point. To eliminate the by-products synthesis, glycerol metabolic pathway was engineered by deleting from the chromosomal DNA the genes involved in the synthesis of by-products. The by-product formation except for acetate was eliminated in the resultant strains. Additionally, 3-hydroxypropionic acid (3-HP) that is one of the most interest among alternative chemicals derived from biomass as precursor of acryl derivatives, could be produced from glycerol by the native and engineered strains of K. pneumoniae. The recombinant strains would be valuable as a platform strain for development of industrial process of production of 1,3-PD or 3-HP from glycerol.

We describe simultaneous assessments of bacterial phenotypes by using an integrated microfluidic system. The microfluidic system enables creating a series of different growth conditions, preserving growth media inside nanoliter reactors, and cultivating multiple bacteria with parallel nanoliter reactors. From a single on chip experiment, we assessed the effect of carbon source compositions on the bacterial phenotypes, i.e. the pattern of growth curves, with a wild Escherichia coli K-12 and lacZ knock-out mutant, as a model bacteria system. Diauxie growth curve, which is the stepwise response of bacteria to mixed carbon sources in a medium, was observed only from wild E. coli K-12. This study provides a new procedure for systematic analyses of phenotypes on effectors from single genotypes or multiple genotypes for microbial technology.

A novel disposable bioreactor system utilizing a pneumatic mixing technology has been developed recently (PBS Biotech, Inc.). Since this technology does not require any external mechanical device for liquid agitation, it can offer a simple, compact, and scalable bioreactor system. Cell culture performance in larger scale units up to 250 L was studied and compared to that of a 300 L stainless steel bioreactor. In addition, 2 L units of this bioreactor system were evaluated to be representative scale-down models that can be used for process development and characterization while maintaining the same mixing mechanism. In this presentation, the new features of this novel disposable bioreactor will be introduced and then the results from biological tests involving CHO cell lines expressing recombinant monoclonal antibodies will be discussed.

Extensive genetic modifications have been made to improve the performance of the bioreactor systems. Although metabolic engineering has been paid much attention with some success in practice, the pathway modifications based on the specific genes knockouts and/or amplifications by plasmids do not necessarily lead to the significant improvement in the cell growth and/or the specific metabolite production without profound insight into the metabolic regulation. Although much information is available on genetic regulation, biochemistry, and physiology of cellular metabolism, we are still far from understanding its overall regulation mechanism. It is strongly desired that different levels of (omics) information be integrated by comparison of the data for different mutants and/or the data obtained at different culture environments with the aid of systems biology approach. Among the different levels of information, the metabolic flux distribution is located on top of them, and it is quite important for understanding the whole cell metabolism in practice. We have, so far, established the 13C-metabolic flux analysis method for the continuous culture using NMR, GC-MS, and/further extended to the batch culture using CETOF/ MS. It was found that the cell metabolism significantly changes with respect to time for specific gene knockout mutants as well as the wild type based on 13C-metabolic flux analysis. We have also attempted to compare the flux data with other levels of information. We have also investigated the effects of different carbon sources, nitrogen levels, phosphate levels, temperature, and pH on the cell metabolism in view of global regulators and metabolic pathway genes. Moreover, we are now developing a model which can simulate the metabolic changes due to specific gene knockout. The goal of our research is to understand the whole cell metabolism, and to realize its mechanism into the computer for the systematic cell design of E.coli for the production of next generation bioenergy.

Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anti-cancer virotherapy. To enhance the NDV vaccine productivity, chicken fibroblast, DF-1 cell, was genetically engineered and rapid virus quantification method was developed. V protein of NDV is well known for its interferon antagonistic activity. The addition of anti-interferon antibody increased NDV virus productivity in cell-based production system. Stable cell lines expressing either lentogenic or velogenic NDV V proteins were established. The overexpression of either lentogenic or velogenic V proteins had an effect of enhancing NDV production but did not increase the productivity of Japanese encephalitis virus (JEV). Our results indicate that NDV V protein have a function of interferon antagonist through inhibiting the increase of type-I interferons rather than incorporating virus particles as a structural protein or degrading STAT proteins. The interferon signals in NDV V-expressing cell lines were weakened by decreased activation or expression of dsRNA-activated enzymes, PKR and OAS. Interferon-antagonist activity seems to help rapid virus replication in the early phase of virus production. A method for rapid detection and quantification of NDV was developed. A SYBR Green I-based real-time RT-PCR was designed using a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection, limit of quantification, and robustness. The validation results satisfied the pre-determined acceptance criteria. The validated method was used to quantify virus samples produced in animal cell culture-based production system. Our results demonstrated that the real-time RT-PCR method is suitable for the rapid detection and quantification of whole virus particles irrespective of viral infectivity.

