DHFR-CHO细胞流加培养过程的开发与优化
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摘要
生物技术类药物包括抗体、重组蛋白、核酸、多肽等药物是目前发展迅速及产业前景看好的医药产业。2010年全球生物药销售额达到1400亿美元,占全球药品市场的16%,其中有一半来自抗体产业的贡献。动物细胞培养技术作为现代生物医药产业的核心技术,近十年来随着抗体药物需求量的不断增加,以及越来越多的新产品进入临床研究,取得了前所未有的发展。如何快速高效地建立细胞生长高密度、产物表达高质高产的动物细胞培养过程及其产品生产工艺,不仅是各国大力发展抗体、重组蛋白质药物等生物技术制药产业的一项重要任务,而且也已成为国际生物医药产业创新和发展的核心竞争力。因此,研究动物细胞培养过程开发和优化的关键技术,建立抗体、重组蛋白质药物等生物医药产品的高效生产工艺,对发展我国生物医药产业、提高国际竞争力具有重要意义。本文将针对表达抗CD20单克隆抗体的DHFR-CHO细胞,研究培养过程中营养物代谢和副产物生成积累与细胞生长和产物表达的关系,并通过培养基组成和流加策略的设计和开发,深入认识以乳酸代谢为核心的细胞物质和能量代谢及其在细胞生长和产物表达中所发挥的重要作用,在此基础上设计开发经济高效的流加培养过程,以期对乳酸代谢实施有效的调控,提高细胞对营养物的利用效率并降低代谢副产物乳酸的生成,提升动物细胞培养过程的产能和经济性,从而为建立抗CD20单克隆抗体的工业化生产工艺奠定基础。
本文首先通过典型的批式培养过程,考察了细胞生长、代谢及产物表达的基本特性。实验发现,批式培养过程的最高活细胞密度为3.6×106cells/ml,最终产物浓度为95mg/L。细胞代谢方面,当初始葡萄糖浓度在40mmol/L时,批式培养结束时葡萄糖尚未耗竭。但在整个批式培养过程中,被细胞消耗的葡萄糖中有58%以上转化为了代谢副产物乳酸,最高乳酸浓度累积至54mmol/L。谷氨酰胺在细胞生长结束时已经耗竭,此时最高氨浓度累积至3.3mmol/L。而其它氨基酸如天冬酰胺和半胱氨酸也在细胞生长结束时耗竭,这些营养物的缺乏可能影响细胞的生长和产物表达。此外,培养过程中各氨基酸的消耗和生成速率不一,现有培养基中氨基酸供给可能导致培养环境中的氨基酸浓度随着培养过程的进行而处于不平衡的状态,有可能影响细胞生长和产物表达。
在上述批式培养基础上,通过对葡萄糖供给方式的研究,发现在培养过程中当以流加方式供给葡萄糖并控制其处于适中浓度时,细胞生长获得显著改善,且细胞对葡萄糖的利用率得到明显提高,从而减少了乳酸的生成和积累。进一步采用Plackett-Burman以及中心组合设计方法优化调整初始培养基中的氨基酸组成,获得了无血清无蛋白PFIA培养基,经生物反应器批式培养实验的验证,最高活细胞密度达到4.6×106cells/ml,最终产物浓度为180mg/L,较优化调整前显著提高,其效果也明显优于两种商业培养基,证明PFIA可作为后续流加培养过程的初始培养基。此外,基于top-down设计原则,通过考察流加培养基中各主要组分对细胞生长和产物表达的影响,初步确定了流加培养基FM-0中各组分的相对浓度。
以流加方式供给葡萄糖,并采用初始培养基PFIA和流加培养基FM-0及相应流加策略,通过生物反应器的流加培养实验对初步开发的流加培养过程予以验证和评价,结果表明,流加培养过程中最高活细胞密度达到6.5×106cells/ml,最终抗体浓度达到270mg/L。但实验也发现,活细胞维持时间短和产物比生成速率低是该流加培养过程的主要不足。通过对营养物代谢的进一步分析发现,该流加培养过程中的营养物供给还不够均衡,大部分氨基酸在培养后期出现累积,同时乳酸出现大量生成和积累,渗透压也有显著提高。
为进一步探明初步开发的流加培养过程存在的不足,并为后续进一步优化指明方向,实验单独考察了代谢副产物乳酸、氨以及渗透压对细胞生长和抗体产物表达的影响,并发现培养过程中乳酸的积累浓度达到并高于35mmol/L时将对细胞生长和产物表达造成非常明显的负面作用。生物反应器及相关实验验证的结果说明,在后续对流加培养工艺的进一步开发和优化过程中,首先需要对流加培养过程中以乳酸代谢为核心的细胞物质能量代谢进行研究、认识,并实现优化调控,以降低乳酸的生成,改善细胞培养环境,从而促进细胞生长、活性维持和抗体产物的表达。
为此,本文继续深入研究了以乳酸代谢为核心的细胞物质与能量代谢,以及其与细胞生长和产物表达的关系,为后续流加培养过程中实现乳酸代谢的优化调控提供合理依据。实验首先根据葡萄糖浓度和碳源种类设计了四种实验条件,并考察了其相应的乳酸及丙酮酸代谢特性。进一步考察乳酸持续生成和乳酸先生成后消耗这两种典型特征下的细胞生长和产物表达特性、糖酵解以及氨基酸物质代谢及能量代谢情况。实验发现,在培养的中后期,伴随着乳酸由生成到消耗的代谢转变,细胞的物质与能量代谢效率相应获得提高,同时产物表达速率和细胞活性也获得了较好维持。此外,本文针对乳酸代谢由生成到消耗发生转变的实验条件,采用在起始点或者中途额外添加葡萄糖或丙酮酸这两种方式,进一步考察了葡萄糖、丙酮酸和乳酸三者之间的关系。实验发现,丙酮酸浓度在乳酸代谢特征发生转变的过程中发挥着核心作用,葡萄糖耗竭将导致丙酮酸的供给受限,从而引起乳酸代谢发生从生成到消耗的逆转,无法从葡萄糖获得足够的丙酮酸,是乳酸从生成到消耗发生代谢特征转变的先决条件。
最后,本文基于对DHFR-CHO细胞乳酸代谢特性的认识,通过调整葡萄糖与半乳糖比例和流加的时间节点,设计优化了葡萄糖-半乳糖联合流加策略,并进一步对原流加培养基FM-0中的主要氨基酸和维生素组成进行了优化,建立了以调控乳酸代谢为核心的流加培养过程。该流加培养过程成功实现了对乳酸代谢的调控和较为均衡的营养供给,大幅延长了细胞的维持时间,其过程IVCC值达到64(109cells·day/L),较批式培养过程提高了170%,较初步开发的流加培养过程提高了45%；产物表达得到显著促进,其产物平均比生成速率达到14.8mg/(109cells·day),较批式培养过程提高了97%,较初步开发的流加培养过程提高了143%；最终抗CD20单克隆抗体浓度得到了大幅提高,终产物浓度达到了944mg/L,较批式培养提高了4.2倍,较初步开发的流加培养过程提高了2.5倍。
