Process and apparatus for isolating protein of interest in heterogeneous tissue culture liquid mixture and purification thereof

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to a process and apparatus for isolating and purifying protein of interest in a stream of a tissue culture liquid obtained from a continuous perfusion fermentation process. The proposed apparatus, in which sterile conditions are maintained, includes a continuous perfusion fermentation system, a continuous particle removal system integrated with the perfusion fermentation system and adapted for continuous reception of tissue culture liquid therefrom and continuous production of clarified tissue culture liquid, and a continuous purification system integrated with the particle removal system and adapted for continuous reception clarified tissue culture liquid therefrom and constant production of the extracted product which contain the protein of interest, where the continuous purification system is an ultrafiltration system. The process involves obtaining heterogeneous tissue culture liquid mixture containing the protein of interest during a continuous perfusion fermentation process, continuous removal of large particle impurities from the liquid mixture to obtain clarified tissue culture liquid containing protein of interest, and purification of the protein from the clarified culture liquid through ultrafiltration. Specific flow rate of the tissue culture liquid mixture during continuous perfusion fermentation, continuous removal of impurities and continuous purification is kept constant.

EFFECT: design of an efficient method of isolating and purifying protein.

9 cl, 17 dwg, 2 tbl

 

The technical field to which the invention relates.

The present invention relates to an improved method and system cleanup molecules of interest from a heterogeneous mixture of molecules. More specifically, the present invention is directed to methods of purification of the protein of interest in the flow of tissue culture fluid obtained during continuous perfusion process of fermentation.

A prerequisite for creating inventions

Specialists in the art it is well known that in recent years, with great commercial success was developed several continuous processes of cell cultivation, which is also called continuous processes perfusion. However, the process of selection carried out directly after continuous perfusion process of fermentation is usually periodic process, separated in the physical and material-technical terms from the previous stages of the continuous process. The main objective of phase selection in the implementation of these processes is the collection of large volumes of relatively dilute the culture supernatant. You should pay special attention to the concentration of the product from the point of view of material-technical and spatial requirements, pragya is appropriated to the specified process since the simultaneous removal of pollutants (clean) plays a very significant role in relation to the information to the minimum required number of additional subsequent stages of refinement.

Figure 1 schematically shows a typical modern process of selection of the product obtained during continuous perfusion fermentation, well known to specialists in this field of technology. The continuous perfusion fermentation includes a device (1) for holding cells, which holds a large part of the cells that produce the product in the fermentation system. A continuous stream of cells grown in culture, from the continuous perfusion fermentation, still containing a number of cells, debris and other particles, is pumped by a pump (2) for pumping cells, grown in culture, prefabricated tanks (3) large volume, for example stainless steel tanks. These tanks for storing collected cells grown in culture, must typically be cooled to keep the loss of product caused by the decomposition, within acceptable limits.

After collecting a certain amount, which usually takes from 1 day to 4 days or more, prefabricated tanks cells grown in culture, disconnected from the sterile fermentation VAT is, and the material collected is marked as one batch of cells grown in culture. The next stage is to remove cell debris and particles (stage 2). On an industrial scale it is, as a rule, is carried out by centrifugation (4) followed by ultrafiltration (5) through a semipermeable membrane with a blind end or surround filter (6) with a blind end, followed by ultrafiltration (7) through a semipermeable membrane with a blind end. Others sometimes used way is with tangential microfiltration (or "cross") thread. In any case, the product of the process of particle removal is the party clarified tissue culture fluid or cTCF (8). More the issue of separating particles from a biotechnological products is discussed in standard textbooks, see, e.g., Biotechnology, vol. 3, Bioprocessing, Wiley-VCH, 2nd edition (1996), ISBN:3527283137.

At the next stage (stage 3), the party clarified tissue culture fluid is subjected to additional processing to concentration and purification of the product. It is, as a rule, is carried out by means of ultrafiltration with cross-flow chromatography in a compact layer.

In the case of ultrafiltration with cross flow cTCF is injected into the recirculation tank (9) of the system. The pump (10) is used for protective the Oia material through an ultra-filter with cross flow. The product retained by the membrane and is recycled in the form of sediment in the recirculation tank, while water and impurities smaller pushed through the membrane in the permeate (11) due to the transmembrane pressure, the resulting differential pressure on the ultrafiltration module. Thus, during each pass through the filter cTCF is becoming more concentrated, and the total amount of cTCF is reduced to achieve the necessary concentration coefficient. After achieving the required ratio of the concentration process is stopped and the remaining amount of concentrate (isolate) is drained from the system and collected. Read more the UF process with cross-stream concentration of biotech products is discussed in standard textbooks, see, e.g., Biotechnology, vol. 3, Bioprocessing, Wiley-VCH, 2nd edition (1996), ISBN:3527283137.

In the case of chromatography in a compact layer cTCF pumped through the chromatographic column (12)containing a compacted layer of resin. The product is associated with the resin and subsequent elution, as a rule, in a concentrated and purified form (isolate, 13) with the corresponding buffer (14) for the elution, followed by purification and regeneration of the column with appropriate buffers and solutions (14) for cleaning.

the other options chromatography proposed for the concentration/purification cTCF, are chromatography in the expanding layer and membrane chromatography. By chromatography in the growing layer can be processed solutions containing particles. Still, however, there remains a need filtering isolate after chromatography, though the area of the filter is reduced. In membrane chromatography instead of compacted layers of resin used packages modified microfiltration membranes. The advantage is that the mass transfer becomes more convective than diffusion, which ensures rapid separation. In other respects the above-mentioned process, as a rule, is equivalent to the standard process chromatography in a compact layer. More process chromatography for concentration and purification of biotechnological products is discussed in standard textbooks, see, e.g., Protein Purification, Principles, High Resolution Methods, and Applications, Wiley-VCH, 2nd edition (1998), ISBN:0-471-18626-0.

The bulk of the isolate in the following often cryopreserved and stored for later use on the additional subsequent steps of purification.

Thus, as described above, the process of allocating, as a rule, is a periodic process, separated in the physical and material-technical terms from the previous system is yunogo process. In addition, while the fermentation should be carried out under sterile conditions, the allocation (i.e. the removal of particles and concentration/purification) is carried out essentially with cleanliness, but not in a sterile environment.

The processes of the prior art described above have several problems:

P1. Loss of output and the potential loss of quality due to the long residence time of material in the plant. Cells, grown in culture in the course of the continuous perfusion fermentation, should be collected and stored for substantial periods of time, as mentioned above, before processing of the selected party. The collected cells grown in culture, though chilled, still represent a harmful environment for complex and inherently unstable protein products. This results in a significant loss of product, which reduces plant productivity and increases the cost of the product. In addition, it may be a negative impact on the quality of the product.

P2. For intermediate storage of large amounts of cells grown in culture, the necessary cooling capacity or refrigerated tanks of considerable size, which results in high capital costs and reducing to nothing the advantages of perfusion fermentors, namely the compactness and mobility.

P3. Traditional methods of concentration/purification (e.g., ultrafiltration, chromatography in a compact layer) have a relatively low volumetric capacity, a significant overall cycle time and labour intensive. As a consequence, during the day is usually not more than 1 batch process.

P4. In addition, modern processes and methods of isolation faced with logistical challenges, because the process for fermentation plants are exposed to varying amounts of material, which implies the use of more than one of the fermenter. In the industrial composition of the continuous perfusion fermentation works a different number of fermenters.

The P5. In addition, the extraction is carried out with observance of cleanliness, however, they may not be subject to sterility. The result is often a significant number of rejected parties due to problems with microbial contamination.

P6. Disposal of disposables such as disposable filter sets (nodes), soft tanks and the like, though very desirable in the production of parenteral drugs for humans (for example, to avoid cleaning, assess the degree of cleaning and other PR the problems), is very expensive and in fact often disadvantageous from an economic point of view.

Accordingly, the present invention is to propose an integrated process for continuous separation of a protein capable of operating for extended periods of time in a sterile environment.

The essence of the invention

The present invention is directed to a new apparatus and process for the purification of molecules from a heterogeneous liquid mixture. More specifically, the present invention is directed to a process of purification of molecules of interest from a heterogeneous clarified liquid mixture from which have been removed coarse impurities. The said process includes a step of filtering heterogeneous clarified liquid mixture by continuous ultrafiltration with a specific flow rate below the phase transition point of the molecule of interest, which depends upon the pressure on the portion of the curve flow-TMD (transmembrane pressure), where a specific flow rate is maintained essentially constant throughout the continuous process of ultrafiltration.

According to specific variants of the implementation process proposed by the present invention includes filtering the clarified liquid mixture through an ultrafiltration membrane with an area in square metres, is equal to PR is approximately 0.1 to 2 volume costs clarified liquid mixture in liters per hour. According to another variant implementation of the process proposed by the present invention includes filtering the clarified liquid mixture through an ultrafiltration membrane with an area in square metres, approximately 0.3 to 1 volume of clarified liquid mixture in liters per hour.

The advantage of the process proposed by the present invention, is that it allows filtering the clarified mixture with a specific flow rate, providing near-wall concentration of less than about 20%, less than 15%, or less than 10% exceeding the concentration of sludge on the membrane, no significant concentration polarization.

According to a more specific variant of implementation of the present invention is directed to an integrated, continuous and sterile process perfusion fermentation, removal of particulate and purification/concentration. According to one aspect of the present invention, the above process includes filtration of tissue culture mixture through a separation process, which is selective separation of the protein of interest from the mixture at the specified operating point is below the phase transition protein-dependent pressure section of the curve flow-TMD (transmembrane pressure), with sterile devoid of particles, concentrated and partially purified the selected product, where a specific flow rate during the allocation process is maintained essentially constant at a level below the phase transition point mentioned protein.

