Positive : Insights

Monoclonal antibodies play a critical role in research, diagnostics and product development and provide numerous benefits to patients in diverse therapeutic areas.

MAB能够识别在抗原上发现的一个表位。因此，它们具有比多克隆抗体（PAB）更高的特异性，这使得它们成为诊断和治疗用途的理想选择。定制单克隆抗体生产需要不同的技能组，而不是多克隆抗体生产，并且经常导致额外的成本和时间。

MAB的特异性使其成为医疗应用的理想选择，例如诊断和治疗使用。那么单克隆抗体生产中的关键考虑因素是什么？

Hybridoma generation

杂交瘤细胞有能力复制and secrete the antibody of interest while continuing to proliferateindefinitely. The unlimited replicative potential of hybridoma cells produces significant levels of mAbs. The杂交瘤的产生和MAb的生产通常需要九到十个月.

传统方法如体内腹水在动物中的产生不太常见;腹水生产在欧洲禁止禁止。使用各种方法可以生产大量的高浓度抗体，例如组织培养烧瓶，旋转辊，辊瓶，搅拌罐发酵罐和中空纤维生物反应器。

抗体噬菌体展示（APD）

科学家们使用APD来针对选定的抗原产生完全人抗体。APD已被利用以产生研究抗体和基于抗体的治疗方法（Nixon，2014）。噬菌体展示技术最初是由乔治史密斯在1985年描述的（史密斯，1985年）。在这一发现之后，科学家们证明了抗体片段可以显示在噬菌体颗粒表面（McCafferty，1990; Barbas，1991）上。这种突破使人抗体可以使用体外processes.

In general, APD involves three major steps. First, a vector is constructed to allow a large number of different light chain and heavy chain genes to be incorporated (Lerner, 2016). Next, the vector is expressed in a host type that allows the coupling of the genotype (i.e., antibody gene) with the phenotype (i.e., antibody molecule expressed outside the host) (Lee, 2007; Lerner, 2016). Once a large collection of phage-bearing antibody molecules has been generated, researchers apply a selection process to isolate phages that bind to a given antigen. The third step, known as “panning,” occurs when phages are exposed to the antigen and only the antigen-bound phages are replicated via infection ofE. colito amplify the monoclonal antibody construct (Hoogenboom; 2005; Lerner, 2016). The antibody genes in these phage particles are used in expression systems to generate purified antibodies (Shukra, 2014; Lerner, 2016).

人源化小鼠

In the 1990’s, scientists disclosed the first genetically engineered mice that expressed fully human antibody repertoires (Lonberg, 1994; Green, 1994). These human immunoglobulin transgenic mice express B cell receptors that are hybrids of mouse and human components, and their B cells develop and mature into seemingly normal B cell subtypes (Lonberg, 2008). IgM (Green, 1994) and IgB (Lonberg, 1994) mAbs, which specifically recognize the antigens of interest, are isolated.

In 2006, the FDA approved the first human mAb generated in a transgenic mouse for epidermal growth factor receptor-expressing colorectal cancers. (Jakobovits, 2007). Recently, Regeneron Pharmaceuticals developed the VelocImmune®mouse, which enabled researchers to rapidly progress ten different fully human antibodies into human clinical trials. (Murphy, 2014).

Another approach is the use of humanized immune system models to generate mAbs. For instance, inoculating newborn immunodeficient mice with human fetal or umbilical cord hematopoietic stem cells, which can result in a robust engraftment of a number of immune cells. (Becker, 2010). However, results generally have been underwhelming. (e.g., Villaudy, 2014).

最佳抗原特异性小鼠MAB生产的方法

Miceare most commonly used for the production of mAbs, in contrast with pAb production which generally usesrabbitsand other larger animals. Researchers must use great care when fusing B-cells with myeloma cells.在融合过程之后，杂交瘤细胞非常脆弱. Fetal bovine serum (FBS) is commonly used to provide cellular factors necessary for growth. Use of FBS has drawbacks, however, such as the potential for contamination with infectious agents. As a result, many commercial serum-free media (SFM) products have been developed to support the growth and maintenance of cells.

When researchers determine that particular hybridoma clones are acceptable for production, they are frozen in cryopreservation media and stored in the vapor phase of liquid nitrogen.

缩放MAB的生产

科学家可以通过各种方式升级MAB的生产，例如中空纤维反应器方法。这些是用于收集组织培养上清液的血管。成千上万的半透明中空纤维在盒内平行布置，该盒子为蜂窝生长提供3D环境，并且在纤维的两侧具有入口和出口（Dewar et al。，2005）。这种方法的一个好处是连续去除废品。

在生产高质量的杂交瘤和MAB时，仔细的规划和制备是至关重要的。通过与经验丰富的合作伙伴合作，研究人员可以消除许多普通陷阱，同时还节省了时间和金钱。

Looking toward the future of antibody optimized production

The production of a custom antigen specific mouse mAb is a time-consuming and technically demanding process. However, once stable hybridomas have been established, they can be expanded and frozen to create a reliable source of the mAb. Scaling up the production of mAbs can be done using a wide range of various methodologies.

需要仔细的规划和准备来生产高质量的杂交瘤和mAb。与深层实践经验的合作伙伴合作可以通过消除最常见的缺陷来确保每一步的优化，并节省时间和金钱。

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