PAC (Process Analytical Chemistry)는 지난 수십년간 각종 산업에서 활용되고있었으며 이를 위하여는 분석기술, 공정기술, chemometrics등의 다자학문의 이해가 필요하다. 이 기술이 다시 각광 받고 있는것은미국 식품의약품안정철(FDA, Food and Drug Administration)에서 2002년 정식으로 발의하고, 2004년 9월에 가이드라인을 발표한 PAT(Process Analytical Technology) 때문이다. FDA에서 정의한 내용을 살펴 보면, "PAT는 원료나 공정 중의 물질을 실시간으로 분석하고, 분석한 결과에 따라 공정을 조절할 수 있는 시스템"을 의미한다. 이러한 시스템은 최적의 품질을 갖춘 의약품을 생산할 수 있도록, 품질에 영향을 미칠 수 있는 각공정의 단계, 단계를 충분히 이해하여, 품질로서 공정을 디자인하고 조절할 수 있도록 한다. 이러한 이유로 많은 다국적 제약회사가 이 기술을 공정에 도입하여 완제품의 불량률과 공정개선등의 효과를 얻었으며 현재 일본 제약회사의 대다수가 이 기술을 도입중이다. 국내인 경우 제약산업의 영세성으로 인하여 이기술의 도입이 아주 미미하며 오히려 농업, 식품, 화학, 반도체등의 많은 분야에서 이 기술을 도입하여 공정개선, 원가절감, 수율향상등 제품의 경쟁력을 향상시키는데 많은 기여를 하고 있다. PAT를 가능하게 하는 분석 기술은 다음과 같은 특징이 요구된다. 먼저, 시료를 파괴하지 않고 분석이 가능해야 하고, 공정중에서 실제 분석을 해야 하므로, 일반적으로 분석에서 수행하는 시료 전처리 과정도 거치지 않아야 한다.그리고, 분석 결과는 매우 빠른 시간에 도출될 수 있어야 한다. 이러한 특징을 갖춘 분석 시스템은 모두PAT에 적용될 수 있고, 현재 많이 사용되고 있는 방법이 근적외분광분석법(Near Infrared Spectroscopy), 적외분광분석법, 자외분광분석법, Raman 분광분석법, RT 등을 사용된다. 또한 측정된 데이터들을 해석하기위하여는 다양한 스펙트럼 전처리기술과 ulticalibration 기술인 MLR(Multiple linear Regression, PLS (Partial Least Square Regression)등의 chemometric 기술이 필수적이다. 이러한 비파괴 실시간 분석 기술이 bioprocess 공정에 어떻게 활용될 수 있는지에 대한 사례와 현재까지 관련 식품, 제약분야에서 적용된 사례등을 중심으로 의 응용사례와 이의 국내 적용가능성에 대하여 발표하고자한다.

We are going to present our research achievements on whole-cell biocatalyst-based oxidative biotransformation with oxygenation of cyclohexanone into ε-caprolactone as a model reaction. Cyclohexanone was efficiently converted into ε-caprolactone by recombinant Escherichia coli expressing the chnB gene encoding cyclohexanone monooxygenase of Acinetobacter calcoaceticus NCIMB9871. ε-Caprolactone was produced to a concentration of 15.3 g/L during fed-batch culture based biotransformation (1). The biotransformation efficiency was significantly improved by using recombinant Corynebacterium glutamicum expressing the same gene. The biocatalyst allowed product accumulated over 22g/L at the reaction conditions similar to those of recombinant E. coli-based process. The higher product concentration and thus higher productivity seemed to be driven by higher solvent tolerance and cofactor regeneration capacity of C. glutamicum.

Algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. Recent development in various algal biotechnology found microalgal mass culture can be a useful solution in treatment of wastewater, fixation of carbon dioxide and production of biofuel. Furthermore, the fact that microalgae can conquer the top three environmental problems in a simultaneous manner is even more exciting. (i) Green house gases such as CO2 in the atmosphere became a serious problem for the whole mankind. One of the possible solutions is a biological CO2 fixation using the photosynthesis of microalgae. (ii) Microalgae have been utilized for several decades for the treatment of municipal and other wastewaters. The removal of heavy metals and/or residual nutrients, specifically N and P, has also been studied with a variety of processes. (iii) Renewable, carbon neutral, liquid fuels for the transportation sector are necessary for environmental and economic sustainability. Approaches for making microalgal biodiesel economically competitive with petrodiesel would be possible in the near future. In this presentation, various photobioreactors and culturing facilities will be presented and analyzed.

바이오촉매란 체내의 갖가지 화학반응이 일어날 때 촉매역할을 하는 물질을 의미하며, 효소가 가장 대표적인 바이오촉매이다. 이외에 특정 효소가 포함된 미생물, 동물세포, 식물세포 등도 촉매로 이용되고 있다. 최근까지 바이오촉매 반응은 상온/상압의 온화한 조건과 수용액 상태에서 일어나는 자연반응을 이용하는 것이 거의 대부분이었으나, 1980년대에 들어서 기존의 바이오촉매 반응의 개념을 벗어난 인위적이고 비자연적인 상태에서의 바이오촉매 반응연구가 활발히 이루어지게 되었다. 최근 들어 효소를 분자촉매의 하나로서 적극적으로 중합촉매로 활용하여 고분자합성분야에 적용한 연구가 교토대학의Shiro Kobayashi 그룹과 미국 Polytechnic Univ.의 Richard A. Gross 그룹에서 체계적으로 진행되어 왔다. 여기에서 효소촉매중합(Enzymatic Polymerization)은 효소를 촉매로 사용한 ｢비 생합성 경로에 의한 in vitro 중합반응｣을 말한다. 근래에는 전자재료용 고분자재료 개발에 있어서 정밀 구조제어의 필요성이 높아지고 있고, 이들에 대응할 수 있는 고분자 합성의 새로운 수법으로써 효소를 활용한 고분자의 중합이 주목되고 있다. 많은 경우에 종래의 합성법으로는 합성이 곤란한 폴리머가 in vitro에서의 효소촉매 작용에 의해 합성되고 있다. 또한, 효소촉매중합은 온화한 조건 하에서의 높은 촉매활성으로 인한 에너지 절약형 특성, 독성이 높은 금속촉매의 비사용에 의한 효소 반응계의 무독성, 많은 경우 천연자원을 출발물질로 활용하는 경우가 많아 생성된 고분자가 생분해성 등 지구환경에 친화적인 고분자 소재의 특성뿐만 아니라 효소를 활용한 중합은 자연계의 물질순환계에서 얻어지기 때문에 고분자 중합에 있어서 그린촉매화학으로서도 기대가 된다. 본 강의에서는 효소 생촉매를 이용하여 바이오매스로부터 유래한 여러 가지 모노머를 이용하여 바이오플라스틱을 합성하는 사례를 소개하고, 이러한 효소 촉매를 이용할 경우의 장단점에 관하여 소개한다.

The purpose of this lecture is to overview the current cellular products and their manufacturing process. Recently, stem cell therapies are considered to be a promising platform for cell and gene therapy for a variety of diseases. However, maintenance of the multipotency and self-renewal expansion of stem cells in vitro are the most difficult operations and essential factors for the clinical application of the stem cells into various patients. Unlike the production of biopharmaceuticals, the cultivation process of stem cells is relevant to the design of bioreactors. Because final products are not proteins but stem cells, accurate characterization of the final products should be performed and also regulatory systems of the cultivation should be developed. After all, novel bioreactors will be needed for the consistent production of stem cells with multidisciplinary systems.