通过本文的研究,建立了以调控乳酸代谢为核心的流加培养过程,为最终建立抗CD20单克隆抗体的工业化生产工艺奠定了基础。本文通过对动物细胞培养过程中乳酸代谢特性的研究,指出了乳酸代谢对过程所具有的重要意义,丰富并加深了人们对代谢副产物乳酸的认识,而本文中采用的流加培养过程的开发方法对其它抗体药物的生产过程开发和优化同样具有重要的借鉴意义。
Biological drugs, including antibodies, recombinant proteins, nucleic acids, peptides and other drugs belong to pharmaceutical industry with rapid development and promising prospects. The global sales of biological drug in2010reachedS140billion, accounting for16%of the global pharmaceutical market, half of which came from the contribution of antibody industry. With the increasing demand for antibody drugs, as well as more and more new products into clinical research, animal cell culture technology, as the core technology of modern bio-pharmaceutical industry, has achieved unprecedented development in the past decade. How to quickly and efficiently establish production processes with high-density cell growth and high-yield-quality product expression, is not only an important task for countries to develop antibodies, recombinant protein drugs, and other bio-products for pharmaceutical industry, but also has become core competency for innovation and development of international bio-pharmaceutical industry. Therefore, it is of great significance for developing China's bio-pharmaceutical industry and improving its international competitiveness to study the key technologies of process development and optimization for animal cell culture and further establish efficient production process for antibodies, recombinant protein drugs and other biological products. In this paper, the relationship between nutrient metabolism and byproducts accumulation with cell growth and production will be studied for DHFR-CHO cells producing anti-CD20monoclonal antibody. And the material and energy metabolism with lactate metabolism at the core as well as their important roles in cell growth and production will be studied deeply through the design and development of medium composition and feeding strategy. On this basis, the cost-effective fed-batch culture will be designed and developed with a view of effective regulation of lactate metabolism, improvement of utilization of nutrient, and reduction of lactate (as metabolic by-product), so as to improve the productivity and economy of animal cell culture process, and lay the foundation for industrial production of anti-CD20monoclonal antibody.
