研究背景：

人活化凝血酶原复合物（activated Prothrombin Complex Concentrate, aPCC）是以健康人的合并血浆为原料制备得到的维生素K依赖性凝血因子混合物，成分复杂，包含多种凝血因子、活化凝血因子及抗凝血因子。aPCC通过补充外源或共同凝血途径中所需的凝血因子形成凝血酶，可以避开凝血因子Ⅷ（Coagulation Factor Ⅷ, FⅧ）从而发挥止血作用，因此，aPCC是治疗A型血友病（Hemophilia A，HA）高滴度抑制物患者出血发作的主要药物之一。目前，aPCC已在80多个国家用于治疗HA抑制物患者超过40年，此外，aPCC对抗凝剂逆转、手术及创伤性出血等患者均有较好的疗效。目前武田公司是全球唯一一家生产和销售aPCC制品的公司，其商品名为FEIBA^®。1985年，我国发布了《关于禁止VIII因子制剂等血液制品进口的通知》，明确了“血液制品除人血清白蛋白以外，其他所有品种均系国家规定禁止进口的药物”，因此我国患者尚无法使用FEIBA^®。目前临床上只能使用价格相对较为昂贵的重组活化人凝血因子Ⅶ（recombinant Activated Coagulation Factor Ⅶ, rFⅦa）或治疗效果并不理想的人凝血酶原复合物（Prothrombin Complex Concentrates，PCC）替代aPCC，以期治疗HA抑制物患者的出血发作。由于aPCC药效成分较为复杂，制备工艺的精准控制是确保制品质量的关键所在，同时也是制约aPCC制品研发的主要因素。

目的：

开发稳定的aPCC实验室制备工艺；利用本研究的制备工艺，制备aPCC浓缩物，并以武田公司的FEIBA^®作对照，分别通过体外和体内实验评价二者的药效差异以及安全性，以期为我国aPCC制品的制备工艺研发和质量标准的建立奠定前期基础。

方法：

1.aPCC制备工艺研究：⑴以去冷沉淀血浆为实验原料，经过蛋白质纯化、病毒灭活、再次纯化、超滤浓缩、活化等试验步骤制备得到aPCC浓缩物。然后以FⅡa活性（FⅡa activity, FⅡa：C）和FEIBA活性作为aPCC浓缩物的主要质量考察指标，分别从激活剂种类、激活温度、激活剂浓度、以及孵育时间等方面对制备工艺过程中凝血因子激活条件进行优化。⑵以FEIBA^®、自制aPCC浓缩物为研究对象，首先采用凝固法比较上述样本中FEIBA活性；然后利用凝固法和发色底物法，检测上述样本中非活化凝血因子、活化凝血因子以及抗凝血因子的活性，比较分析上述样本的主要药效成分活性差异。

2.自制aPCC浓缩物与FEIBA^®的主要药效差异研究：通过体外实验，在aPCC样品未添加/添加鱼精蛋白的情况下，比较其对血液凝血功能初筛4项和凝血酶生成能力的影响。同时以未添加药物处理的抑制物血浆、正常标准血浆和FEIBA^®处理血浆作为对照。具体包括：⑴通过凝血酶原时间(Prothrombin Time，PT)、活化部分凝血活酶时间(Activated Partial Thromboplastin Time，APTT)、纤维蛋白原（Fibrinogen，Fib）、凝血酶时间（Thrombin Time，TT）检测，初步评价自制aPCC浓缩物对外源性凝血途径、内源性凝血途径、共同凝血途径等凝血初筛4项的影响；⑵通过自动标准化凝血酶生成试验(Calibrated Automated Thrombin Generation, CAT)，以进一步评价自制aPCC浓缩物对凝血酶生成能力的影响。

3.自制aPCC浓缩物与FEIBA^®的体内安全性比较研究：首先，建立FeCl₃诱导大鼠颈动脉血栓形成模型；其次，经颈静脉对大鼠进行生理盐水或不同aPCC样品给药处理。同时以生理盐水和FEIBA^®作为对照，比较上述成分对大鼠血栓形成、凝血功能、以及凝血酶生成能力的影响，具体包括：⑴激光散斑血流成像监测观察大鼠颈动脉血栓的生成情况；⑵ PT、APTT、Fib、TT检测；⑶CAT检测，以期评价自制aPCC浓缩物的安全性。