Hepatitis B virus (HBV) is one of the main pathogens of hepatitis and hepatocarcinoma. Human plasma-derived Hepatitis B immune globulin (HBIG) is being used for prophylactic and liver transplantation currently. However, it is required to replace a HBIG with a recombinant one because of limited availability of human plasma with high anti-HBs antibody titer and possible contamination of human pathogens. In order to meet this requirements, we selected human antibody to the HBsAg from an anti-HBs enriched phage-display library and a Chinese hamster ovary (CHO) cell line, HB-C7A, was established which produces a fully human anti-HBs IgG1 antibody. Mass production and purification processes were established and this mAb was characterized in terms of HBV neutralization and safety for the purpose of human trial for liver transplantation and chronic hepatitis B treatment. Phase I clinical trial of this mAb for healthy volunteers was completed without any serious adverse events and current stage is in clinical phase II/III for liver transplantation in Korea. This HB-C7A mAb has several advantages compared to plasma-derived Hepabig® such as activity, safety and availability.

Transgenic livestock animals such as goat and sleep have been generated for the production of recombinant protein in the milk. Chicken has also attracted great attention as an alternative animal for establishing so called transgenic bioreactor, since they have relatively short breeding time for sexual maturation. We previously reported production of erythropoietin (1) and a single chain immunoglobulin (2) in egg white of genetically manipulated chicken using a retroviral vector. However, we found that terminal sialic acid and galactose were barely detected in N-linked glucan of these protein deposited in egg white, which may considerably reduce the biological activity of these biopharmaceuticals. In order to overcome this drawback in chicken transgenic technology, we firstly analyzed galactosyltransferase in the chicken organs, and found that the expression of a major galactosyltransferase (GalT1) was low in magunum portion of the oviduct where majority of egg-white proteins are produced. Therefore, GalT1 gene was introduced to a transgenic chicken producing the single-chain immunoglobulin, and the expression of GalT1 in the golgi fraction of magnum cells was observed. It was also confirmed that the antibody deposited to the egg white of the GalT1-introduced chicken was heavily galactosylated. These results indicate that N-linked glucan of recombinant proteins produced by chicken can be modified by gene manipulation.

The protein therapeutics are mostly parenteral formulations due to the characteristics of protein with a low stability or fragility. It results the frequent injection of medication to treat the patients for long period. To satisfy the unmet medical need of patients such as reducing the number of injection or changing the route of administration, the new formulations of products are developed such as a long-acting form of product utilizing DDS(Drug Delivery System) technique. The recent achievement of DDS techniques derives the products available to satisfy the unmet medical needs of less frequent injection, emerging as a new technologyf protein therapeutics development by adding a higher value on the patent-off biopharmaceuticals. There are few cases of microparticle formulations used as long-acting or slow releasing protein or peptide therapeutics developed using biodegradable and biocompatible polymers as matrix material. Depending on the action mechanisms of targeted therapeutic proteins, the products are to be designed by control of biodegradation rate with selection of polymers in terms of molecular weight, the ratio of mixing ingredients, and hydrophilic property. The achievable technique of long-acting or slow releasing microparticle formulation for development of protein or peptide therapeutics is limited so far. The desire and expectation for a better hGH(human growth hormone) product has driven a number of pharmaceutical companies to develop SR-hGH (sustained-release hGH) products. LB03002 is the biocompatible hyaluronic acid-based formulation with promising features such as, complete release of hGH, structural integrity/activity of hGH, easy administration with 26 gauge needle, and no significant injection site reaction. Moreover, the clinical data obtained at present demonstrate the bioavailability, efficacy and safety of LB03002 comparable to those of daily injection products. The efficacy and safety of LB03002 administered once-aweek in adults with GHD(growth hormone deficiency) were evaluated in a phase III clinical study, completed recently. The clinical study results will be presented to discuss how well be designed based on its action mechanism.