According to another aspect of the present invention, the above process is a continuous process of purification of the protein of interest from a heterogeneous tissue culture fluid mixture, including:

(a) receiving through a continuing process of perfusion fermentation heterogeneous tissue culture fluid mixture containing the protein of interest;

(b) the use of the above tissue culture fluid mixture in a continuous process of removing particles, integrated continuous process perfusion fermentation;

(c) removal of coarse contaminants from tissue culture fluid in the ongoing process of removal of particles from getting the clarified tissue culture fluid containing the protein of interest;

(d) the use of the above clarified tissue culture fluid in a continuous treatment process, integrated with the continuous process of removal of particles; and

(e) purification of the protein of interest from the clarified tissue culture fluid during continuous the aqueous cleaning process;

where a specific flow rate mentioned mixture in the course of the continuous perfusion fermentation, continuous process of removal of particles and continuous cleaning process is maintained essentially constant.

According to another aspect of the present invention, the above process is a semi-continuous process of purification of the protein of interest from a heterogeneous tissue culture fluid mixture, including:

(a) receiving through a continuing process of perfusion fermentation heterogeneous tissue culture fluid mixture containing the protein of interest;

(b) the use of the above tissue culture fluid mixture in a continuous process of removing particles that are integrated with the system of continuous perfusion fermentation;

(c) removal of coarse contaminants from tissue culture fluid in the ongoing process of removal of particles from getting the clarified tissue culture fluid containing the protein of interest;

(d) moving mentioned the clarified tissue culture fluid in the surge tank, integrated in a continuous process of particle removal;

(e) periodic use of the clarified tissue culture fluid in the cleaning process, integration is arowana from the surge tank; and

(f) purification of the protein of interest from the clarified tissue culture fluid in the cleaning system of obtaining sterile, devoid of particles, concentrated and partially purified the selected product containing the protein of interest;

where a specific flow rate mentioned mixture in the course of the continuous perfusion fermentation and continuous process of removal of particles is maintained essentially constant.

The present invention is also directed to apparatus for separating a protein of interest from a heterogeneous tissue culture fluid mixture. According to one aspect of the present invention, the above apparatus includes: (a) a system of continuous perfusion fermentation; (b) a system for continuous removal of particles that are integrated with the system perfusion fermentation; and (C) the continuous purification system that is integrated with a system of removal of particles, where the said apparatus is adapted to maintain sterile conditions.

According to another aspect of the present invention, the above apparatus includes: (a) a system of continuous perfusion fermentation; (b) a system for continuous removal of particles that are integrated with the system perfusion fermentation; and (C) a system of periodic cleaning, integrated with the system-the Oia particles, where the above-mentioned apparatus is adapted to maintain sterile conditions.

Mentioned cleaning system may be, for example, ultrafiltration or the system of convective adsorption/desorption or any other system capable of purification or partial purification of the protein of interest from a heterogeneous mixture in an integrated, continuous or semi-continuous sterile system, as described in the present description.

Process and apparatus proposed by the present invention, adapted for continuous processing of heterogeneous liquid mixtures, such as cell culture fluid, with essentially constant rate of flow. According to a particular aspect of the present invention a process and apparatus proposed by the present invention, adapted for continuous processing of heterogeneous cell or tissue culture liquid mixture essentially constant rate of flow below the phase transition point is dependent on the pressure curve flow-TMD (transmembrane pressure) for a continuous period of time during the cleaning process.

These and other aspects of the invention are described in detail hereinafter in the following detailed description of the invention.

Brief description of figures

The accompanying drawings, which are included in the description and which constitute part of, illustrate embodiments of the invention and, together with the detailed description of the variant of the invention, serve to explain the principles of the present invention and its advantages.

Figure 1: Schematic diagram of the conventional continuous process perfusion with subsequent 3 separated in the physical and logistical sense stages of the allocation process (periodic collection of cells, grown in culture, periodic removal of particles and periodic concentration/purification).

Figure 2: Schematic diagram of the 2 options proposed by the present invention device for continuous, integrated and sterile production. A schematic representation of a variant of implementation A1 presents on the left side, and a schematic representation of a variant of implementation of A2 is represented on the right side.

Figure 3: Schematic diagram of the 2 options proposed by the present invention device for continuous, integrated and sterile production. A schematic representation of a variant of implementation of the B1 presents on the left side, and a schematic representation of a variant of implementation of the B2 presents on the right side.

Figure 4: Schematic diagram of the options proposed by the present invention an integrated system the volumes (100) continuous removal of particles, which is the item as proposed by the present invention devices a and proposed by the present invention device C.

Figure 5: Schematic diagram of the additional options proposed by the present invention device which combines several elements to improve or overall performance of the unit (A3), or the efficiency of concentration and separation (A4).

6: Schematic diagram of the additional options proposed by the present invention device C.

Fig.7: Optional implementation of the present invention device, combining successively arranged elements of the device a and device for increasing the overall efficiency of concentration and separation.

Fig: Comparison of examples of the full load capacity in litres per 10" (254,00 mm) filter capsules for traditional periodic process and the options proposed by the present invention device and method for the continuous removal of particles (integrated continuous filtration process, which use identical commercial filter capsules. As the example shows a method of production of recombinant factor VIII blood clotting system.

Fig.9: Example of curve Yes the split-stream (specific flux of dissolved substances in LMH=liters/hour/m 2depending on the transmembrane pressure) and determination of the working point. The circle shows the typical operating point, which will be regulated by the transmembrane pressure for traditional periodic processes. The rectangle shows the preferred work area, which will be regulated by a pump for pumping the solute, for example, in the application method proposed by the present invention device A.

Figure 10: Example of the distribution of the residence time of material in the plant and the average residence time of the material in the installation of an integrated system (300) continuous ultrafiltration, for example, for application method proposed by the present invention device A. was Determined for a single continuous system module 290 cm2(length 62,5 cm), cross-flow 120 LMH, stream sediment of 0.2 LMH, stream solute 2 LMH.

11: Example of allocation of rFVIII (recombinant factor VIII blood clotting) from the result of the continuous perfusion fermentation product, not containing plasma protein, using the options proposed by the present invention device A. a Comparison of the average output allocation proposed by the present invention a continuous way with the average output periodic discharge, includes one standard variance mean-square deviation. To determine the periodic output used 3 consecutive periodic process, while for the continuous process used 3 consecutive points (days).

Fig: Examples of performance offered by the present invention device A. Shows the transmembrane pressure and specific flux integrated system (300) continuous ultrafiltration as a function of the length of a continuous process for 3 different examples. Triangles =100 kDa membrane, recombinant factor VIII blood clotting system (rFVIII); Squares = 10 kDa membrane, recombinant interleukin-2; Circle =50 kDa membrane, genetic engineering glycoprotein (Mr>100 kDa). All the examples refer to the result of the continuous perfusion fermentation product, not containing plasma protein.

Fig: an Example of long-term performance offered by the present invention device And directly applied in a continuous process perfusion fermentation cell line, coexpression 2 protein product (green fluorescent protein GFP and IL-2SA (interleukin 2SA)). Shown transmembrane pressure and specific flux proposed by the present invention system (300) complianc is Noah ultrafiltration as a function of the length of a continuous process. Used 10 kDa membrane.

Fig: an Example of long-term performance offered by the present invention device And directly applied in a continuous process perfusion fermentation cell line, coexpression 2 protein product (green fluorescent protein GFP and IL-2SA). Used 10 kDa membrane. The ratio of the concentrations of both protein products, certain relevant analyses, and the ratio of the volume concentration is presented as a function of the length of a continuous process.

Fig: Example of performance offered by the present invention device C. the product Yield and the pressure drop within about 100 consecutive cycles of adsorption/desorption with convective adsorber (target protein: genetically engineered version of factor FVIII coagulation system of the blood; convective adsorber, the adsorber, Mustang Q, the company Pall Corporation).

Fig: Example of performance offered by the present invention device Century. UV profile and the profile of electrical conductivity during a typical cycle of adsorption/desorption with convective adsorber (target protein: genetically engineered version of factor FVIII coagulation system of the blood; convective adsorber, the adsorber, Mustang Q, the company Pall Corporation).

Fig: Sample performance of the proposed the CSOs present invention device C. Shows data related to SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate) (silver staining) load = sweetlenny solution of cells grown in culture, continuously leaving the system (100) to remove particles and semi-continuous input into the system (400) convective adsorber, and a typical eluate obtained from the adsorption/desorption. (Target protein: genetically engineered version of factor FVIII coagulation system of the blood; convective adsorber, the adsorber, Mustang Q, the company Pall Corporation). The eluate was diluted to a concentration of load before conducting a series of experiments on polyacrylamide gel.

Detailed description of the invention

Definition

The terminology used in this application, except when explicitly stated otherwise, is a standard for this technology. The following definitions of some terms are presented in this description for clarity and certainty in the understanding of the meaning of the claims.

Units, prefixes and symbols can be expressed in the form adopted for them in the SI system. Numeric ranges that are referenced in this description, include numbers that define the range, and also include and support each integer within a certain range. In the absence of ins the instructions the singular shall be construed as meaning "at least one". The section titles used in this description are purely organizational purpose and should not be construed as limiting the described subject matter. All documents or portions of documents referred to in this application, including (but without limitation) patents, patent applications, articles, books and scientific works (monographs), included here in full solely as a reference for any purpose.

The term "whitening" and "clarified" means the removal of particles from the solution, allowing the remaining solution passes through a 0.2 μm membrane.

The term "continuous perfusion fermentation" refers to a stationary system or process of fermentation, which (who) operates without interruption and in which (where) cells or microorganisms are maintained in culture in exponential growth phase by the continuous addition of fresh medium, which is balanced by the removal of a suspension of cells from the bioreactor.

