阅读说明：本技术 一种超滤渗滤过程中控制微粒产生的方法 (Method for controlling generation of particles in ultrafiltration and diafiltration process ) 是由 肖志华 罗映 于 2019-10-28 设计创作，主要内容包括：本发明涉及蛋白纯化技术,尤其涉及一种超滤渗滤过程中控制微粒产生的方法。所述方法包括：样品浓缩前往样品中加入吐温。通过加入表面活性剂吐温,有效的防止了超滤膜包堵塞,提高了产品的得率,整个过程无肉眼可见颗粒,透过通量未衰减,能够保证超滤渗滤过程顺利进行。(The invention relates to a protein purification technology, in particular to a method for controlling the generation of particles in an ultrafiltration and diafiltration process. The method comprises the following steps: the sample was concentrated before adding tween to the sample. By adding the surfactant Tween, the blockage of the ultrafiltration membrane package is effectively prevented, the yield of the product is improved, no visible particles exist in the whole process, the permeation flux is not attenuated, and the smooth proceeding of the ultrafiltration and diafiltration process can be ensured.)

1. A method for controlling particulate generation during ultrafiltration diafiltration, comprising: the sample was concentrated before adding tween to the sample.

2. The method of claim 1, wherein the tween is tween 20 or tween 80.

3. The method of claim 1, wherein the tween content of the sample after concentration is not more than 0.01% (w/v).

4. The method of claim 1, wherein the tween is added and then concentrated to a target concentration using an ultrafiltration membrane module to obtain sample a.

5. The method of claim 4, wherein the ultrafiltration membrane is polyethersulfone.

6. The method of claim 4, wherein sample A is exchanged into a target buffer to obtain sample B.

7. The method of claim 6 wherein the step of ultrafiltration diafiltration is completed by concentrating and recovering sample B to yield the product.

8. The method of claim 1, wherein the sample is a crude and purified protein sample.

9. The product obtained by the process according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1-8 in the field of protein purification.

Technical Field

The invention relates to a protein purification technology, in particular to a method for controlling the generation of particles in an ultrafiltration and diafiltration process.

Background

The current stage of downstream process platform for protein purification mainly comprises the steps of fermentation liquor clarification and harvesting, affinity chromatography and capture, low pH virus inactivation, fine purification, virus inactivation, filtration and ultrafiltration concentration. Ultrafiltration diafiltration is a concentration change of the protein of interest using a tangential flow filtration membrane module. FIG. 1 shows an installation diagram of a UF/DF system. Selecting a membrane with a proper pore diameter according to the size of molecular weight, intercepting target protein, allowing micromolecular salt to permeate the membrane, continuously adding ultrafiltration buffer solution into the Feed Tank, and uniformly stirring and mixing the sample to realize liquid change.

The complex molecular structure of proteins makes them susceptible to forming aggregates of varying sizes, from dimers to microparticles. Although microparticles typically account for very little and do not affect drug activity, there are many reports that both protein aggregates and microparticles will elicit an immune response, and therefore, the production of microparticles must be controlled during both purification and formulation development. There are many routes for particle formation, the simplest route is that proteins with a tendency to form polymers form oligomers, which then grow gradually into particles; it is also possible that the protein itself forms microparticles due to conformational instability or the influence of external pressure; proteins can adhere to gas or liquid-solid surfaces and when proteins accumulate on these surfaces or these surfaces undergo conformational changes, particulates are formed. In other purification steps, particles can be removed by filtration, but an ultrafiltration system is continuously circulated, the whole process usually lasts for 2-6 hours, in order to ensure that the Feed Tank is in a uniform state after backflow and fluid infusion, the whole process needs to be stirred and uniformly mixed, proteins are subjected to external pressure such as stirring, shearing force and the like, in the prior art, a plurality of particles visible to naked eyes are formed after stirring for 30 minutes, membrane packages are blocked, the permeation flux is reduced, the time of the whole process is prolonged, the inlet pressure of the system is increased, the system is difficult to carry out, and the blocked membrane packages are difficult to clean and cannot be reused, so that the cost is increased.

