KR20110085981A - Clarification process - Google Patents

Abstract

The present invention relates to a method for purifying cell broths containing cells by the following steps: contacting the cell broth with particulate anion exchange material having an intrinsic particle density of 1.4 to 3 g / ml, sufficient incubation time Allowing, and separating the resulting cell pellet from the supernatant layer. The present invention also relates to a method for recovering a desired secreted biological material from a cell broth containing cells producing a desired secreted biological material by the following steps: a cell broth and an intrinsic particle density of 1.4-3 g. / Ml contacting the particulate anion exchange material, allowing sufficient incubation time to form the cell pellet and supernatant layer, separating the resulting cell pellet from the supernatant layer, and the desired secreted biological material from the supernatant layer Step to extract. Optionally, residual biological material can be further extracted from the resulting cell pellet in one or more washing steps.

Description

Purification method {CLARIFICATION PROCESS}

The present invention relates to a method for purifying cell broth and a method for recovering a desired secreted biological material from a cell broth containing cells producing a desired secreted biological material.

Fermentation production of biological materials, such as pharmaceuticals, in particular monoclonal antibodies, yields complex cell broths in which the biological material must be isolated and purified in multiple steps.

As a first step, solid materials such as cells and cell debris must be separated from the cell broth fluid-this step is called purification. In many cases, the biological material is extracellular and therefore will be present in the cellular broth fluid.

Examples of purification methods used to date include centrifugation, filtration (eg, microfiltration, deep filtration, and filtration through absolute pore size membranes) and expanded bed chromatography. Agglomeration can be used to enhance any of the above purification methods, especially those combined with filtration.

Known flocculants for this purpose can range from simple electrolytes to synthetic poly-electrolytes (eg DEAE dextran, acrylic-based polymers, polyethylene amines) or inorganic materials (eg diatomaceous earth or pearlite). Recently, the use of chitosan has been developed for this purpose.

The disadvantages of using multiple flocculants are, in particular, that they can bind the desired biological material of interest, that they can deactivate the desired biological material of interest, that the flocculation process takes too long and / or that the flocculant is medical It can be difficult or expensive to manufacture with the high quality required for the application. In addition, flocculants must be removed from the final purified molecule, which requires expensive and time-consuming assays to confirm their removal.

The method according to the invention has none of the above disadvantages.

It is an object of the present invention to provide a method for cost-effective purification of cell broth harvested from a bioreactor and containing mammalian cells as well as secreted biological material.

It is a further object of the present invention to provide a method of recovering a desired secreted biological material from a cell broth containing mammalian cells producing a desired secreted biological material.

In certain embodiments, the present invention relates to a method of purifying a cell broth containing the desired secreted biological material having overall positive charge in the cell broth as well as mammalian cells and culture medium by the following steps:

a. Contacting the cell broth with particulate anion exchange material having an intrinsic particle density of 1.4 to 3 g / ml,

b. Allowing sufficient incubation time to form cell pellet and supernatant layers, and

c. Separating the resulting cell pellet from the supernatant layer.

In the context of the present invention, "cell broth" means a cell culture inoculated with intact mammalian cells, which may further contain secreted biological material as well as culture medium as defined below. In the process of the invention, it may also be desirable to dilute the starting material from the bioreactor to the desired cell density, especially when the cell density is very high. For the purposes of the present invention, such diluted substances are still included in the term cell broth.

As a practical upper limit, the method according to the invention can be carried out at a cell density of 175 × 10 ⁶ cells / ml or less, more preferably 130 × 10 ⁶ cells / ml or less.

Cell density can be measured using a cell counter such as Vi-CELL ™ (using trypan blue exclusion method), but other suitable methods include cell count, compressed cell volume measurement or Coulter counter (using Electrical Sensing Zone Method). do.

If the initial cell density is greater than 130 × 10 ⁶ cells / ml, it is preferred to first dilute the cell broth. Indeed, it is preferred that the cell broth with an initial cell density greater than 100 x 10 ⁶ cells / ml be first diluted. Preferably it can be diluted to a cell density of 80 x 10 ⁶ cells / ml or less. The cell broth may be diluted with a solution that does not significantly change the environment of the cell, ie, an isotonic solution such as PBS, so as not to cause lysis of mammalian cells.