The term "cultivation", "growing", "maintenance" and "reproduction" are synonymous in the sense that the cells remain viable and capable of producing offspring.

The term "concentration" in verb form means removal the water from the solution, so the number of molecules of interest, per unit volume of the remaining solution increases.

The term "concentration polarization" means the accumulation of detainees molecules (layer) on the surface of the membrane, due to a combination of factors: the transmembrane pressure, the speed of the cross flow, the viscosity of the sample and the concentration of the dissolved substance.

The term "continuous" means not interrupted in time, sequence, and/or operation over extended periods of time. In the case of application in relation to the processes of fermentation, clarification and filtration in the present invention, the term "continuous" means that the processes in the physical and logistical integrated, allowing continuous operation over a prolonged period of time sufficient to produce sterile, devoid of particles, concentrated and partially purified the selected product containing the protein of interest. The term "continuous", in the case of its application in relation to the processes of the present invention, it should also be understood as indicating that the process is not periodic manner or in a continuous way in the literal sense of the word. Processes offered this image is Itanium, can be carried out continuously, for example for extended periods of time ranging from 1 day to several months without interruption implementation or sequence of processes. According to the present invention processes are carried out during a continuous period of time, the duration of which exceeds 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks or 3 months, 4 months, 5 months, 6 months or more.

The terms "semi-continuous" and "periodic" means that one or more processes or elements of the integrated system are intermittent or periodic way, while other processes or elements of the integrated system are carried out in a continuous way. For example, according to some variants of implementation of the present invention, the cleaning process is a process of convective adsorption/desorption, which usually requires adsorption heterogeneous mixture adsorbing substrate, resulting, ultimately, is the saturation of the substrate, and which requires completion of the adsorption process and the desorption or selection associated faction. This process has an inherently periodic, although suitable for integration with the next one, which is presumably innymi, processes.

The term "convective adsorption/desorption means chromatographic process in which the mass transfer is mainly by convection. Convective adsorption/desorption is a process in which a portion of the mixture containing the molecule of interest is separated from another part of the mixture through the adsorption of one part of the substrate, followed by desorption of this part of the substrate.

The term "cross-flow or cross-flow fluid" means the fluid flow passing through the upper part of the membrane surface.

The term "integrated", applicable to multiple systems and/or processes, means that the system and/or processes are integrated into the physical and material-technical terms so that they form a single system that can operate continuously. In the context of the system proposed by the present invention which is directed to an integrated continuous or semi-continuous system for the production devoid of particles, concentrated and partially purified protein of interest, the integrated system will directly integrate the various constituent elements of the way sufficient to maintain sterile conditions between the various component elements of the system.

The terms "nutrient medium" (plural) and "culture medium" (singular) are synonyms and are used herein interchangeably, and the use of the above term in one form does not imply the exclusion of other forms.

The term "mixture" means a heterogeneous combination of molecules and compounds containing a molecule of interest, such as protein, and various impurities. The preferred mixture, suggested by the present invention is a tissue culture fluid, representing a heterogeneous mixture of proteins, including exogenous protein of interest, which initially receive as a result of the continuous perfusion fermentation.

The term "layer" means a microscopically thin layer of molecules that can be formed on top of the membrane. It can have a negative impact on the retention of molecules due to blockage of the membrane surface and thereby reduce the flow of filtrate or, in the mode of constant flow, increasing transmembrane pressure.

The term "molecule of interest" means particles or molecules of other species which must be separated from a solution or suspension in the fluid (e.g. liquid). Particles or molecules, predstavlyayushie the interest separated from the fluid and, in most cases, from other particles or molecules in said fluid environment. The size of the subject to separate the molecules of interest will be determined by the pore size of the used membrane. Preferably the molecules of interest are biological or biochemical origin, or get them through transgenic or in vitro processes and include proteins, peptides, polypeptides, antibodies or antibody fragments. Examples of the origin of the flows of nutrients, which are preferred are mammalian cell culture and cell culture of microorganisms, such as bacteria, fungi and yeast. You should also pay attention to the fact that species subject to removal by filtering, are undesirable polypeptides, proteins, components of cells, DNA, colloids, Mycoplasma, endotoxin, viruses, carbohydrates and other molecules representing biological interest, regardless of whether glycosylated or not.

The term "solute" is used as a synonym of the term "filtrate".

The term "selected product" means devoid of particles, concentrated and partially purified product containing the protein of interest. The isolated product is a product that achieved the purification and concentration, the comparison is with my degree, achieved through a process of ultrafiltration or convective adsorption/desorption. The selected product is not necessarily homogeneous, but it will be essentially cleaned compared to the original main body of the product obtained by the fermentation process.

The term "low flow rate" is used interchangeably with the term "stream of filtrate", as it relates to the filtrate. The specific flow rate of sludge means the flow velocity of sediment, normalized to the applied area of the membrane.

The term "essentially constant"applied flow means that the flow is maintained generally at a constant level over a significant period of time during filtering.

The term "tissue culture fluid" means a heterogeneous mixture of components derived from tissue culture medium. According to preferred aspects of the present invention the tissue culture fluid get through a continuing process of perfusion fermentation. "Clarified" tissue culture fluid is a tissue culture fluid, which was subjected to pre-filtration to remove cell debris and other large macromolecules.

The term "transmembrane pressure" and its acronym "TSR" means the average pressure applied to the membrane from the feed side of the filtrate. TSR is calculated by the formula TMP[bar]=[(PF+PR)/2]-Pf, where PF is the pressure, PR is the pressure of the sludge, Pf is the pressure of the filtrate.

The term "allocation" means the number of molecules of interest that can be collected and processed. Is usually expressed in the form of a percentage of the original material or exit.

The term "sediment" means the portion of the sample, which has not passed through the membrane, also known as concentrate.

The term "ultrafiltration" refers to a form of filter in which the use of microporous or semi-permeable membranes are preferred for separating fluids or ions on the basis of different size or molecular weight. Ultrafiltration, typically used for separation by filtration of molecules of molecular weight greater than about 10,000 daltons.

The present invention is directed to an integrated, continuous and sterile process involving continuous perfusion fermentation, removal of particles and purification/concentration. According to one aspect of the present invention, the above process includes filtration of tissue culture mixture through a separation process, which is selective separation of the protein of interest from the mixture at the specified operating point is below the phase transition protein depending on the m from the pressure section of the curve flow-TMD (transmembrane pressure), obtaining sterile, devoid of particles, concentrated and partially purified the selected product, where a specific flow rate during the allocation process is maintained essentially constant at a level below the phase transition point mentioned protein.

According to another aspect of the present invention, the above process is a continuous process, comprising: (a) a continuous by continuous perfusion fermentation heterogeneous tissue culture fluid mixture containing the protein of interest; (b) continuous use of the above tissue culture fluid mixture in the process of removing particles that are integrated with the system of continuous perfusion fermentation; (C) continuous removal of coarse contaminants from tissue culture fluid during the process of removal of particles with continuous receive the clarified tissue culture fluid containing the protein of interest; (d) continuous use of the above clarified tissue culture fluid in the process clean, integrated with a system of particles removal; and (e) the permanent separation of the protein of interest from the clarified tissue culture fluid in the cleaning system for permanent production of sterile, devoid of the hour is CI, concentrated and partially purified the selected product containing the protein of interest.

According to another aspect of the present invention, the above process is a semi-continuous process, comprising: (a) a continuous by continuous perfusion fermentation heterogeneous tissue culture fluid mixture containing the protein of interest; (b) continuous use of the above tissue culture fluid mixture in the process of removing particles that are integrated with the system of continuous perfusion fermentation; (C) continuous removal of coarse contaminants from tissue culture fluid during the process of removal of particles with continuous receive the clarified tissue culture fluid containing the protein of interest; (d) continuous movement mentioned the clarified tissue culture fluid in surge tank, integrated in the process of removing particles; (e) periodic use of the clarified tissue culture fluid in the cleaning process, integrated with the surge tank; and (f) separating the protein of interest from the clarified tissue culture fluid in the cleaning system of obtaining sterile, devoid of particles, kontsentrirovannej and partially purified the selected product, containing the protein of interest, where a specific flow rate mentioned mixture in the course of the continuous perfusion fermentation and continuous process of removal of particles is maintained essentially constant and is equal to the average time-averaged throughput integrated semi-continuous purification process.

Device for the practical implementation of the methods of the present invention

The present invention is also directed to apparatus for separating a protein of interest from a heterogeneous tissue culture fluid mixture. In General, the apparatus includes: (a) a system of continuous perfusion fermentation; (b) a system for continuous removal of particles that are integrated with the system perfusion fermentation; and (C) the continuous purification system that is integrated with a system of removal of particles, where the said apparatus is adapted to maintain sterile conditions. According to another aspect of the present invention, the device includes: (a) a system of continuous perfusion fermentation; (b) a system for continuous removal of particles that are integrated with the system perfusion fermentation; and (C) the periodic cleaning system that is integrated with a system of removal of particles, where the said apparatus is adapted to maintain a sterile condition the conditions. Mentioned cleaning system may be, for example, ultrafiltration or the system of convective adsorption/desorption or any other system can purify or partially purify the protein of interest from a heterogeneous mixture in an integrated continuous or semi-continuous sterile system as described.

Process and apparatus proposed by the present invention, adapted for continuous processing of heterogeneous tissue culture liquid mixture essentially constant rate of flow. According to a particular aspect of the present invention a process and apparatus proposed by the present invention, adapted for continuous processing of heterogeneous tissue culture liquid mixture essentially constant rate of flow below the phase transition point of the protein, which depends upon the pressure of the curve flow-TMD (transmembrane pressure) for a continuous period of time and during the entire cleaning process.

According to specific embodiments the present invention provides two new devices (a, b), each of which consists of 3 different, but fully integrated elements, all of which play a significant role and together form a particularly efficient working platform for continuously is the selection of the protein, which solves the problems of prior art described above.