结果：

1. 以去冷沉淀血浆为原料，经过蛋白质纯化、病毒灭活、二次纯化、超滤浓缩、以及因子活化等步骤制备得到aPCC浓缩物，并通过正交实验，筛选出aPCC实验室制备工艺的最佳活化条件。具体如下：⑴本研究最佳活化条件为4℃条件下、以10mM的钙离子活化12小时，该条件下制备得到的aPCC浓缩物FEIBA活性约40IU/ml，且FⅡa：C不超过1IU/ml。⑵自制aPCC浓缩物FEIBA活性与FEIBA^®无统计学差异（p>0.05），其FⅡ、FⅩ、FⅦa、FⅩa、PC活性均显著高于FEIBA^®（p均<0.05），FⅡa、PS活性及肝素含量高于FEIBA^®，FⅨa活性显著低于FEIBA^®（p<0.001），AT-Ⅲ活性低于FEIBA^®，FⅦ、FⅨ活性与FEIBA^®没有统计学差异（p均>0.05）。

2.自制aPCC浓缩物具有较好的凝血作用。具体如下：⑴体外实验凝血四项检测结果显示：添加鱼精蛋白中和肝素后，自制aPCC浓缩物组与FEIBA^®处理组的PT、APTT、Fib、TT检测结果均无显著差异（p>0.05）。⑵体外实验CAT检测结果显示：无论是否添加鱼精蛋白，2个aPCC实验组内源性凝血酶生成潜力（Endogenous Thrombin Potential，ETP）和凝血酶峰值(Peak Height，Peak)检测值均显著高于2个对照组（p均<0.001）；未添加鱼精蛋白时，FEIBA^®组ETP、Peak最高，添加鱼精蛋白处理后，自制aPCC浓缩物组ETP、Peak检测值最高。

3.自制aPCC浓缩物的致血栓风险较低，具有较好的安全性。具体如下：⑴激光散斑血流成像监测结果显示：2个aPCC组的血栓发生率相同，均为28.6%。自制aPCC浓缩物处理组形成的血栓发生自溶，血流恢复；FEIBA^®处理组形成的血栓均较为稳定，在血流监测结束时血流仍未恢复。⑵大鼠血浆凝血四项检测结果显示：自制aPCC浓缩物组与FEIBA^®处理组的PT、APTT、Fib、TT检测结果均无显著差异（p>0.05）。⑶大鼠血浆CAT检测结果显示：自制aPCC浓缩物组与FEIBA^®处理组的ETP、Peak检测值均无显著差异（p>0.05）。

结论：

1.本研究初步建立了一套较为稳定的aPCC浓缩物制备工艺。

2.自制aPCC浓缩物（FEIBA活性约40IU/ml）具有较好的凝血作用和较低的致血栓风险。

Background:

Activated prothrombin complex concentrate (aPCC) is a mixture of vitamin K-dependent coagulation factors prepared from pooled plasma of healthy individuals, it contains a variety of coagulation factors, activated coagulation factors, and anticoagulation factors. By supplementing the clotting factors required in the exogenous or co-coagulation pathway to form thrombin, aPCC can bypass coagulation factor Ⅷ (FⅧ) and thus exert a hemostatic effect, aPCC is therefore one of the main drugs for the treatment of bleeding episodes in patients with high-titer inhibitors of hemophilia A (HA). Currently, aPCC has been used in more than 80 countries to treat patients with HA inhibitors for more than 40 years. In addition, aPCC has been shown to be effective in patients with anticoagulant reversal, surgical and traumatic bleeding. Currently, Takeda is the only company globally that produces and sells aPCC products, marketed under the brand name FEIBA^®.In 1985, China issued the Circular on the Prohibition of the Import of Blood Products such as Factor VIII Preparations, which clearly stated that “all varieties of blood products, except for human serum albumin, are drugs prohibited from being imported by the state”, so FEIBA^® is not available to patients in China. Currently, only the relatively expensive recombinant activated coagulation factor Ⅶ (rFⅦa) or the less effective prothrombin complex concentrates (PCC) can be used to replace aPCC in patients with HA inhibitors, with a view to treating bleeding episodes in patients with HA inhibitors. Due to the complexity of the pharmacodynamic components of aPCC, the precise control of the preparation process is the key to ensure the quality of the products, and it is also the main factor restricting the research and development of aPCC products.

Objective:

To develop a stable laboratory preparation process for aPCC; to prepare aPCC concentrate using the preparation process in this study, and using Takeda's FEIBA^® as a control to evaluate the differences in efficacy and safety of the two by in vitro and in vivo experiments, respectively, with a view to laying a preliminary foundation for the development of the preparation process and the establishment of the quality standard for the preparation of aPCC products in China.