Firstly, the characteristics of cell growth, metabolism and monoclonal antibody production were investigated in batch culture. It was found in batch culture that the maximal viable cell concentration reached3.6×106cells/ml, and the final antibody concentration was95mg/L. Glucose was not exhausted in the end of batch culture with initial concentration of40 mmol/L, but above58%consumed glucose was converted into lactate in batch culture and the peak concentration of lactate reached54mmol/L. It was observed that glutamine was depleted in the end of cell growth stage, and the peak concentration of ammonium reached3.3mmol/L. In addition, other amino acids like aspartate and cysteine were also exhausted in the end of cell growth stage. The deficiency of these nutrients might have adverse influence on cell growth and productivity. Moreover, the unsatisfied supply of amino acids in the current medium might also cause the unbalanced concentrations during the culture process, with various consumption and production rates of different amino acids, which further inhibited cell growth and productivity.
On the basis of atch culture results, through the study of glucose supply approach, it was found that when g(?)cose was fed and maintained at mediate concentration, cell growth was significantly impr(?)ed and the utilization efficiency of glucose was increased remarkably with reduced acc(?) nulation concentration of lactate. Through Plackett-Burman and central composite design method, the formulation of amino acids in the initial medium was then optimized to obtain serum-and protein-free medium PFIA. When verified in batch culture carried out in bioreactor, cell growth and productivity were both improved with maximal viable cell concentration of4.6×106cells/ml and the final antibody concentration of180mg/L, better than two commercial medium, demonstrating that PFIA could be used as the initial medium for fed-batch culture. Furthermore, by investigating the different influence of components in feeding medium on cell growth and productivity, the relative concentrations of amino acids and vitamins in FM-0was determined based on the top-down approach.
With PFIA as the initial medium and with feeding of glucose and FM-0as feeding medium in corresponding feeding strategy, the fed-batch culture was carried out in bioreactor for validation and evaluation. It was found in fed-batch culture that maximal viable cell concentration reached6.5×106cells/ml, and the final antibody concentration reached270mg/L. However, it was also observed that the short cell maintenance and low specific production rate of antibody were the major deficiency of the current fed batch culture process. Further investigation of nutrient metabolism indicated that the supply of nutrient was not balanced enough with great accumulation of most amino acids during the later cultivation. Meanwhile, lactate was found significantly produced and accumulated, and the osmotic pressure was also remarkably increased.
To further explore the deficiencies of current fed-batch culture process and provide the direction for further optimization, a series of separate experiments were carried out to investigate the influence of metabolic by-products i.e. lactate and ammonia, and the osmotic pressure on cell growth and productivity. It was found that when the accumulation concentration of lactate reached above35mmol/L during the cultivation, cell growth and productivity was greatly inhibited. The results of fed-batch culture in bioreactor and relative experimental verification indicated that it was essential and necessary to study and understand material and energy metabolism with lactate metabolism at the core in the fed-batch culture process and finally realize the regulation of lactate metabolism for further development and optimization. Therefore, the production of lactate could be reduced for better culture environment, and cell growth, maintenance and production of monoclonal antibody could be further improved.