Disclosure of Invention

The problem to be solved by the present invention is to provide a method for controlling the generation of particles during UF/DF, preventing the clogging of the membrane pack, and making the whole ultrafiltration and diafiltration process smoothly performed.

Specifically, the technical scheme of the invention is as follows:

a method of controlling particulate generation during ultrafiltration diafiltration, comprising: the sample was concentrated before adding tween to the sample.

Preferably, the tween is tween 20 or tween 80.

More preferably, because tween 20 or tween 80 does not permeate the membrane and micelles easily form, the amount of tween 20 or tween 80 in the final sample is not more than 0.01% (w/v) by calculating the concentration factor that needs to be performed on the protein.

Specifically, different buffer systems and ultrafiltration effects of different additives are screened through comparison, and finally, tween serving as the additive is screened out. According to the method, Tween is needed, the Tween is needed to be used as a final auxiliary material during prescription development, the addition amount before UF/DF can be properly adjusted according to the Tween content in the final auxiliary material, the Tween is detected after the UF/DF is finished, and the Tween is added to the final auxiliary material concentration.

Preferably, tween is added and then concentrated to the target concentration using an ultrafiltration membrane pack to obtain sample a.

More preferably, the ultrafiltration membrane is made of polyether sulfone.

It should be understood that the ultrafiltration membrane package of the present invention is not limited to polyethersulfone materials, and one skilled in the art can select any suitable ultrafiltration membrane package to accomplish the present invention and is within the scope of the present invention.

More preferably, the ultrafiltration membrane package is a 30KD ultrafiltration membrane package.

It is to be understood that the ultrafiltration membrane package of the present invention is not limited to a 30KD ultrafiltration membrane package, and that any suitable ultrafiltration membrane package may be selected by one of skill in the art to carry out the present invention and is within the scope of the present invention.

Preferably, the sample A is subjected to liquid exchange into a target buffer solution to obtain a sample B.

Preferably, sample B is concentrated and recovered to yield the product, i.e., the ultrafiltration diafiltration step is completed.

Preferably, the sample is a protein sample that has been both coarsely purified and finely purified.

In a second aspect of the invention, the product obtained by the above method is disclosed.

The third aspect of the invention discloses the application of the method in the field of protein purification.

On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.

Compared with the prior art, the invention has the following remarkable advantages and effects:

according to the invention, the Tween is added into the sample before the ultrafiltration and diafiltration step, so that the generation of particles in the ultrafiltration and diafiltration process is controlled, the blockage of an ultrafiltration membrane pack is effectively prevented, the product yield is improved, no visible particles exist in the whole process, the permeation flux is not attenuated, and the smooth operation of the ultrafiltration and diafiltration process can be ensured.

Drawings

FIG. 1 is a diagram of the UF/DF system (in the diagram, 1-material liquid tank, 2-sample pump, 3-valve, 4-filtering module, 5-reflux valve, P)_R: reflux pressure, P_P: through pressure, P_F: inlet pressure, Q_R: flow rate of reflux, Q_P: through flow rate, Q_F: inlet flow rate);

FIG. 2 is a graph showing a comparative experiment of permeation flux of each buffer system in the ultrafiltration process according to the example of the present invention;

FIG. 3 is a graph comparing the agitation of different buffers in examples of the present invention.

Detailed Description

The technical solution of the present invention is clearly and completely described below. According to the technical scheme, the influence of the buffer solution and the additive is compared, the reason for generating the particles is found out, and an optimal solution is invented. The embodiments described below are some, but not all embodiments of the present invention, and all other embodiments obtained by those skilled in the art without inventive work are within the scope of the present invention.

The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

In the following examples of the present invention, the final formulation was confirmed by prescription development to be 20mM phosphate, 10mg/mL arginine, 300mg/mL trehalose, 0.02% PS20 (Tween 20), pH 6.0. During UF/DF development, 20mM phosphate, 10mg/mL arginine, pH6.0 was used as the initial buffer for changing solutions.