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Applications of protein engineering in pharmaceutical industry
Synthetic Biology Journal
2025, 6 (1):
65-86.
DOI: 10.12211/2096-8280.2024-061
Protein engineering performs specific designs and modifications on proteins through directed evolution, semi-rational or rational design, computer-assisted design, and so on. The engineered proteins, with improved properties, have significant applications in food, medicine, fuel, and material industries. For the chemical and pharmaceutical industry, engineered enzymes can serve as efficient biocatalysts for the synthesis of active pharmaceutical ingredients (API) and their intermediates, aligning with the concepts and principles of green chemistry and manufacturing. For the biopharmaceutical industry, the engineering of peptide or protein modifying enzymes can boost the efficiency in preparing drug candidates, while engineered diagnostic enzymes can make detection more accurate and sensitive. Moreover, protein engineering can improve the bioactivities of biological drugs such as therapeutic enzymes and antibodies, increase stability, and mitigate immunogenic response for their safety and efficacy. Here, we review the tremendous progress in protein engineering, elucidate its importance in the research and development of chemically derived drugs and biologics, and provide examples of its applications. These examples encompass the discovery of enzymes or antibodies, the process of protein engineering, and the subsequent economic advantages. We aim to showcase the practical implementation of protein engineering in the pharmaceutical industry and facilitate technology transfer, thereby fostering seamless integration between research, development, and industrial production. Furthermore, we discuss challenges such as cost-effectiveness and market changes in the synthesis of API, and multi-target optimization, long cycle and high risk in the discovery and development of biopharmaceuticals. Finally, we look forward to the prospects of protein engineering in pharmaceutical industry. In the future, automated pipelines consisting artificial intelligence and self-driving laboratories will accelerate the design-build-test-learn cycle, leading to rapid progress in molecular design and discovery.
Fig. 1
Optimization of the manufacturing process of sitagliptin and the directed evolution of ATA-117
Extracts from the Article
基于绿色化学和原子经济性的原则，西格列汀的化学合成工艺经过了多次优化改良，从用手性辅基控制引入手性中心到通过不对称的催化氢化引入手性［47-50］。其中，采用以铑盐为手性催化剂的第二代化学合成路线［51］生产西格列汀减少了合成步骤，提高了对映体过剩率（enantiomeric excess， ee），减少了80%以上的废弃物，并完全消除了工业废水［50］（图1）。Merck公司的西格列汀化学工艺团队因此荣获美国环境保护署（United States Environmental Protection Agency，EPA）颁发的“总统绿色化学挑战：2006年更绿色合成途径奖”［52］。然而，该工艺仍然存在一定的缺陷，反应需要高压设备，而且铑作为一种极为罕见的金属，稀缺不可再生，价格波动大。因此，Merck公司和Codexis公司借助于CodeEvolver?平台［53］合作开发了通过转氨酶引入手性氨基的生物催化路线［54］。
转氨酶（transaminases，EC 2.6.1）催化氨基从胺供体到酮受体的可逆转移，产生手性胺［55］或氨基酸［56-57］。转氨酶都需要相同的辅酶，5'-磷酸吡哆醛（pyridoxal 5′-phosphate， PLP），所有的转氨酶都催化同一类型的反应，但具有不同的底物特异性，大部分转氨酶仅能催化酮羰基的邻位取代基比甲基更小的底物。研究团队测试了各种商业化的转氨酶，没有酶对西格列汀酮（prositagliptin ketone）表现出可检测的转氨活性。Arthrobacter sp.来源的转氨酶ATA-117对甲基酮和小环酮具有R构型选择性的转氨活性［58］，适合作为酶工程的起始酶。同源模建和分子对接分析发现，ATA-117的活性腔不具备结合底物西格列汀酮的能力，因此研究团队首先使用底物游走（substrate walking）策略［59］对ATA-117进行改造，先以截短的西格列汀酮为底物（图1中结构式蓝色部分）进行生物催化，将大活性口袋中与其有相互作用的氨基酸残基饱和突变建库，使转氨酶的活性向所需目标底物方向进化；之后再设计三氟苯基侧（图1中结构式红色部分）的突变文库打开小的活性口袋。将上述获得的所有有益突变组合，最终首次获得对西格列汀酮有催化活性的转氨酶ATA-117Rd1b。第二轮基本完成了对活性腔形状的改造，获得的ATA-117Rd2的活性相比于ATA-117Rd1b提高了75倍。基于ProSAR、随机突变和理性设计等策略的3～11轮改造侧重于提高酶活性和过程稳定性以满足生产工艺要求。具体实施方式为在越来越有挑战性的反应条件下进行筛选：增加底物负载，增加i-PrNH2浓度，增加有机溶剂浓度，提高pH和温度等。最终获得的含27个突变的ATA-117Rd11在底物转化率、有机溶剂的耐受程度、耐受氨基供体的浓度、高温下的稳定性和产物的对映体纯度等多个维度均实现了性能提升。
收率↑，产量↑ ... Recent achievements in developing the biocatalytic toolbox for chiral amine synthesis 1 2014 ... 转氨酶（transaminases，EC 2.6.1）催化氨基从胺供体到酮受体的可逆转移，产生手性胺［ Transaminase biocatalysis: optimization and application 1 2017 ... 转氨酶（transaminases，EC 2.6.1）催化氨基从胺供体到酮受体的可逆转移，产生手性胺［ Transaminases for industrial biocatalysis: novel enzyme discovery 1 2020 ... 转氨酶（transaminases，EC 2.6.1）催化氨基从胺供体到酮受体的可逆转移，产生手性胺［ Microbial synthesis of chiral amines by (R)-specific transamination with Arthrobacter sp. KNK168 1 2006 ... 转氨酶（transaminases，EC 2.6.1）催化氨基从胺供体到酮受体的可逆转移，产生手性胺［ Rapid creation of a novel protein function by in vitro coevolution 1 2005 ... 转氨酶（transaminases，EC 2.6.1）催化氨基从胺供体到酮受体的可逆转移，产生手性胺［ Presidential green chemistry challenge: 2010 greener reaction conditions award 1 ... 相比于铑催化的工艺路线，生物催化工艺避免了高压加氢操作和金属催化剂回收，总收率增加了10%～13%，增产53%［以kg/（L·d）计］，降低了生产成本，还减少了19%的废料产生［ Engineering a transaminase for the efficient synthesis of a key intermediate for rimegepant 2 2022 ... 用于治疗急性偏头痛的口服降钙素基因相关肽（calcitonin gene-related peptide，CGRP）受体拮抗剂rimegepant的制备过程涉及类似的立体选择性转氨反应，其化学合成也涉及过渡金属催化的两步高压还原氨化反应.2022年，凯莱英生物合成技术研发中心使用底物游走、定向进化和迭代饱和突变等策略，得到了活性提高近50 000倍的转氨酶突变体M20，其在优化的工艺条件下对底物的转化率达99%［
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