In order to provide reasonable basis for regulation of lactate metabolism in the follow-up fed-batch culture process, an in-depth study of material and energy metabolism with lactate metabolism at the core, as well as their relationship with cell growth and productivity was carried out in this paper. Four conditions were firstly designed according to the concentration of glucose and category of carbon sources to investigate the metabolic characteristics of lactate and pyruvate. Then cell growth, productivity, glycolysis, amino acids and energy metabolism was furtuer explored under two distinct metabolic characteristics of lactate. It was found during the later cultivation, accompanied by the metabolic shift of lactate metabolism from production to consumption, the efficiency of cellular material and energy metabolism were both improved and cell productivity as well as cell maintenance was kept stable. Moreover, the relationship among glucose, pyruvate and lactate was also investigated via addition of glucose or pyruvate at the beginning or midway of cultivation with metabolic shift of lactate metabolism from production to consumption. It was found that the concentration of pyruvate played critical role in the metabolic shift of lactate metabolism. The depletion of glucose caused limited supply of pyruvate and finally led to the consumption of lactate from production. Therefore, inadequate pyruvate from the dried-up supply of glucose was the prerequisites for metabolic shift of lactate from production to consumption.
Finally, based on the understanding of lactate metabolism of DHFR-CHO cells, a combined glucose and galactose feeding strategy was designed and optimized through adjusting the ratio of glucose and galactose and feeding time. Meanwhile, the formulation of amino acids and vitamins in the current feeding medium FM-0was also optimized to obtain feeding medium FM-Opt suitable for the combined glucose and galactose feeding strategy. With feeding of FM-Opt and with the combined glucose and galactose feeding strategy, a fed-batch culture process was established with regulation of lactate metabolism at the core. Due to the effective regulation of lactate metabolism as well as the balanced supply of nutrients, cell maintenance was remarkably prolonged with IVCC value of64(109cells·day/L), increased by170%compared with batch culture, and increased by45%compared with the initially-developed fed-batch culture; antibody productivity was improved with specific production rate of antibody of14.8mg/(109cells·day), increased by97% compared with batch culture, and increased by143%compared with the initially-developed fed-batch culture. The final concentration of anti-CD20monoclonal antibody reached at944mg/L, greatly increased by4.2-fold compared with batch culture, and increased by2.5-fold compared with the initially-developed fed-batch culture.
Through this research, a fed-batch culture process was established with regulation of lactate metabolism at the core, laying the foundation for eventual industrial production of anti-CD20monoclonal antibody. Based on the study of metabolic characteristic of lactate during the animal cell culture process, the importance of lactate metabolism in process has been pointed out, which enriched and deepened people's understanding of lactate as metabolic by-product. Moreover, the development approaches for the fed-batch culture process in this paper also provide important reference for other production processes of antibody drugs.