By "secreted biological material" is meant herein a biological material that is mainly released (actively or passively) into the culture medium upon production by mammalian cells.

"Want" means herein that the biological material is intentionally produced using mammalian cells.

By "total positive charge" of the desired secreted biological material is meant herein the electrostatic contribution of the positive and negatively charged ionizers on the biological material under solvent conditions in the cell broth to produce a net positive charge. The overall charge of the biological material is based on the pKa of acidic and basic residues and the pH of the solution-in this case the pH of the cell broth. In order for a biological substance to have a net positive charge in the cell broth, the pi (the pH at which the net charge is zero) of the substance must be higher than the pH of the cell broth.

"Culturing medium" refers herein to the extracellular environment of a cell, which contains nutrients and other ingredients that support the growth and production of cells, but also from waste products or host cell proteins (HCP) or lysed cells It may contain. The composition of the culture medium may vary during the culturing process of the cells, and at least one original component may be greatly reduced in the clarification step.

"Contact" herein means introducing an anion exchange material into a cell broth and settling the cell (eg, under gravity or by centrifugation).

"Anion exchange material" means herein a particulate weak or strong anion exchange chromatography medium. Anion exchange materials generally comprise a carrier, which may be an organic or inorganic material or a mixture of organic and inorganic materials. Suitable organic materials are agarose based media and methacrylates. Suitable inorganic materials are silica, ceramics and metals. The particles may preferably be 15 to 150 μm in size. More preferably its size is from 15 to 70 μm. The particles may have a density suitable for sedimenting cells relatively quickly from the cell broth, but the density should not be too high because it has been observed that too dense particles do not settle.

The method of the present invention applies particulate anion exchanger material having an intrinsic particle density of 1.4 to 3 g / ml. Preferably, the particle density is about 2 g / ml. Suitable methods for determining the particle density of anion exchange material are described in the Examples section. Suitable anion exchange materials that meet these requirements are, for example, materials having particles consisting of or containing silica, ceramic materials or metal cores.

“Moderate incubation time” means herein the time for cell precipitation to produce a clear cell pellet volume and supernatant layer.

"Separation" means herein any method for removing the supernatant from cell pellets, such as by decanting or drawing out the supernatant, or withdrawing pellet from a tube, for example, through a port in the bottom.

A "supernatant layer" is a volume of liquid superimposed as a result of sedimentation. The supernatant layer may still contain (usually will) cells at a cell density significantly lower than the initial cell density.

According to a further embodiment, the invention recovers the desired secreted biological material from mammalian cells and culture broth as well as from the cell broth containing the desired secreted biological material with the total positive charge in the cell broth by the following steps: It's about how:

a. Contacting the cell broth with particulate anion exchange material having an intrinsic particle density of 1.4 to 3 g / ml,

b. Allowing sufficient incubation time to form the cell pellet and supernatant layer,

c. Separating the resulting cell pellet from the supernatant layer, and

d. Isolating the desired secreted biological material from the supernatant layer.

The present invention further provides a method of recovering a desired secreted biological material from mammalian cells and culture broth as well as from a cell broth containing the desired secreted biological material having a total positive charge in the cell broth, the resulting precipitate further comprising: The method is treated by the following steps:

e. Resuspending the resulting precipitate,

f. Allowing sufficient incubation time to form the cell pellet and supernatant layer,

g. Separating the resulting cell pellet from the supernatant layer, and

h. Isolating the desired secreted biological material from the supernatant layer.

In a further method according to the invention, steps e to h of the method are repeated one or more times.

In a further preferred method according to the invention, the resulting cell pellets are added to a solution which does not significantly change the environment of the cells such as to cause lysis of mammalian cells, such as an aqueous solution of salt (preferably isotonic), more preferably PBS. Resuspend.

Preferably the supernatant layer is collected and the desired secreted biological material is extracted from the pooled supernatant.