TNFα is a important mediator of inflammatory diseases including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn’s disease, psoriatic arthritis, and ankylosing spondylitis. In recent years, the biologics that inhibit the activity of the TNFα have become a major target for drug developers and it has been recognized that successful treatment strategies for RA. We produced and characterized a murine monoclonal antibody (mAb) TSK114 against human TNFα, and further developed the humanized mAb YHB1411-2 by complementary-determining region (CDR) grafting procedure. The neutralizing activity of YHB1411-2 to inhibit human TNFα-induced cytotoxicity in vitro was tested using WEHI cell based assay system. The neutralizing activity of YHB1411-2 showed 2~3 fold higher than that infliximab and adalimumab. The TNFα neutralizing activity of YHB1411-2 in vivo was evaluated using galactosamine-sensitized mouse model. YHB1411-2 demonstrated a dose-dependent inhibition of mortality and a more potency than infliximab on the same dosage. Epitope mapping was conducted by yeast display technology and site directed mutagenesis. YHB1411-2 has two predominant binding regions located on the apical bottom and top of the subunit surfaces of trimeric TNFα. The therapeutic efficacy of YHB1411-2 was assessed by using transgenic mouse polyarthritis model. In the results, YHB1411-2 dose-dependently prevented the progress of polyarthritis. Especially, the severity of arthritis in Tg mice treated with YHB1411-2 was significantly reduced in comparison with infliximab treated Tg mice at the same dosage in the histopathological evaluation. In summary, our study showed that YHB1411-2 has higher TNFα-inhibitory activity in vitro and in vivo than other TNFα blockers.

Thiazolidinedione insulin sensitizing agent is oral diabetic medicine of glitazone class with a novel mechanism showing an activity of improving insulin resistance, notably different from previous drugs that stimulate insulin secretion such as sulfonylurea or biguanide and α-glucosidase inhibitor. The mechanism of action of such glitazone class is to boost the response for insulin in the target tissues of insulin. The molecular target of glitazone class was reported to be the PPARγ and it elicits its pharmacological action by binding and activating nuclear receptor PPARγ. CKD Research Institute developed lobeglitazone featuring greater hypoglycemic activity than rosiglitazone and strengthened lipid lowering activity that tends to be pointed out as a weakness for PPARγ–acting drugs. Lobeglitazone is evaluated as a phramacokinetically appropriate drug in terms of high bioavailability and the availability of once a day administration regimen to enhance the convenience of patients, which represent necessary features for oral medications. Phase II clinical study of lobeglitazone showed efficacy and safety in human diabetic patients and it was commenced to Phase III clinical study recently.

There can be no doubt that oncology drug development is entering a critical phase. This phase is driven by the ever expanding knowledge of genomics and proteomics, breakthrough advances in drug discovery/drug design, and new in vitro and in vivo testing methodologies that allow drugs faster entry into clinical trials. The industry is on the brink of introducing a range of targeted therapeutics that promise to revolutionise cancer treatment. However, developing Oncology products is full of major challenges that need to be met head on in order to bring safe and effective treatments to market. Kinases are perhaps the most druggable class of targets, and yet researchers have only begun to scratch the surface. The ubiquity of kinases as potential targets means that eventually they will be exploited for virtually every human disease, particularly diabetes, inflammatory disorders, and especially cancer. The success of Novartis' Gleevec, Roche's Herceptin, AsraZeneca’s Iressa and OSI’s Tarceva has demonstrated that kinase inhibitors for cancer that are effective and well-tolerated. However, these successful drugs are just the top of an emerging line of therapy. Nonetheless, challenges remain, including: ensuring target relevance and specificity, overcoming resistance. There are today more than 245 protein kinase inhibitors for the treatment of cancer, from early preclinical to marketed drugs. Developmental stage contains more than 245 PKI drugs in development or more than 450 clinical projects for cancer: > 25 projects in Phase III > 160 projects in Phase II > 110 projects in Phase I > 180 projects in Preclinical The Oncology market is currently valued at $34 billion/year and is set to top $55 billion in 2009. It is no surprise that big pharma is investing up to 20% of its annual R&D budget on the development of cancer treatment. Given the fact that they all target kinases and that kinase mutations can cause patients to become resistant to these drugs, there is plenty of room for more competition. Since virtually all the big pharmas have kinase R&D programs ongoing, we need to stay on top of the R&D and commercial developments in the area of kinase therapeutics.