Three different elements of each device are, first, the integrated system (100) continuous removal of particles, secondly, sterile surge tank (200) and, thirdly, an integrated system of concentration/purification (300, 400, respectively). All three elements are, thus, represent a new developed devices and methods of use of these devices are described in detail below.

To provide integrated, continuous or semi-continuous concentration/purification of the protein product proposed by the present invention device And two options for the implementation of which is shown in figure 2) includes an integrated system (300) continuous sterile ultrafiltration, as proposed by the present invention the device (option 2 implementation of which is shown in Figure 3) includes an integrated system (400) semi-continuous convective adsorption/desorption.

The device proposed by the present invention, is directly integrated with one or more perfusion of the fermenters continuous action and, thus, form a new integrated working space of continuous operation.

Device And

Integrated system (100) continuous-the Oia particles

Figure 2 shows 2 options exercise device As proposed by the present invention. Integrated system (100) continuous removal of particles directly connected to the side of the selection grown in cell culture system (1) continuous perfusion fermentation.

4 shows a more detailed schematic illustration of the options proposed by the present invention of the integrated system (100) continuous removal of particles, which includes a pump (101), a pressure gauge or sensor (107), respectively, of the connecting line (102) and the node (103), comprising multiple units of the consecutive filters. All component parts are connected by hoses and/or pipes.

Pump (101) is a common peristaltic pump providing accurate pumping cells, grown in culture, without any rotating parts or seals in contact with the sterile product. The value of the parameters of the pump and pipeline is selected so as to ensure the necessary flow rate of cells grown in culture obtained from fermentation, cell culture, which is up to 15 volumes of the bioreactor in the day, for example to 9.4 l/h in the case of the fermenter with a capacity of 15 liters and up to 125 l/h in the case of the fermenter with a capacity of 200 liters

Design gauge Il the pressure sensor (107) provides for the possibility of sterilization by autoclaving or irradiation. In modern constructions are used or piezo-resistive sensor reusable stainless steel or gauge reusable stainless steel. Additional improvements, however, may include the use of disposable sensors, which can easily be sterilized by irradiation.

According to a variant of implementation of the Interconnector (102) made of tubes, where the tube clamps (or valves) and the corresponding sterile connectors allow connection of additional sites, including several units of the consecutive filters, without compromising the sterility of the system. Preferably the pipe diameter is selected in such a way as to provide a linear velocity of the fluid within approximately 2 m/s or less at the required flow rate, which allows you to avoid high pressure and shear forces. In presents another embodiment, instead of the sterile connectors are special flexible pieces of pipes that can be made using industrial pipe welding machines without breaking sterility. Such pieces of pipes are made of PVC skin or other suitable polymers.

The composition of the host (103), including a few the to units of consecutive filters includes at least two, preferably greater number of identical units of consecutive filters (as shown in the schematic representation), and at any given time is open only one of the units of the consecutive filters, as shown in the example in figure 4 (105).

The composition of each unit of the consecutive filters includes at least one filter, preferably connected in series one filter for pre-treatment and one fine filter (as shown in Figure 4). If necessary increase the bandwidth for a specific case for each unit of the consecutive filters (105, 106, etc.) itself may also include a large number of parallel-connected filters or units of consecutive filters (not shown).

According to a variant implementation of the present invention, shown in figure 4, the second unit of the consecutive filters node (106) overlaps sensitive to the pressure of the bursting disk or discontinuous pin (104), respectively. In the process, the function of the rupture disc or rupture pin is in the automatic opening of the passage to the second unit (107) of consecutive is introw in case if the pressure on the first unit (105) of the consecutive filters reaches a certain limit, thus ensuring uninterrupted continuation of the filtration process. In the system proposed by the present invention, used commercially available rupture discs or frangible pins, which, otherwise, are used to ensure safe pressure relief. In the present embodiment is applied has proved very useful rupture discs or frangible pins with the limit specified burst pressure of not more than 16 lb/in2(1.125 kg/cm2). There are, however, the range limit of the set pressure.

Each additional unit of the consecutive filters node that includes multiple units of consecutive filters, separated by manual or automatic valve and the other bursting bursting disk or pin. After the entry into operation of the second unit (106) of the consecutive filters opens the valve for the next rupture disk or breaking the pin, respectively, making the next unit of the consecutive filters can act as a backup, etc.

In an alternative embodiment, only apply automatically the s valves, and in the process control system actuates the valve under the influence of the input signal piezoresistive sensor (107) pressure, which can also be sterilized by autoclaving. Applicants have found, however, that the present design, including a rupture disk or breaking the pin, respectively, provides a very stable long term operation.

The nominal limit of the filter is at least 3 μm or less, preferably 0.45 μm and more preferably 0.2 μm. Thus, the unit (6) of the consecutive filters found in the work, delays all other cells and the corresponding cell debris and other particles, resulting in getting free from particles of the output stream (9) the clarified tissue culture fluid (cTCF).

You can apply various commercially available filter materials. The filters used in modern construction is used disposable filter capsules, for example capsules for pre-treatment Sartopure or Sartoclear (company Sartorius, Goettingen) and capsules slim clean Sartobran (company Sartorius, Goettingen), which can be sterilized by autoclaving or irradiation.

As an example of the suggested implementation of the device is TBA, proposed by the present invention, designed for a flow rate of 1 l/min, each unit (105, 106 and so on) of the consecutive filters node (103) includes 3 30" (761,99 mm) capsules for pre-treatment (Kleenpak Ultipleat, Pall Corp., nominal permissible limit of 4.5 μm, each area of 0.75 m2), followed by a 3 20" (507,99 mm) capsule polisher Sartobran P, Sartorius, nominal permissible limit of 0.45 μm/0.2 μm, each area of 1.3 m2). It was found that this particular exercise is particularly useful for industrial production of recombinant factor VIII blood clotting, as well as genetically engineered variant FVIII, including FVIII with a deletion In the domain.

Applicants, however, also found that when using the device and method of the present invention, the removal efficiency of particles using a variety of available filter materials and configurations from different manufacturers (company Pall, Sartorius, Cuno) consistently and significantly improved compared to the traditional batch processes.

Thus, the new device and the process proposed by the present invention are also advantageous in the case of applications with filters new types and configurations, for example with filters such types, which increases the t size of the filter/membrane and with filters such types, which provide education cross-threads or other information, education precipitate filtered to a minimum level, for example, by vibration or rotation of the filter elements.

In another embodiment, the process proposed by the present invention, the host (103), comprising multiple units of the consecutive filters is just one sterile backup unit of the consecutive filters, closed bursting bursting disk or pin, respectively, but there are a large number of units of the consecutive filters. The first unit of the consecutive filters mentioned site works before completion of the processing of a specific pre-determined volume of the load, after which the process switches (manually or automatically) to the next unit of the consecutive filters mentioned site. A certain amount of load is set so that under normal operating conditions, the limit pressure of the rupture disc or rupture pin is not exceeded. If, however, the process of filtration pressure increases more than usual, for example, due to unusually low filterability cells, arossa in culture, reserve unit of the consecutive filters again provides continuous continuous filtration, opening immediately after exceeding the predetermined pressure. After the opening of the reserve unit of the consecutive filters filtering is switched to another unit of the consecutive filters mentioned site, and other reserve unit of the consecutive filters with bursting disk or discontinuous pin is installed without compromising the sterility of the system.

Specialists in the art it is well known that to maintain as the cost of filtering and processing time in the case of periodic processes of removal of particles at a minimum value of the parameters periodically operating units of the consecutive filters should be chosen so that the size of the filter required to provide the desired absolute flow rate (l/h) and the maximum pressure was possibly as low as possible. The required absolute flow velocity, in turn, should be high enough to ensure an acceptable time batch processing of the required amount. This inherently requires high specific flow rate (l/h/m2square Phil is travunia).

In contrast to other comparable optimized periodic filtering device proposed by the present invention, in constructive terms, intended for several times lower specific flow rate, which is kept at a constant level (l/h/m2established square filtering), so that the absolute flow velocity equal to the velocity of the stream grew up in a cell culture obtained by a process of continuous fermentation.

Applicants unexpectedly found that the amount that can be passed through the filter at such low flow rates, is disproportionately higher than the flow rates set at periodic processes.

It is important to note that in the traditional periodic allocation processes such low specific flow rate unattainable due to either limit squares filtering (and therefore costs)or too low absolute flow rate. Similar mainly due to the fact that the equipment for the periodic removal of particles most of the time idle, while it is collecting grown in cell culture for the next batch. Moreover, strikingly disproportionate increase in achievable bandwidth of the filter is in the way, proposed by the present invention enables a significant reduction in the degree of wear of the filter and thus production costs.

Surge tank (200)

The outlet of the integrated system for continuous removal of particles directly and permanently connected to the surge tank (201), as shown in figure 2. This surge tank is sterile container, such as a disposable soft tank or stainless steel tanks with at least one inlet and one outlet, the latter being preferably located at the bottom of the tank. Can be applied to tanks of different sizes and designs. However, the magnitude of the parameters of the surge tank preferably is selected so that it was small compared with the volume throughput of the system to maintain the residence time of product in the tank at a minimum level, i.e. within less than 24 hours, preferably within less than 8 hours and more preferably within less than 4 hours

Applicants have found that such a short time of the product in the system, possible only thanks to the device proposed by the present invention, provides a significant increase in output is unstable in his the essence of protein products, that solves one of the problems of the prior art.

In some embodiments, the exercise device of the present invention, the surge tank is placed on the measuring Mendoza or torque element (202), as shown in the case of devices B1 and B2 in figure 3. This measurement Mendoza or torque element ensures delivery weight signal to a computerized control system (not shown).