本文首先通过典型的批式培养过程,考察了细胞生长、代谢及产物表达的基本特性。实验发现,批式培养过程的最高活细胞密度为3.6×106cells/ml,最终产物浓度为95mg/L。细胞代谢方面,当初始葡萄糖浓度在40mmol/L时,批式培养结束时葡萄糖尚未耗竭。但在整个批式培养过程中,被细胞消耗的葡萄糖中有58%以上转化为了代谢副产物乳酸,最高乳酸浓度累积至54mmol/L。谷氨酰胺在细胞生长结束时已经耗竭,此时最高氨浓度累积至3.3mmol/L。而其它氨基酸如天冬酰胺和半胱氨酸也在细胞生长结束时耗竭,这些营养物的缺乏可能影响细胞的生长和产物表达。此外,培养过程中各氨基酸的消耗和生成速率不一,现有培养基中氨基酸供给可能导致培养环境中的氨基酸浓度随着培养过程的进行而处于不平衡的状态,有可能影响细胞生长和产物表达。
在上述批式培养基础上,通过对葡萄糖供给方式的研究,发现在培养过程中当以流加方式供给葡萄糖并控制其处于适中浓度时,细胞生长获得显著改善,且细胞对葡萄糖的利用率得到明显提高,从而减少了乳酸的生成和积累。进一步采用Plackett-Burman以及中心组合设计方法优化调整初始培养基中的氨基酸组成,获得了无血清无蛋白PFIA培养基,经生物反应器批式培养实验的验证,最高活细胞密度达到4.6×106cells/ml,最终产物浓度为180mg/L,较优化调整前显著提高,其效果也明显优于两种商业培养基,证明PFIA可作为后续流加培养过程的初始培养基。此外,基于top-down设计原则,通过考察流加培养基中各主要组分对细胞生长和产物表达的影响,初步确定了流加培养基FM-0中各组分的相对浓度。
以流加方式供给葡萄糖,并采用初始培养基PFIA和流加培养基FM-0及相应流加策略,通过生物反应器的流加培养实验对初步开发的流加培养过程予以验证和评价,结果表明,流加培养过程中最高活细胞密度达到6.5×106cells/ml,最终抗体浓度达到270mg/L。但实验也发现,活细胞维持时间短和产物比生成速率低是该流加培养过程的主要不足。通过对营养物代谢的进一步分析发现,该流加培养过程中的营养物供给还不够均衡,大部分氨基酸在培养后期出现累积,同时乳酸出现大量生成和积累,渗透压也有显著提高。
为进一步探明初步开发的流加培养过程存在的不足,并为后续进一步优化指明方向,实验单独考察了代谢副产物乳酸、氨以及渗透压对细胞生长和抗体产物表达的影响,并发现培养过程中乳酸的积累浓度达到并高于35mmol/L时将对细胞生长和产物表达造成非常明显的负面作用。生物反应器及相关实验验证的结果说明,在后续对流加培养工艺的进一步开发和优化过程中,首先需要对流加培养过程中以乳酸代谢为核心的细胞物质能量代谢进行研究、认识,并实现优化调控,以降低乳酸的生成,改善细胞培养环境,从而促进细胞生长、活性维持和抗体产物的表达。
为此,本文继续深入研究了以乳酸代谢为核心的细胞物质与能量代谢,以及其与细胞生长和产物表达的关系,为后续流加培养过程中实现乳酸代谢的优化调控提供合理依据。实验首先根据葡萄糖浓度和碳源种类设计了四种实验条件,并考察了其相应的乳酸及丙酮酸代谢特性。进一步考察乳酸持续生成和乳酸先生成后消耗这两种典型特征下的细胞生长和产物表达特性、糖酵解以及氨基酸物质代谢及能量代谢情况。实验发现,在培养的中后期,伴随着乳酸由生成到消耗的代谢转变,细胞的物质与能量代谢效率相应获得提高,同时产物表达速率和细胞活性也获得了较好维持。此外,本文针对乳酸代谢由生成到消耗发生转变的实验条件,采用在起始点或者中途额外添加葡萄糖或丙酮酸这两种方式,进一步考察了葡萄糖、丙酮酸和乳酸三者之间的关系。实验发现,丙酮酸浓度在乳酸代谢特征发生转变的过程中发挥着核心作用,葡萄糖耗竭将导致丙酮酸的供给受限,从而引起乳酸代谢发生从生成到消耗的逆转,无法从葡萄糖获得足够的丙酮酸,是乳酸从生成到消耗发生代谢特征转变的先决条件。
最后,本文基于对DHFR-CHO细胞乳酸代谢特性的认识,通过调整葡萄糖与半乳糖比例和流加的时间节点,设计优化了葡萄糖-半乳糖联合流加策略,并进一步对原流加培养基FM-0中的主要氨基酸和维生素组成进行了优化,建立了以调控乳酸代谢为核心的流加培养过程。该流加培养过程成功实现了对乳酸代谢的调控和较为均衡的营养供给,大幅延长了细胞的维持时间,其过程IVCC值达到64(109cells·day/L),较批式培养过程提高了170%,较初步开发的流加培养过程提高了45%；产物表达得到显著促进,其产物平均比生成速率达到14.8mg/(109cells·day),较批式培养过程提高了97%,较初步开发的流加培养过程提高了143%；最终抗CD20单克隆抗体浓度得到了大幅提高,终产物浓度达到了944mg/L,较批式培养提高了4.2倍,较初步开发的流加培养过程提高了2.5倍。
通过本文的研究,建立了以调控乳酸代谢为核心的流加培养过程,为最终建立抗CD20单克隆抗体的工业化生产工艺奠定了基础。本文通过对动物细胞培养过程中乳酸代谢特性的研究,指出了乳酸代谢对过程所具有的重要意义,丰富并加深了人们对代谢副产物乳酸的认识,而本文中采用的流加培养过程的开发方法对其它抗体药物的生产过程开发和优化同样具有重要的借鉴意义。
Biological drugs, including antibodies, recombinant proteins, nucleic acids, peptides and other drugs belong to pharmaceutical industry with rapid development and promising prospects. The global sales of biological drug in2010reachedS140billion, accounting for16%of the global pharmaceutical market, half of which came from the contribution of antibody industry. With the increasing demand for antibody drugs, as well as more and more new products into clinical research, animal cell culture technology, as the core technology of modern bio-pharmaceutical industry, has achieved unprecedented development in the past decade. How to quickly and efficiently establish production processes with high-density cell growth and high-yield-quality product expression, is not only an important task for countries to develop antibodies, recombinant protein drugs, and other bio-products for pharmaceutical industry, but also has become core competency for innovation and development of international bio-pharmaceutical industry. Therefore, it is of great significance for developing China's bio-pharmaceutical