Drug molecules usually bind to target proteins controlling their functions to exert pharmacological effects. The binding between a drug molecule and a protein is initiated by mutual recognition of their three-dimensional (3D) structures which implicate physicochemical properties. Thus, exploring the structural aspects of the recognition is important for understanding drug action mechanisms. Moreover, it enables us to discover new chemicals that fit the active site and mediate the function of the target protein. Such chemicals become drug candidates if they demonstrate proper pharmacokinetics and pharmacological effects without significant toxicity events. Studies for the recognition consist of determination and analysis of the 3D structures of target proteins with bound ligands, more importantly drug molecules. In detail, most 3D structures of iso-type proteins in a family, if not all, have been collected and/or determined for structural analysis. Complex structures have been compared to identify main structural features such as non-bonding interactions, pocket structures, and conformational changes between the proteins and the chemicals in their binding sites. The purpose of this study is conceptually different from that of the “structural proteomics” although we use the same research tools as it does. The structural proteomics generally identifies molecular functions of proteins through the determination of entire protein structures and the comparison of their functional domains. On the other hand, our approach takes advantage of the 3D aspects of the recognition between proteins and ligands, and applies them to drug discovery, being, therefore, named “structural chemoproteomics”. During last several years, CrystalGenomics has applied this method for the discovery of various drug candidates. Specifically we focused on proteins in the epigenetics and signal transductions. They include families of histone deactylases (HDACs), histone methyl transferases (HMTs) and kinases. In this presentation, we would like to introduce the ethodology of structural chemoproteomics based drug discovery and discuss recent results obtained from their applications for the discovery of molecular targeted anticancer drug candidates.

The oxazolidinones represent a novel chemical class of synthetic antimicrobial agents. DA-7218 (re-coded as TR-701 by Trius Therapeutics after in-licensing) is a second generation oxazolidinone antibacterial prodrug. It is a potent inhibitor of Gram-positive bacterial pathogens such as methicillin-resistant Staphylococccus aureus, penicillin-resistant Streptococcus pneumoniae and vancomycin-resistant Enterococcus spp. DA-7218 is also active against pathogens that have developed resistance to first generation oxazolidinones such as linezolid-resistant S. aureus and linezolid-resistant Enterococcus spp. With a long half-life, high bioavailability and solubility and high potency, DA-7218 will provide clinicians with flexible dosing and a possible shorter course of therapy thereby enabling the early transition of hospitalized patients from IV to oral treatment and subsequent early discharge from the hospital. DA-7218 successfully completed Phase I and Phase II clinical trials in the US. In a recent Phase II clinical trail for treatment of complicated skin and skin structure infections (cSSSI) caused by Gram-positive bacteria, DA-7218 was well tolerated and achieved high clinical cure rates in all doses evaluated. In this presentation, overall progress of the development of DA-7218 will be discussed.

가장 첨단 산업인 의약산업에서 기술선진국이 되기 위하여서는 의약기술이 상업화되는 과정에서의 독특한 점을 이해하여야 할 것이다. 의약시장은 소수의 global 기업에 의해 독점되어 있으며 이런 독점을 뚫고 시장진입을 하기 위하여서는 신약, 개량신약, generic의 독점권에 대한 이해가 필요하며, 특히 Biopharmaceutical의 비중이 커지고 있는 이때 Biosimilar의 상업화 전략도 중요해지고 있다.

In this presentation, my recent years' interaction with KSBB colleagues will be briefly summarized. Succeeding to my last year's presentation at KSBB spring meeting about biotechnology of medicinal higher fungi, our new progresses in purification of ganoderic acids (antitumor compound) from Ganoderma lucidum and their bioactivity study will be mentioned. Comparative study on individual ganoderic acids (GAs) production and identification of differentially expressed genes between liquid static and shaking cultures of G. lucidum will be mainly demonstrated. The content of individual GAs was higher in liquid static culture than that in conventional shaking culture and the higher production was related to increased transcription of biosynthetic genes. A total of 147 up-regulated ESTs were identified in hyper-production of GA conditions and this study provided interesting information on possible genes functions related to cell differentiation, GA biosynthesis and its regulation. In addition, enhancement of GAs production and biosynthetic gene expression was achieved by addition of phenobarbital in liquid static cultures of G. lucidum. The demonstrated molecular biochemical engineering approach will be useful to the industrialization of the antitumor compound production process.