In addition, in the embodiment, devices (B2)of the present invention, the buffer tank (204) is connected to the surge tank through the peristaltic pump (203). Under the operating conditions of such a device is used to control properties without particles flow cells, grown in culture, such as conductivity (ionic strength or pH, by adding the corresponding buffer or diluent. In such case, use the optional system (205) mixing and sensors to control the necessary conditions (206), for example, pH or conductivity. In the proposed design solution applied magnetic stirrer; to apply, however, also other mixing system, such as vibrators or pulsating device.

Integrated system (300) continuous concentration/purification

p> The structure of the device And, option 2 implementation which is presented in figure 2, includes an integrated system (300) continuous sterile ultrafiltration. The composition of embodiments of the system of continuous sterile ultrafiltration includes a recirculation pump (301) and recirculation bypass conduit (306), one or more sterile ultrafiltration modules (303) cross-flow pump (305) for pumping the solute, sterile receiving vessel (307) of a dissolved substance on the measuring Mendoza or torque element (309) and the pump (311) for pumping sludge. In addition, it includes instrumentation in the form of the input pressure gauge or sensor (302), a pressure gauge permeate or sensor (304), the output of the pressure gauge or sensor (308), and the recirculation flow (310). In the process, the hole (312) of the system produces a continuous stream of concentrated and partially purified protein product, which can continuously collect, freeze, or be subjected to additional processing.

The composition of the case for A2, proposed by the present invention additionally includes a tank (314) for a buffer or diluent, peristaltic pump (313) for the extra supply buffer/diluent, as well as the flow sensors to monitor the state of the concentrate recirculation bypass pipeline, for example, the sensors (315, 316) pH and conductivity. In the process, a similar device is used to control properties without particles flow cells, grown in culture, such as conductivity (ionic strength or pH, by adding the corresponding buffer or diluent. This device can also be used to add protein stabilizers. Although in the embodiment, A2, proposed by the present invention, himself recirculation bypass pipeline performs the role of the mixing chamber, additional modifications can also be carried out using a surge tank, which is shown for the device In (an implementation option B2), which consists of the elements(203, 204, 205, 206), what will be discussed later in this description (see device description).

The composition of embodiments of the device proposed by the present invention, also includes a programmable control system and data logging, which logs incoming data signals from the measuring devices (for example, but without limitation, pressure, flow rate, mass of the tank, pH, conductivity) and regulates the speed of the pumps according to a predetermined control algorithm.

All pumps (301, 305, 311, 313) are peristaltic the ski pumps, allowing for the pumping flow of the respective liquids without any rotating parts or seals in contact with a stream of sterile product. Applicants have found that such an option is preferred to provide a reliable long-term operation under sterile conditions. In principle, however, can be used sterile pumps of other designs. The value of the parameters of the recirculation pump (301) and related piping is selected so as to ensure reliable adjustment required speed cross-flow within 80-800 l/h to m2the square of the installed membrane, depending on the characteristics of mass transfer applied ultrafiltration module. The value of the parameters of the pump for pumping the solute is set so as to ensure reliable and accurate adjustment of specific flux of dissolved substances in the range 90-99% of the speed of the stream grew up in a culture of cells resulting from the continuous perfusion fermentation. The value of the parameters of the pump for pumping sludge is set so as to ensure reliable and accurate adjustment of the flow of sludge substances within 1-10% of the speed of the stream grew up in a culture of cells resulting from continuous is Noah perfusion fermentation.

Pressurized ultrafiltration modules (303) used to ensure reliable sterile work and sterilised by autoclaving or irradiation. Optimal bandwidth nominal molecular weight is selected based on the molecular weight of the protein product of interest, and it must be confirmed by standard experiments, well-known specialists in this field of technology. A variety of membrane materials, such as polyethersulfone, hydrophilizing polyethersulfone or regenerated cellulose, can be used up until the membrane module as a whole may be sterilized by irradiation and/or autoclaving without damaging the membrane. It is expected that hydrophilic materials can increase efficiency by giving them less prone to contamination.

Applicants have found that the device And is very effective in that case, if the total area of installed ultrafiltration membrane in square metres is within 0.1-2 volumetric flow rates raised in a culture of cells resulting from the continuous perfusion fermentation, in l/H. for Example, if the flow velocity raised in a culture of cells resulting from the continuous perfusion fermentation, composing the th 1 l/h, the total area of installed membrane must be 0.1-2 m2. Applicants have found that the device And operates more efficiently if the area of the prescribed ultrafiltration membrane in square metres is within 0.3-1 flow rate was raised in a culture of cells resulting from the continuous perfusion fermentation, in l/h

In one of the embodiments of the present invention are applied commercially available "disposable" hollow-fiber membrane modules (GE Healthcare, formerly Amersham Biosciences). You can, however, be used sealed membranes and modules of various structures, such as spiral modules, sealed cartridge or capsule with enhanced mass transfer due to adverse currents (for example, vortex flow rotating elements (e.g., rotary disc type filters) or vibrating the filters. It is expected that particularly advantageous in devices of the present invention is the use of pressurized ultrafiltration cassettes, as they provide high mass transfer coefficients at a relatively low desired speed of the cross flow, which leads to a decrease pump performance while reducing complexity and cost of capital invested is I.

The device proposed by the present invention, provides not only continuous, but also sterile in the true sense of the word work, as opposed to working only under aseptic conditions. The applicants were able to achieve this through the design of all system components in contact with the product, so that they stood not only cleaning, but also sterilization by autoclaving, treatment with live steam in situ or gamma irradiation. In the present embodiments implement disposable sealed modules are used for the continuous removal of (100) particles, as well as for continuous ultrafiltration (300). Peristaltic pumps are used to avoid any contact of the product with rotating elements and mechanical seals. Moreover, in the present embodiments, the implementation instead of the rigid pipes are disposable units, composed of flexible pipelines and soft tanks. Disposable items that come in contact with product (e.g., hoses, soft tanks, modules, or groups of elements pre-assembled and sterilized as a unit, thus facilitating the start-up and operation. The system is designed in such a way (e.g., fume hood laminar flow)to the extent of any potential to the of ntact sterile system with the environment, for example for sampling, replacement soft tanks or instrumentation, was reduced to a minimum level. In the present embodiments, the implementation of this device distribution manifold is designed with redundancy for switching from one sterile item (for example, soft tank for receiving product to the next without opening. Additional replacement hoses, modules or soft tanks preferably by using a sterile tube welding machines, not sterile connectors.

Other additional embodiments of the device of the present invention may also include items such as stainless steel tanks, housings filters or hoses that can be sterilized in place, either alone or in combination with disposable items until then, while maintaining the integrity and sterility of long-term operation.

Additional embodiments of the device As proposed by the present invention, designed to handle material from multiple fermenters, larger production units (A3). An example of a schematic image presented on Figure 5. Additional options for implementation are the La increase of the total concentration coefficient and efficiency of the Department through the progressive unification of the 2-speed system (300) continuous ultrafiltration (A4, schematically presented in Figure 5).

Description of the application of the device And

Continuous process perfusion fermentation is carried out for an extended period of time (one campaign), the duration of which usually ranges from 2 weeks to 6 months or more. Tissue culture fluid (TCF), containing the product, cells and debris cells continuously treated with device A. Receiving a stream of sterile, devoid of particles, concentrated and partially purified product ("selected product"), which is continuously released from these devices through the output hole (312). Using a pump (101) of the system (100) continuous sterile removal of particles, cells, grown in culture, continuously pumped through the filter unit (103) at the required rate Qh of the stream grew up in a culture of cells resulting from the continuous perfusion fermentation.

The output of the system of continuous filtration, i.e. the clarified tissue culture fluid (cTCF), continuously enters the surge tank (201). cTCF from the surge tank is subjected to a continuous processing system (300) continuous sterile ultrafiltration at a flow rate equal to the velocity of the flow exiting the continuous perfusion fermentation. Following the journey of the small size of the surge tank in comparison with the established flow rates the average residence time of product in the tank is kept to a minimum, i.e. less than 12 hours, preferably less than 4 hours and even more preferably less than 2 hours

The corresponding cross-flow and, thereby, the mass transfer is governed in ultrafiltration module using a recirculating pump (301). The flow rate of sludge is regulated and controlled by a pump (311) for pumping sludge, which provides a constant and continuous rate Qi flow of concentrated dedicated product coming out of the device And through the outlet (312). Pump (305) for pumping the solute is used for controlling and monitoring the speed of Qp flow of the solute, which is continuously sampled at the output side of permeate of the ultrafiltration module(s) and which consists of water and components of the solution that are small enough to pass through the ultrafiltration membrane (e.g., salts, small proteins).

The flow rate of the solute (Qp) and sediment/selected product (Qi) are carefully controlled and monitored to ensure that it meets the speed Qh flow grew up in a culture of cells resulting from the continuous perfusion fermentation, so that:

Qp+Qi=Qh.

At the same time, the flow rate is regulated and controlled so h is ordinary achieved the required concentration factor cf by satisfying the conditions:

Qi=1/cf·Qh.

For example, to obtain the necessary factor (10 in the selected product) concentration of the product in comparison with the initial concentration of cells grown in culture, Qi stand-level Qi=1/10·Qh with a pump (311) for pumping sludge/selected product, while Qp stand-level Qp=0,9·Qh with a pump (305) for pumping the solute.

Since the speed of the output flow is controlled by means of pumps (305) and (311), the ultrafiltration system automatically selects the flow Qp+Qi from a small surge tank (201).

In the case of the use case for A2 (see right part of Figure 2) sterile stream buffer or water for injection from the reservoir 314 is continuously added to the system of continuous ultrafiltration with constant speed Qb flow through the pump (313) for the extra supply buffer. Thus, the selected product can be freely and continuously be adjusted, for example, from the viewpoint of ionic strength, pH, addition of stabilizers, etc. the flow Rate, therefore, is controlled at the level

Qp+Qi=Qh+Qb.