industry and improving its international competitiveness to study the key technologies of process development and optimization for animal cell culture and further establish efficient production process for antibodies, recombinant protein drugs and other biological products. In this paper, the relationship between nutrient metabolism and byproducts accumulation with cell growth and production will be studied for DHFR-CHO cells producing anti-CD20monoclonal antibody. And the material and energy metabolism with lactate metabolism at the core as well as their important roles in cell growth and production will be studied deeply through the design and development of medium composition and feeding strategy. On this basis, the cost-effective fed-batch culture will be designed and developed with a view of effective regulation of lactate metabolism, improvement of utilization of nutrient, and reduction of lactate (as metabolic by-product), so as to improve the productivity and economy of animal cell culture process, and lay the foundation for industrial production of anti-CD20monoclonal antibody.
Firstly, the characteristics of cell growth, metabolism and monoclonal antibody production were investigated in batch culture. It was found in batch culture that the maximal viable cell concentration reached3.6×106cells/ml, and the final antibody concentration was95mg/L. Glucose was not exhausted in the end of batch culture with initial concentration of40 mmol/L, but above58%consumed glucose was converted into lactate in batch culture and the peak concentration of lactate reached54mmol/L. It was observed that glutamine was depleted in the end of cell growth stage, and the peak concentration of ammonium reached3.3mmol/L. In addition, other amino acids like aspartate and cysteine were also exhausted in the end of cell growth stage. The deficiency of these nutrients might have adverse influence on cell growth and productivity. Moreover, the unsatisfied supply of amino acids in the current medium might also cause the unbalanced concentrations during the culture process, with various consumption and production rates of different amino acids, which further inhibited cell growth and productivity.
On the basis of atch culture results, through the study of glucose supply approach, it was found that when g(?)cose was fed and maintained at mediate concentration, cell growth was significantly impr(?)ed and the utilization efficiency of glucose was increased remarkably with reduced acc(?) nulation concentration of lactate. Through Plackett-Burman and central composite design method, the formulation of amino acids in the initial medium was then optimized to obtain serum-and protein-free medium PFIA. When verified in batch culture carried out in bioreactor, cell growth and productivity were both improved with maximal viable cell concentration of4.6×106cells/ml and the final antibody concentration of180mg/L, better than two commercial medium, demonstrating that PFIA could be used as the initial medium for fed-batch culture. Furthermore, by investigating the different influence of components in feeding medium on cell growth and productivity, the relative concentrations of amino acids and vitamins in FM-0was determined based on the top-down approach.
