Application of Hollow Fiber Filtration Technology in Baibai Break Vaccine

Application of Hollow Fiber Filtration Technology in Baibai Break Vaccine

Wang Yijun General Electric Medical Group

Foreword

The pertussis, diphtheria, and tetanus vaccine is referred to as the Baibai Breaking Vaccine. It is prepared from the pertussis vaccine, refined diphtheria and tetanus toxoid in an appropriate proportion to prevent pertussis, diphtheria and tetanus. At present, there are a mixed vaccine for adsorbing pertussis vaccine, diphtheria and tetanus toxoid (adsorption whitening) and adsorption of acellular pertussis vaccine, diphtheria and tetanus poisonous drugs (adsorption cell-free whitening). At present, the domestic process basically belongs to the process of the 1970s and 1980s. The dialysis steps used in the whole process and the routine filtration steps are very numerous, which greatly affects the process production process of the whole product.

GE's hollow fiber is a tangential flow membrane filtration product designed as an open channel. It has low shear force and high activity yield in the whole biological downstream separation. It is especially suitable for high concentration, high viscosity and high particle size. Separation of the sample. According to the unique advantages of hollow fiber, we made some useful attempts in the process of breaking the white, and obtained good results.

Original pertussis rough process

Figure 1: Pertussis process

Throughout the process, we have made a beneficial attempt in the step (3), dialysis to remove ammonium ions, and step (6) of the process, and dialysis to remove the antidote glutaraldehyde. Good experimental results.

Hollow fiber deammonium ion experiment

Figure 2 Quixstand system

Experimental membrane type: 10KD filter column (UFP-10-C-4MA).

Experimental conditions: pump speed - 200 rpm, loading volume 1000 ml, membrane pressure 18 psi.

Experimental liquid: The liquid in the (3) step of the pertussis process, before the ammonium ion is removed (the liquid after high-speed centrifugation).

Experimental procedure: Pertussis process (3) salting out precipitation and buffer extraction of pertussis toxin and filamentous hemagglutinin, followed by dialysis to remove ammonium ions. The traditional process is high-speed centrifugation and then dialysis to remove ammonium ions (before high-speed centrifugation) The liquid is very turbid, and the whole dialysis time is very long. It usually takes about 2 days. It is completely a speed limit step of the process. Here, the experiment attempts to replace the dialysis with a hollow fiber with a molecular weight of 10k, and has obtained good experimental results. Basically, when the filtered volume is about 3-4 times of the original volume, the ammonium ion can be inspected to meet the process requirements. The entire experimental process takes only about 2 hours, and since the hollow fiber can handle the characteristics of high-particle and high-viscosity particles, We also continue to try to concentrate the turbid liquid solution 2-3 times and then wash it again. It is also the result that the ammonium ion is qualified when the filtered volume is 2-3 times the original volume, which can save the subsequent centrifugation. The volume also saves time in the process.

Hollow fiber de- glutaraldehyde experiment

Experimental system: Quixstand system

Experimental membrane type: 10KD filter column (UFP-10-E-4MA) Experimental conditions: pump speed - 260 rpm, loading volume 1000 ml, membrane pressure 15 psi.

Experimental solution: Step (6) of the pertussis solution process for detoxification by adding glutaraldehyde.

Experimental process: The experiment process is carried out by first concentrating the feed liquid about 5 times, and then immediately washing and filtering. After washing the filter volume to 8 times volume, the glutaraldehyde can be obtained. The dialysis removal step of glutaraldehyde is carried out. It belongs to a multi-floc-like state. In the traditional process, dialysis bags remove glutaraldehyde, which usually takes more than three days, which brings unstable factors to the whole process. If the dialysis time is too long, it is easy to stain the bacteria. The aspect also has a certain influence on the yield activity of the protein. Here, try to replace the dialysis with 10k hollow fiber. When the volume of the raw material is 8 times or more, the glutaraldehyde can be removed. Try to experiment the process by first concentrating the feed liquid about 5 times, and then immediately washing the filter. After the filtered volume is 4-5 times the volume of the filter, the glutaraldehyde can be qualified. The whole process time is basically 2-3 hours, and the operation is fully enclosed, which greatly increases the safety of the entire biological product.

Original diphtheria or tetanus toxin general process

Figure 3: Diphtheria or tetanus toxin process

In the process (2), the supernatant is collected by crepe filtration, which takes 10 hours or more, and the external time is placed in the air for a long time, which brings the risk of contamination of the liquid on the other hand. The clarity of the liquid is also poor, which will affect the properties of the final product to a certain extent. We tried to replace the original steps with hollow fiber in this process, and got a good pass, not only the liquid is very clear And there is almost no loss of activity.

Diphtheria toxin experiment

Experimental system: Quixstand system

Experimental membrane type: 0.45um filter column (CFP-4-E-4MA)

Experimental conditions: pump speed -320 rpm, loading volume 1000 ml, membrane pressure 5.5 psi

Experimental liquid: Decolorized liquid after adding activated carbon, process step (2)

Experimental procedure: The method of first concentrating the feed liquid to 900 ml, followed by washing and filtering, and stopping the experiment by filtering the volume of the liquid solution into a volume of 2.5 times the volume of the raw material liquid, and basically filtering the target toxin in the fermentation liquid after the activity detection Washed out, reached a good experimental result, the entire experiment took about two hours, greatly saving the process time.

Tetanus toxin experiment

Experimental system: Quixstand system.

Experimental membrane type: 0.45um filter column (CFP-4-E-4MA).

Experimental conditions: pump speed - 320 rpm, loading volume 1000 ml, membrane pressure 3 psi.

Experimental liquid: Step (2) of the process flow for decolorization after adding activated carbon.

Experimental procedure: The method of first concentrating the feed liquid to 600 ml, followed by washing and filtering, and stopping the experiment when the filtration volume is 2 times the original volume, the filter can be completely filtered out in the fermentation broth, and the reaction is very good. Good results, the entire experiment took about two hours, greatly saving the process time.

In addition to this step, the diphtheria tetanus toxin process also attempts to remove ammonium ions from diphtheria and tetanus toxins using 30k hollow fibers, which also yields very good results. It can be put in a very short time. Ammonium ion removal.

Due to its unique open structure, GE's hollow fiber can process high-concentration, high-viscosity, high-particle samples, replacing the original extensive filtration and dialysis process to remove bacteria and glutaraldehyde. The purpose of ammonium ion has been well applied in the process of pertussis tetanus diphtheria toxin.

Figure 4: Fully automatic membrane filtration system

GE's hollow fiber series filter columns (HF) and systems offer the following benefits:

● The cost of purchase/use is reasonable;

● Various microfiltration pore sizes and ultrafiltration molecular weights are optional;

● Most filter columns can be autoclaved or steamed online;

● The inner diameter of the fiber tube is optional, suitable for processing samples with high concentration, viscosity and granularity;

● Easy installation and operation;

● Fully enclosed operation;

● Comply with cGMP/FDA strict safety and controllable certification requirements;

● GE also has a fully automated membrane process development and an automated membrane system for production, ensuring process optimization and linear amplification from the laboratory.

Figure 5 Grandstand 550 production system