In addition, the ratio of flow rates can be selected so that was the desired concentration factor cf by satisfying the conditions Qi=1/cf·(Qh+Qb). According to Alta the native variant of this process can be applied only to changes in conditions (e.g., pH, conductivity) by setting Qi=Qh+Qb.

A new way of using the device And also differs from periodic processes ultrafiltration (prior art) in relation to a given operating point of the ultrafiltration. Traditional periodic processes of ultrafiltration is designed for a specific bandwidth through a small area of membrane in a short period of time. Periodic process of ultrafiltration, therefore, is, as a rule, the phase transition point is dependent on the pressure plot in the control area of mass transfer (see Fig.9). The consequence of this is getting a wish high initial specific flux, which, however, significantly and rapidly (over a period of time ranging from seconds to minutes) decreases as the concentration polarization leads to the rapid emergence of osmotic pressure and the formation of a limiting layer of gel (secondary membrane). The consequence of this high near-wall concentration of macromolecules is increased adsorption of the compounds to the inner and outer surfaces of the membrane, i.e. the contamination of the membrane. This contamination over time further reduces the flow of the dissolved substance.

Applicants unexpectedly discovered that in case the e device And many times higher total permissible area load installed ultrafiltration membrane is achieved when operating at the lower end of curve flow from the pressure (see Fig.9).

The normalized near-wall concentration of cwallfully detainee component can be described as follows:

where J is the specific flux of dissolved substances in l/h/m2;

kdthe mass transfer coefficient in l/h/m2;

Cbulkthe concentration of the component in the bulk solution.

Like periodic continuous ultrafiltration ultrafiltration is carried out with the optimized mass transfer coefficient for the information of the concentration polarization to a minimum level. However, in contrast to the periodic ultrafiltration, the applicants set the flux J of a dissolved substance at the low end of curve flow from the pressure (see Fig.9). Due to the exponential dependence of near-wall concentration of cwallon the membrane surface, thus, is significantly lower than it would be in the case of periodic ultrafiltration. For example, shows a variant of the method proposed by the present invention, a target specific flux of dissolved substances at the level of approximately 1/10 achievable coefficient of mass transfer, thereby establishing the near-wall concentration, only 10% exceed the concentration of the main volume (or wasp is ka).

The following Table 1 shows an example of application of the device And an implementation option A1) for a constant release of protein product from the fermenter, in the process of development:

Table 1
Example usage presents a variant of implementation of the device for continuous allocation of the protein product in continuous perfusion fermentation
WorkGoal
Speed Qh flow grew up in a culture of cells resulting from the continuous perfusion fermentation (controlled by the pump 101)5 l/h (120 l/day)
Rate Qp of the flux of dissolved substances (controlled by the pump 305)4,75 l/h
The speed of Qi flow sediment (separate product) (controlled pump 311)0.25 l/h
The specific flow J solute2 l/h/m2
The concentration coefficient cfFold

For each molecule of product to which iterei durability can decide to install sterile continuous ultrafiltration, for example, on the basis of the transmembrane pressure. After exceeding the limit transmembrane pressure continuous sterile ultrafiltration is replaced with another identical unit without compromising the integrity and sterility of the system. This can be done by analogy with the continuous sterile ultrafiltration through use or distribution mains and sterile connectors, or by use of the disposable hose and sterile pipe welding machines.

Device

Integrated system (100) continuous particle removal

Figure 3 shows 2 options exercise of the device proposed by the present invention. Integrated system (100) continuous removal of particles directly connected to the side of the selection grown in cell culture system (1) continuous perfusion fermentation. This part of the device is identical to the device A (see above detailed description of the device and Figure 4).

Surge tank (200)

The outlet of the integrated system for continuous removal of particles directly and permanently connected to the surge tank (201), as shown in Figure 3. This surge tank is sterile container, such as a disposable soft tank or cutting is the storage tank is stainless steel with at least one inlet and one outlet, the latter preferably is located at the bottom of the tank. Can be applied to tanks of different sizes and designs. However, the magnitude of the parameters of the surge tank preferably is selected so that it was small compared with the volume throughput of the system to maintain the residence time of product in the tank at a minimum level, i.e. within less than 26 hours, preferably within less than 12 hours and more preferably within less than 4 hours

In the device In the surge tank is placed on the measuring Mendoza or torque element (202), as shown for the embodiments B1 and B2 in figure 3. This measurement Mendoza or torque element ensures delivery weight signal to a computerized control system (not shown).

In addition, in the embodiment, the device (B2), proposed by the present invention, the buffer tank (204) is connected to the surge tank through the peristaltic pump (203). Under the operating conditions of such a device is used to control properties without particles flow cells, grown in culture, such as conductivity (ionic strength or pH, by adding components to modify the properties of clarified tissue to lauralei fluid, retrieved from removal system of particles, for example the corresponding buffer or diluent or of the corresponding protein stabilizer. In this case, the composition of the presented implementation is also part of the system (205) mixing and sensors to control the necessary conditions (206), such as pH or conductivity. In the proposed embodiment, applied magnetic stirrer; but can also be applied to other mixing system, such as vibrators or pulsating device.

In another embodiment, the device proposed by the present invention, applied 2 surge tank. At any given moment one surge tank directly connected to the system (100) continuous particle removal, taking, thereby clarified liquid, while the other surge tank connected to the system (400) semi-continuous concentration/purification, supplying, as a result, the flow in the convective cycle of adsorption/desorption. Switching between the surge tanks is carried out by the control system, using the weight of the receiving tank to turn on the switch after reaching the said tank maximum volume of the fill.

Integrated system (400) semi-continuous concentration/purification/p>

The structure of the device, 2 variants of the implementation is shown in Figure 3, is an integrated system (400) semi-continuous convective adsorption/desorption.

Integrated semi-continuous convective adsorption/desorption is constructed and the value of its parameters are set so that the flow rate of loading (Qloadthis system was significantly higher flow velocity raised in a culture of cells resulting from the continuous perfusion fermentation, and the process of continuous filtration (Qh), i.e. Qload>>Qh.

The composition of embodiments of the system (400) semi-continuous concentration/purification is bootable pump (401), a multipath valve unit (402) and several buffer tanks (404), a three-way valve (403)connected with sterile tank (413) for receiving waste and one or more convective adservername modules (406), the input and output pressure gauges or sensors (405, 408), additional instrumentation, such as UV-sensor (409), sensors (409, 410) pH and conductivity, flow meter (412)and another three-way valve (407), also connected with the tank (413) for receiving waste and the outlet (414) of the eluate of product.

The composition of embodiments of the device proposed in the present invention is, also includes programmable control system and a data logger (not shown)that registers incoming data signals from the measuring devices (for example, but without limitation, pressure, UV radiation, pH, conductivity, flow rate, mass surge tank) and manages automated valves and pump in accordance with the programmed methods.

Boot pump (401) is preferably a peristaltic pump to avoid direct contact of the product or sterile buffers with any seals or mechanical parts. Applicants have found that such an option is preferred to provide a reliable long-term operation under sterile conditions. In principle, however, can be used sterile pumps of other designs. The magnitude of the loading pump should be chosen depending on the amount of installed matrix of convective adsorber (406) to ensure reliable control within at least 12 volumes matrix/min for Example, in one of the presented embodiments are applied membrane adsorber capsules Mustang (firm Pall Corp.), the volume of the matrix which is approximately 0.3 L. Therefore, the value of the loading pump is set so that to ensure that the flow rate of loading up to 3.6 l/min

The function of the multipath valve unit (402) is to provide a switch between the load-containing product obtained from the surge tank (201), and each of the sterile buffers and lightening solutions from reservoirs (404)containing sterile buffer. In the presented variants of implementation of the device applied to a series of automatic spring-loaded valves that pinch the outside of the hose connected to each reservoir buffer overlap and the opening of each line. Applicants have discovered that these spring-loaded valves provide a particularly advantageous solution for the device In, because they avoid any contact with the product and, therefore, do not require cleaning or sterilization. You can, however, be applied to a variety of commercially available valves, suitable for sterile processing and known to experts in the art, such as diaphragm valves.

In the present embodiment, three-way valves (403, 407) are suitable for autoclaving diaphragm valves. In principle, however, can be used a variety of commercially available valves, suitable for sterile processing, including, for example, spring-loaded valves.

Convecti the hydrated adderbury module (406) contains a chromatographic matrix predominantly by convective mass transfer of the product on the adsorption surface, and in contrast to traditional chromatography sterilized before working through autoclaving, treatment with live steam or irradiation. Predominantly convective mass transfer allows, in contrast to traditional chromatography in a compact layer, to work with very short cycles of adsorption/elution/regeneration, which is used by applicants in the device proposed by the present invention, for the implementation of semi-continuous operation.

According to a variant of implementation of the device proposed by the present invention, the composition of the convective adsorber (406) includes one or more commercially available membrane adsorption capsules with ion exchange chemistry (Mustang, Pall Corporation, or Sartobind, Sartorius). In the above device, however, can be applied to other membrane adsorption material and other configurations, as well as new convection matrix, for example a monolithic matrix, because, in contrast to traditional chromatography excluded gasket resin and the matrix, as a rule, can be sealed in the form of ready-to-use modules.

Moreover, the chemical interaction of other species, including convective affine matrix, composed of specific binding product ligands, also provide unique high eff is aktivnosti device, proposed by the present invention.