Examples of mammalian cells include CHO (Chinese hamster ovary) cells, hybridomas, BHK (cub hamster kidney) cells, myeloma cells, human cells such as HEK-293 cells, human lymphoblasts, E1 immortalized HER cells, Mouse cells, such as NS0 cells, are included. More preferably, El immortalized HER cells, most preferably PER.C6 cells, are used.

In a preferred embodiment, the cells of the methods of the invention are E1-immortalized HER cells, more preferably PER.C6 cells (see US Pat. No. 5,994,128, the contents of which are incorporated herein by reference). PER.C6 cells are exemplified as cells deposited under ECACC No. 96022940 (see, eg, US Pat. No. 5,994,128, EP 0833934 B1, the contents of which are incorporated herein by reference).

Cell broth for clarification can be obtained by any cell culture method suitable for reaching cell densities of mammalian cells of at least 15 × 10 ⁶ cells / ml. Suitable methods for this are described for example in WO2005095578, WO2004099396 and WO2008006494. These contents are incorporated herein by reference.

Thus, for example, a biological material that can be produced by a cell by expressing (recombinant) gene coding is for example a blood such as a virus or (recombinant) protein, in particular a protein from a receptor, enzyme, fusion protein, blood aggregation cascade Proteins, for example multifunctional proteins such as erythropoietin, for example viral or bacterial proteins for vaccines; For example immunoglobulins such as IgG or IgM. Preferably the protein, more preferably the immunoglobulin or part thereof, is produced by the cell. Preferably, biological substances such as proteins or vaccines produced by the cells can be used as active ingredients in pharmaceutical preparations. In the context of the present invention, the terms 'product' and 'biological material' are interchangeable.

Suitable methods for extracting the desired secreted biological material from the supernatant layer include, for example, filtration (eg, deep filtration, microfiltration, ultrafiltration, diafiltration), chromatography (eg, size exclusion chromatography, affinity chromatography, cation). Exchange chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography), aqueous two-phase extraction, precipitation or centrifugation. Advantageously, the desired biological material can be extracted very effectively by cation exchange chromatography. For immunoglobulins as desired biological materials, affinity chromatography, in particular Protein A chromatography and cation exchange chromatography, are particularly suitable separation methods.

1. Supernatant cell density over time for various anion exchange materials. Cell density was measured by Vi-CELL.
2. Supernatant volume over time for various anion exchange materials. In each case the total volume was 24 ml.

Example

sign:

X _t = total cell density, cell / ml

Si-PEI = Bakerbond Wide-Pore PEI Prep LC Packing Grafted Silica Beads (JT Baker)

DEAE Hyper D = diethylaminoethyl grafted ceramic beads (Pall)

Super Q = quaternary amino functional group (Tosoh) on methacrylate support.

TP DEAE = diethylaminoethyl functional group (Tosoh) on methacrylate support.

PBS = phosphate buffered saline

The following clarification experiments were performed using PER.C6® cells prepared according to the procedure shown in WO2008006494. PER.C6® cells produced antibodies.

Method of Determination of Particle Density of Anion Exchange Material

Particle density (g dry / ml) was determined by specific gravity bottle. The volume of the 10 ml specific gravity bottle (# 15123R-10 Kimble Glass, Inc., Vineland, NJ) was determined as follows:

1. Weigh a clean, dry, empty pycnometer ( W _i ) assembly.

2. Fill the pycnometer completely with water at room temperature.

3. Insert the thermometer in the overflow tube and wipe off excess water. Cover the lid of the overflow tube.

4. Reweigh the pycnometer ( W _f ) assembly.

5. Determine the volume of pycnometer by the following formula:

Where ρ _H2O (T) is the density of water with temperature.

The particle density was then determined as follows:

a. Weigh the clean, dry, empty pycnometer ( W _i ) assembly.

b. Weigh dry the pycnometer ( W _r ) (dried at 50 ° C. for 3 hours) and weigh the anion exchange material.

c. Fill the pycnometer with water completely at room temperature.

d. Insert the thermometer in the overflow tube and wipe off excess water. Cover the lid of the overflow tube.

e. Reweigh the pycnometer ( W ' _f ) assembly.

f. The particle density ρ _d is determined by the following formula:

The measurement was performed three times.