With PFIA as the initial medium and with feeding of glucose and FM-0as feeding medium in corresponding feeding strategy, the fed-batch culture was carried out in bioreactor for validation and evaluation. It was found in fed-batch culture that maximal viable cell concentration reached6.5×106cells/ml, and the final antibody concentration reached270mg/L. However, it was also observed that the short cell maintenance and low specific production rate of antibody were the major deficiency of the current fed batch culture process. Further investigation of nutrient metabolism indicated that the supply of nutrient was not balanced enough with great accumulation of most amino acids during the later cultivation. Meanwhile, lactate was found significantly produced and accumulated, and the osmotic pressure was also remarkably increased.
To further explore the deficiencies of current fed-batch culture process and provide the direction for further optimization, a series of separate experiments were carried out to investigate the influence of metabolic by-products i.e. lactate and ammonia, and the osmotic pressure on cell growth and productivity. It was found that when the accumulation concentration of lactate reached above35mmol/L during the cultivation, cell growth and productivity was greatly inhibited. The results of fed-batch culture in bioreactor and relative experimental verification indicated that it was essential and necessary to study and understand material and energy metabolism with lactate metabolism at the core in the fed-batch culture process and finally realize the regulation of lactate metabolism for further development and optimization. Therefore, the production of lactate could be reduced for better culture environment, and cell growth, maintenance and production of monoclonal antibody could be further improved.
In order to provide reasonable basis for regulation of lactate metabolism in the follow-up fed-batch culture process, an in-depth study of material and energy metabolism with lactate metabolism at the core, as well as their relationship with cell growth and productivity was carried out in this paper. Four conditions were firstly designed according to the concentration of glucose and category of carbon sources to investigate the metabolic characteristics of lactate and pyruvate. Then cell growth, productivity, glycolysis, amino acids and energy metabolism was furtuer explored under two distinct metabolic characteristics of lactate. It was found during the later cultivation, accompanied by the metabolic shift of lactate metabolism from production to consumption, the efficiency of cellular material and energy metabolism were both improved and cell productivity as well as cell maintenance was kept stable. Moreover, the relationship among glucose, pyruvate and lactate was also investigated via addition of glucose or pyruvate at the beginning or midway of cultivation with metabolic shift of lactate metabolism from production to consumption. It was found that the concentration of pyruvate played critical role in the metabolic shift of lactate metabolism. The depletion of glucose caused limited supply of pyruvate and finally led to the consumption of lactate from production. Therefore, inadequate pyruvate from the dried-up supply of glucose was the prerequisites for metabolic shift of lactate from production to consumption.
Finally, based on the understanding of lactate metabolism of DHFR-CHO cells, a combined glucose and galactose feeding strategy was designed and optimized through adjusting the ratio of glucose and galactose and feeding time. Meanwhile, the formulation of amino acids and vitamins in the current feeding medium FM-0was also optimized to obtain feeding medium FM-Opt suitable for the combined glucose and galactose feeding strategy. With feeding of FM-Opt and with the combined glucose and galactose feeding strategy, a fed-batch culture process was established with regulation of lactate metabolism at the core. Due to the effective regulation of lactate metabolism as well as the balanced supply of nutrients, cell maintenance was remarkably prolonged with IVCC value of64(109cells·day/L), increased by170%compared with batch culture, and increased by45%compared with the initially-developed fed-batch culture; antibody productivity was improved with specific production rate of antibody of14.8mg/(109cells·day), increased by97% compared with batch culture, and increased by143%compared with the initially-developed fed-batch culture. The final concentration of anti-CD20monoclonal antibody reached at944mg/L, greatly increased by4.2-fold compared with batch culture, and increased by2.5-fold compared with the initially-developed fed-batch culture.
Through this research, a fed-batch culture process was established with regulation of lactate metabolism at the core, laying the foundation for eventual industrial production of anti-CD20monoclonal antibody. Based on the study of metabolic characteristic of lactate during the animal cell culture process, the importance of lactate metabolism in process has been pointed out, which enriched and deepened people's understanding of lactate as metabolic by-product. Moreover, the development approaches for the fed-batch culture process in this paper also provide important reference for other production processes of antibody drugs.
引文
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