According to one of embodiments of the device proposed by the present invention, numerous convective adsorption modules are used in the device as a node, comprising several parallel-connected units of consecutive convective adsorbers, like the system (100) continuous particle removal. The entire site with all by its constituent units of consecutive convective adsorbers sterilized as a single unit, so that the process provides the ability to switch from one unit of consecutive adsorbers to another if the first is over the normal operating period, which is determined, for example, pre-established criteria, such as backpressure when uploading or maximum number of completed cycles. The composition of each unit of consecutive adsorbers includes one or more parallel and/or series-connected convective adsorption modules to increase the binding capacity and/or increase throughput.

It is important to emphasize that the device proposed by the present invention, provides not only continuous, but also sterile the th, in the true sense of the word, in work, in contrast to the work only under aseptic conditions. The applicants were able to achieve this through the design of all system components in contact with the product, so that they stood not only cleaning, but also sterilization by autoclaving, treatment with live steam in situ or gamma irradiation. In the present embodiments implement disposable sealed modules are used for the continuous removal of particles (100), as well as for semi-continuous sterile convective adsorption/desorption (400). Peristaltic pumps are used to avoid any contact of the product with rotating elements and mechanical seals. Moreover, in the present embodiments, the implementation instead of the rigid pipes are disposable units, composed of flexible pipelines and soft tanks. Disposable items that come in contact with product (e.g., hoses, soft tanks, modules, or groups of elements pre-assembled and sterilized as a unit, thus facilitating the start-up and operation. The system is designed in such a way (e.g., fume hood laminar flow)to the extent of any potential contact sterile system with the environment, for example, for selection of PR is b, replacement soft tanks or instrumentation, was reduced to a minimum level. In the present embodiments, the implementation of this device distribution manifold is designed with redundancy for switching from one sterile item (for example, soft tank for receiving product to the next without opening. Additional replacement hoses, modules or soft tanks preferably by using a sterile tube welding machines, not sterile connectors.

Other additional embodiments of the device of the present invention may also include items such as stainless steel tanks, housings filters or hoses that can be sterilized in place, either alone or in combination with disposable items until then, while maintaining the integrity and sterility of long-term operation.

Additional embodiments of the device proposed by the present invention, designed to handle material from multiple fermenters, larger production units (B3). An example of a schematic image presented on Fig.6. Additional embodiments of device offered hereby and the acquisition, designed to improve total factor concentration and efficiency of the Department through the serial connection of multiple systems (400) convective adsorption/desorption with in between appropriate sterile surge tanks (200) (see Fig.6, B4).

Developed additional embodiments of the device of the present invention, to increase the total concentration coefficient and efficiency of the Department through the serial connection of the system (300) continuous ultrafiltration system (400) semi-continuous convective adsorption/desorption through additional surge tank. A schematic representation of a variant example of implementation presented on Fig.7.

Description of the application of the device

Continuous process perfusion fermentation is carried out for an extended period of time (one campaign), the duration of which usually ranges from 2 weeks to 6 months or more. Tissue culture fluid (TCF), containing the product, cells and debris cells continuously treated using a device Century. Receive a stream of sterile, devoid of particles, concentrated and partially purified product ("selected product"), which continuously you the result of the above-mentioned device through the outlet (414). Using a pump (101) of the system (100) continuous sterile removal of particles, cells, grown in culture, continuously pumped through the filter unit (103) at the required rate Qh of the stream grew up in a culture of cells resulting from the continuous perfusion fermentation.

The output of the system of continuous filtration, i.e. the clarified tissue culture fluid (cTCF), continuously enters the surge tank (201).

As soon as the surge tank is filled to a predetermined level, the signal mass or level automatically starts the cycle of adsorption/desorption of the integrated system sterile semi-continuous concentration/purification. The material collected in the equalization tank, quickly loaded the adsorption unit, i.e. within 4 hours, preferably for 2 h and even more preferably within 1 h or less, emptying, thereby surge tank.

In the present embodiments, the implementation shown in Figure 3, the collection devoid of particles in the clarified tissue culture fluid (cTCF) continues all the time, in one and the same surge tank small size. Volume in the surge tank small in size, thus, varies between the minimum and maximum value. In another variant of the implementation the description of which was given above, collecting alternately switches between 2 identical surge tanks.

While cTCF continues to be collected in a surge tank on the loaded adsorber is a series of stages of pre-defined methods chromatography intended for deformirovaniya target product in a concentrated, purified form and preparation of the adsorber to the next loading cycle. The complete cycle thus includes the loading, washing, elution, regeneration and re-balancing, and each of these operations is carried out with one or more corresponding buffers.

Since the flow velocity in the implementation of these stages can be high due to the convective nature of the adsorbers, the total duration of the cycle is kept low, i.e. less than 6 h, preferably less than 3 h and even more preferably less than 1.5 hours Thus, the integrated system is designed in such a way that adsorbent installation ready for the next loading cycle before surge tank is again filled, which allows semi-continuous operation.

The following Table 2 shows an example of the method of application presents options for the implementation of the ing device, proposed by the present invention, for the selection of recombinant human factor VIII blood clotting system (data presented large-scale production). The above method proved to be extremely beneficial. The output and performance of each cycle of adsorption/desorption were similar to the corresponding figures of the periodic process, and the total yield of product is increased by more than 10% due to the short residence time of product in the installation and thereby minimize its collapse. The same method has proved very useful for the selection of genetically engineered FVIII variants, including FVIII with a deletion of the b domain, which is significantly different from reprezentirovannoe FVIII in size and other characteristics. We hope that this method will be also suitable for other proteins and biomolecules.

By itself, the technique of chromatography (chemistry and consistency of application buffers, the amount of loading and flow rate) can be developed in experiments on periodic chromatography for each individual molecules and subsequently easily converted for use with the variants of implementation of the device proposed by the present invention.

Table2
Example usage presents a variant of implementation of the device for continuous allocation of FVIII and FVIII variants in continuous perfusion fermentation
Goal
Speed Qh flow cells grown in culture under continuous perfusion process of fermentation [l/day]2000
Surge tank: maximum working volume Vs [l]200
Volume loading [volumes matrix]600
The amount of adsorption matrix installed in the device In [ml]260
The flow rate of loading [volumes matrix/min]12
Volume loading [l]156
The time of loading [min]50
Total time chromatography [h] (method including loading, washing, elution and several stages of regeneration/re-equilibration)1,5
The downtime of the adsorber/cycle [h] 0,372
Cycles/period of time lasting 24 hours12,8
Collection time [h]1,872
The approximate average duration of stay of the material in the device acquisition time + loading + elution)2,7

For each molecule of product criteria durability can be defined for convective adsorption installation, for example, on the basis of the pressure in the process of loading or discharge. As a rule, is estimated and the maximum number of cycles nmax. After adsorption plant was used for semi-continuous operation for nmaxcycles, it is replaced with another identical adsorption installation without compromising the integrity and sterility of the system. In the presented variants of implementation this is done by analogy with the continuous sterile filtration through the use or distribution mains and sterile connectors, or by use of the disposable hose and sterile pipe welding machines.

In the case of the use case for the device proposed by the present invention, is shown in Figure 3 on the right side is s, sterile flow of the buffer solution to regulate the pH of the stabilizing solution or water for injection is added continuously or periodically from the sterile tank (204) using a pump (203) for additional buffer. Thus, the state of cTCF can be freely adjusted, for example, from the viewpoint of ionic strength, pH, addition of stabilizers, etc.

Advantages of the invention

The device proposed by the present invention, and appropriate ways of using these devices solve the problems of the conventional processes of selection described above (see General background of invention).

In all variants of the implementation of the devices and corresponding methods of using these devices, the time of the product in a potentially harmful environment is minimized, which greatly increases the yield and quality of the unstable inherently complex biological products. The plant capacity can be increased, and the cost of the product is reduced.

Moreover, devices and related methods eliminate the need for refrigeration containers or refrigerated tanks of considerable size for the intermediate storage of large amounts of cells grown in culture, which reduces the cost of capital investment in the installation and allows you to identify who realize the benefits of perfusion fermentation, consisting of compactness and mobility.

Embodiments of the devices of the present invention, and appropriate ways to reduce labor costs compared to traditional time-consuming periodic processes with a high degree of automation. The new devices provide the possibility of continuous operation 24 hours a day, for extended periods of time, achieving maximum performance and hardware utilization.

In addition, the device proposed by the present invention, eliminates the logistical difficulties in plants, comprising one or more fermenters. Options for implementation may handle the material received through one or more processes perfusion fermentation.

It is important to note that as new devices and methods enable the implementation of work in completely sterile conditions, eliminates the problems associated with microbial contamination and endotoxin, which cannot be achieved by aseptic processing with subsequent simple sterile filtering.

In addition, the device proposed by the present invention, to avoid or minimize the need to assess the level of treatment through the use of agnor the basic details. Due to the unique characteristics of the devices and methods of the present invention, each single module, as well as hoses, soft tanks and units can be used for an extended period of time (during the campaign)that dramatically reduces costs and greatly increases the attractiveness of recycling of disposable items from an economic point of view.

Presents embodiments of the devices a and b, proposed by the present invention, and corresponding methods proved to be very suitable for the production of recombinant factor VTII of the blood coagulation system, as well as genetically engineered FVIII variants, including, but without limitation, FVIII with a deletion In the domain. It is assumed, however, that the present invention is equally suitable for other proteins and biological molecules, in particular complex and inherently unstable proteins, such as factor VII, factor IX, factor X, etc.

The advantages of the device and corresponding method

On Fig shows an example of an unexpected increase in the bandwidth of the filter, which was discovered by applicants for integrated system (100) continuous particle removal.