Methods:

1.Research on the preparation process of aPCC: (1)Using cryoprecipitate-depleted plasma as the experimental material, it undergoes protein purification, viral inactivation, re-purification, ultrafiltration concentration, and activation to obtain aPCC concentrate. Then FⅡa activity (FⅡa:C) and FEIBA activity were used as the main quality indicators of aPCC concentrate, and the conditions of coagulation factor activation in the preparation process were optimized in terms of the type of activator, activation temperature, activator concentration, and incubation time, respectively. (2)Using FEIBA^® and homemade aPCC concentrate as research objects, firstly, the coagulation method was used to compare the FEIBA activity in the above samples;  then, using the coagulation method and the color-emitting substrate method, the activities of non-activated coagulation factors, activated coagulation factors, and anticoagulant factors were detected in the above samples to comparatively analyze the differences in the activities of the main pharmacodynamic components in the above samples.

2.Study on the main pharmacodynamic differences between homemade aPCC concentrate and FEIBA^®: Through in vitro experiments, the effects of aPCC samples with and without added protamine on the initial screening of four blood coagulation functions and thrombin generation capacity were compared. Inhibitor plasma without added drug treatment, normal standard plasma and FEIBA^®-treated plasma were also used as controls. Specifically includes: (1)Through the detection of Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), Fibrinogen (Fib), and Thrombin Time (TT), initially evaluate the impact of the homemade aPCC concentrate on the extrinsic coagulation pathway, intrinsic coagulation pathway, common coagulation pathway, and other initial screening tests for coagulation; (2)evaluate the impact of the homemade aPCC concentrate on thrombin generation capacity through the calibrated automated thrombin generation (CAT) assay.

3.In vivo safety comparison study of homemade aPCC concentrate versus FEIBA^®: First, establish a rat carotid artery thrombosis model induced by FeCl₃; second, administer physiological saline or different aPCC samples via the jugular vein to the rats. Comparing the effects of the aforementioned components on thrombus formation, coagulation function, and thrombin generation capacity in rats, with physiological saline and FEIBA^® as controls, specifically including: (1)monitoring thrombus formation in the carotid arteries of rats using laser speckle flow imaging; (2)PT, APTT, Fib, and TT tests; (3)CAT test, aiming to evaluate the safety of the homemade aPCC concentrate.

Results：

1.Using cryoprecipitate-depleted plasma as raw material, aPCC concentrate is prepared through protein purification, viral inactivation, secondary purification, ultrafiltration concentration, and factor activation. An orthogonal experiment was conducted to screen out the optimal activation conditions for laboratory preparation of aPCC. Specifically as follows: (1)The optimal activation condition in this study were activation with 10 mM calcium ions at 4°C for 12 h, the FEIBA activity of the aPCC obtained from the preparation under this condition was approximately 40 IU/ml, and FⅡa:C did not exceed 1 IU/ml. (2)The FEIBA activity of homemade aPCC was not statistically different from FEIBA^® (p>0.05), its FⅡ, FⅩ, FⅦa, FⅩa, and PC activities were significantly higher than FEIBA^® (each p <0.05), FⅡa, PS activities and heparin content were higher than FEIBA^®, FⅨa activity was significantly lower than FEIBA^® (p<0.001), AT -Ⅲ activity was lower than FEIBA^®, and FⅦ and FⅨ activities did not differ from FEIBA^® (each p>0.05).

2.Homemade aPCC concentrate has good coagulation properties. Specifically as follows: (1)The results of four coagulation tests in vitro showed that there was no significant difference in PT, APTT, Fib and TT test results between homemade aPCC concentrate group and FEIBA^® treatment group after adding protamine and heparin (p>0.05). (2)CAT test results in vitro showed that: The endogenous thrombin potential (ETP) and peak height (Peak) of the two aPCC groups were significantly higher than those of the two control groups regardless of the addition of protamine (each p <0.001). The ETP and Peak values of FEIBA^® group were the highest when no protamine was added, and the ETP and Peak values of homemade aPCC concentrate group were the highest when protamine was added.

3.The homemade aPCC concentrate has a lower risk of thrombosis and a better safety profile. Specifically as follows: (1)The results of laser speckle blood flow imaging monitoring showed that the incidence of thrombosis in the two aPCC groups was the same, 28.6%. The thrombus formed in the homemade aPCC concentrate treatment group was autolysis and the blood flow recovered. The thrombus formation in FEIBA^® treated group was relatively stable, and the blood flow still did not recover at the end of the blood flow monitoring. (2)The results of four tests of rat plasma coagulation showed that there was no significant difference in PT, APTT, Fib and TT between homemade aPCC concentrate group and FEIBA® treatment group (p>0.05). (3)The results of rat plasma CAT detection showed that there was no significant difference in ETP and Peak detection values between homemade aPCC concentrate group and FEIBA^® treated group (p>0.05).

Conclusion:

1.In this study, a set of relatively stable preparation process of aPCC concentrate was established.

2.Homemade aPCC concentrate (FEIBA activity about 40IU/ml) has good coagulation properties and a lower risk of thrombosis.