Figure 10 shows a typical distribution of the residence time of material in the plant and the average residence time of p is oduct in the system (300) continuous ultrafiltration option exercise device And, proposed by the present invention defined by the adsorption of UV radiation sediment at 280 nm with a model protein under typical conditions. As shown, the average residence time of product in the system is only about 40 minutes So the total time of the product in the present embodiment, device a, a pipeline for the discharge from the fermenter cells grown in culture, to the end of the concentrated sludge (isolate), 1-2 h or less. This is less than 1/10 of the time of stay of the material (28 hours or more) in the traditional periodic allocation process, in which the product (cells grown in culture) are collected for at least 24 hours (up to several days), after which the product is treated, usually for at least 4-10 hours

Figure 11 shows the comparison of the obtained total yield of recombinant factor of the blood coagulation system (rFVIII) in the case of continuous perfusion fermentation without plasma protein as in the case of the traditional process of periodic selection (periodic filtering plus periodic ultrafiltration), and in the case of use of the device As proposed by the present invention and a corresponding method. As shown on the above figure, a continuous process proposed by the present invention, both the accounts for a significantly higher yield, the result may be to increase production capacity and reduce production costs.

In the case of use of the device And according to the method corresponding to the present invention, the transmembrane pressure integrated continuous ultrafiltration increases over time, while the specific thread on the membrane (l/h/m2/bar) decreases at a constant volumetric throughput. This is true for all ultrafiltration processes and due to effects such as concentration polarization, formation of a layer of gel and pollution. However, in contrast to the periodic ultrafiltration, as can be seen from the example in Fig, changes of pressure and specific flux in the case of the device And happen very slowly, allowing continuous operation for weeks before they need cleaning or replacement of the membranes. In addition, the degree of change and efficiency are completely insensitive to produce the product or cell line used for continuous perfusion fermentation (Fig). Thus, the device As proposed by the present invention and the corresponding method is also ideal as a General framework for the rapid production of various proteins, as they reliably and p is escanuela work with different target proteins from different cell lines.

Unexpectedly, applicants have found that the negative effects of the formation of the layer of gel and contamination in the case of the device And actually reduced to such a low level that can be handled much more on the size of the installed ultrafilter before they need cleaning or replacing filters. Fig demonstrates reliable long-term performance of the device As proposed by the present invention. After about 25 days transmembrane pressure unexpectedly showed a tendency to stabilize in the quasi-stationary state, which suggests an even higher long-term availability. On day 27, the flow rate of sludge was purposefully doubled to test the effect of a higher bandwidth. After 34 days produced a short rinse with sterile 0.5 M NaOH solution without opening the system, thereby maintaining the integrity and sterility of the entire system. After that, the transmembrane pressure has stabilized again, or at least increased, however, with an extremely low speed. After 70 days of continuous sterile work recirculation flow rate was purposefully reduced by half to test the impact on system performance. As expected, the transmembrane pressure start led is to rise at a slightly greater speed due to reduced mass transfer, thus, increased parietal concentration on the membrane surface. However, before the system was turned off, she successfully and reliably worked for 95 days. 1 m2square membrane as a whole accounted for about 4500 processed litres with a minimum expenditure of manual labor (daily sampling). For comparison, the optimized traditional periodic ultrafiltration with the same version of application is 45 times smaller allowable load (approximately 100 l/m2) and requires at least a full time job 1-2 operators.

The surprise is that the selectivity of the device As proposed by the present invention, in particular its integrated system (300) continuous ultrafiltration was significantly higher selectivity traditional batch process. Specialists in the art it is well known that in conventional periodic ultrafiltration of the detainees macromolecules at the initial stage of the process produces secondary membrane (layer), which reduces bandwidth average molecular weight. The result is delayed as the target molecule, and contaminating proteins smaller, which makes it almost impossible substantial simultaneous cleaning. Thus, if Tr is traditional periodic ultrafiltration is rarely possible separation of proteins, with a coefficient differences with respect to their molecular weight, less 10. However, as shown in Fig, in the case of an integrated process for continuous ultrafiltration, proposed by the present invention, it is possible to adjust the conditions for effective separation of IL-2SA (approximately 16 kDa) and green fluorescent protein GFP (27-30 kDa). This separation efficiency higher than expected, and allows for simultaneous concentration and purification.

The advantages of the device and corresponding method

Fig illustrates operational characteristics of a device proposed by the present invention. Using commercial convection adsorber (Mustang Q, Pall Corporation, 15-layer module), made about 100 consecutive cycles of adsorption/desorption, concentrating and purifying recombinant FVIII variant from the culture obtained by a process of continuous perfusion fermentation. Average the resulting output was approximately 95% (the variation is a consequence of the analytical differences), while the pressure remained relatively constant over the total number of executed cycles. Thus, it can be accurately stated that to replace adsorbing installation can be carried out at least 100 consecutive cycles.

As the show is but a detailed description of the application of the device, proposed by the present invention, the total average residence time of the product in the present embodiment, is less than 3 h before it is elyuirovaniya in concentrated, purified and stabilized form in the appropriate buffer. This is significantly less time in traditional periodic allocation process (more than 24 hours), carried out once a day, resulting in a significantly higher yield of labile inherently protein products. In the present embodiment described above, during the day time is approximately 13 cycles, which in the context of Fig means that adderbury site semi-continuous steps will need to be replaced only every 7-8 days, which is carried out without compromising the sterility and business continuity.

On Fig shows an example of UV-profile and profile of electrical conductivity during one typical cycle of adsorption/desorption and regeneration devices Century Shows that can be downloaded over 450 volumes adsorber (CVs), while the product eluted as a very sharp peak. The content of pollutants in the stream is significantly reduced during the phase of loading, as well as for flushing and cleaning (regeneration phase).

Fig illustrates the efficiency of cleaning item is ocess, proposed by the present invention, which includes semi-continuous convective adsorption/desorption. As an example polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate isolate variant FVIII. As can be seen, the fraction of the eluate, containing 95% loaded variant FVIII (as determined by a separate analysis of the activity), contain much less protein than the load, and are thus purified. In the eluate (isolation) does not show additional bands of decomposition, indicating the excellent quality of the product.

Summing up, the device proposed by the present invention can provide the same cleaning efficiency that is comparable periodic processes, at the same time reducing to a minimum level of loss of output unstable inherently protein products, as well as problems with the quality of the product due to information residence time of product in the installation to a minimum level. Simultaneously, greatly reduced labor costs due to the high degree of automation inherent in the process corresponding to the present invention, requiring minimal operator intervention.

Although the present invention has been described with some detail to illustrate and example of order p is thinking, professionals in this area of technology will be obvious opportunity to practice, certain changes and modifications. Therefore, the description and examples should not be construed as limiting the scope of invention, which is defined by the attached claims.

Accordingly, it is understood that embodiments of the present invention described in the description and offering advanced filtering method for obtaining high yield of the molecule of interest from the flow of nutrients, perform illustrative only role the application of the principles of the present invention. From the above description it will be apparent ability to make changes in the form, methods of application and use cases disclosed elements of the invention without departure from the invention or the scope of the attached claims.

1. The process of separation of the protein of interest from a heterogeneous tissue culture fluid mixture and its purification, including:
(a) receiving through a continuing process of perfusion fermentation heterogeneous tissue culture fluid mixture containing the protein of interest;
(b) the use of the above tissue culture fluid mixture in a continuous process of removing particles, and the ting that is integrated with the continuous process perfusion fermentation;
(c) removal of coarse contaminants from tissue culture fluid in the ongoing process of removal of particles from getting the clarified tissue culture fluid containing the protein of interest;
(d) the use of the above clarified tissue culture fluid in a continuous process of purification by ultrafiltration, integrated with the process of removing particles; and
(e) purification of the protein of interest from the clarified tissue culture fluid during continuous cleaning process;
where a specific flow rate mentioned mixture in the course of the continuous perfusion fermentation, continuous process of removal of particles and continuous cleaning process is maintained essentially constant.

2. The process according to claim 1, further comprising filtering the clarified tissue culture mixture with a specific flow rate, providing near-wall concentration of less than approximately 20% exceeds the concentration of the precipitate on the membrane.

3. The process according to claim 1, further comprising filtering the clarified tissue culture mixture with a specific flow rate, providing near-wall concentration of less than about 15% exceeds the concentration of the precipitate on the membrane.

4. The process according to claim 1, optionally in the with filtering the clarified tissue culture mixture with a specific flow rate, providing a wall surface concentration of less than about 10% exceeding the concentration of the precipitate on the membrane.

5. The process according to claim 1, further comprising filtering the clarified tissue culture mixture through an ultrafiltration membrane with an area in square meters, equal to approximately 0.1-2 volumetric costs of the product obtained by a process perfusion fermentation, in l/h

6. The process according to claim 1, further comprising filtering the clarified tissue culture mixture through an ultrafiltration membrane with an area in square meters, equal to approximately 0.3-1 volumetric flow rate of the product obtained by a process perfusion fermentation in litres per hour.

7. Apparatus for separation of the protein of interest from a heterogeneous tissue culture fluid mixture and its purification, including:
(a) a system of continuous perfusion fermentation, which is adapted for continuous production of tissue culture fluid containing the protein of interest, with essentially constant volumetric flow rate;
(b) a system for continuous removal of particles that are integrated with the system perfusion fermentation and adapted for continuous reception of tissue culture fluid from it and continuous production clarified the th tissue culture fluid; and
(c) a continuous purification system that is integrated with a system of removal of particles and adapted to continuously receive the clarified tissue culture fluid from it and continuous production of the selected product containing the protein of interest, and this continuous purification system is a system ultrafiltration;
moreover, the above-mentioned apparatus is supported in a sterile environment.

8. The apparatus according to claim 7, characterized in that the system includes ultrafiltration ultrafiltration membrane with an area in square meters, equal to approximately 0.1-2 volumetric costs of the product obtained by a process perfusion fermentation, in l/h

9. The apparatus according to claim 7, characterized in that the system includes ultrafiltration ultrafiltration membrane with an area in square meters, equal to approximately 0.3-1 volumetric flow rate of the product obtained by a process perfusion fermentation, in l/H.



 

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