Particle Density of Anion Exchange Material

The particle density of various anion exchange materials was determined using the method described above.

The results are summarized in the table below:

Example 1 Comparison of Purification by Different Density Anion Exchange Materials

Si-PEI (15 and 40 μm), ToyoPearl Super Q (35 and 65 μm), ToyoPearl DEAE (35 and 65 μm) and DEAE Hyper D containing PER.C6® cells producing monoclonal antibodies (MAbs) Evaluated with cell broth. PER.C6® cells were prepared according to the method outlined in WO20088006494. X _t was 98.8 × 10 ⁶ cells / ml on Day 13 of the reactor. The material was diluted to 75 × 10 ⁶ cells / ml with Dulbecco's PBS (5 mS / cm) to a final volume of 20 ml. Anion exchanger was added as a 50/50 slurry (total volume was 24 ml) and the cells were allowed to settle until the pellet volume was constant (60 minutes).

Subsamples of harvest and supernatants were analyzed by analytical Protein A chromatography to determine product recovery. I decided to recover:

The assay was calibrated for biomass if necessary, ie when the cell density was very high, and the cells contributed significantly to the working volume.

1 shows the supernatant cell density over time for each anion exchange material as well as a control to which no anion exchange material was added. In each case, accelerated cell sedimentation was observed compared to the control. Smaller particle sizes reduce the supernatant cell density to less than 10 × 10 ⁶ cells / ml, while larger particle sizes appear to reduce the cell density to 11-15 × 10 ⁶ cells / ml.

2 shows the supernatant volume versus time for each anion exchange material. The addition of Si-PEI material produces the highest amount of supernatant volume, which corresponds to the densest pellets. In this case the pellets accounted for 40% of the total volume, whereas in the control group the pellets accounted for 63% of the total volume. Si-PEI materials have higher densities than methacrylate and agarose based materials, which apparently allow for denser pellets and faster settling rates. Ceramic Hyper D materials have a medium density and a corresponding medium sedimentation rate and pellet volume.

Example 2 Purification of Desired Biological Materials

The initial cell density of 175 × 10 ⁶ cells / ㎖ the cell culture harvest was diluted to ~ 75 × 10 ⁶ cells / ㎖ in PBS (the initial volume of 1.7 ℓ). After dilution Si-PEI chromatography media was added to the harvest (0.1 L of Si-PEI resin per 1 L of diluted harvest). Cells were allowed to settle for ˜60 minutes. The product containing the supernatant was decanted and the precipitated cells were washed twice with PBS. Initial supernatant was pooled with two washes to maximize product recovery (˜95%). The pooled pools contained less than 5 x 10 ⁶ cells / ml and the HCP content was reduced by 59%.

The product recovered after Si-PEI sedimentation was further purified by deep filtration. Deep filtration removes the primary filter (typically 10 or 5 μm pore size) used for further reduction of cell volume, followed by removal of smaller particles and clarified harvest for aseptic filtration, usually through a gradient 0.8 / 0.2 μm filter. Resulting secondary filter (usually 3 or 1 μm pore size). The deep filtration train may be a Millipore Millistak + HC filter containing a medium such as D0HC (primary) followed by X0HC (secondary) or a CUNO ZetaPlus filter containing a medium such as 10M02 (primary) followed by 60ZA05A (secondary). Can be. In this case, the clarified harvest is further filtered through a 0.8 / 0.2 μm filter (Supor, Pall).

A 85% HCP reduction was also observed through the secondary filter during deep filtration. The reduction of HCP through the secondary filter could contribute to the charge characteristics of this filter, see Yigzaw Y, Piper R, Tran M, Shukla AA. 2006. Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal during Monoclonal Antibody Purification.Biotechnology Progress 22 (1): 288-296).

The clarified material was further purified by cation exchange chromatography such as GigaCap S (Tosoh). Monoclonal antibodies (products) were immobilized to the resin at a volume of chromatographic media of greater than 95 g / l. The conditions used for antibody immobilization were somewhat acidic (pH-5.3) and the conductivity was ˜4.5 mS / cm. After binding the antibodies were washed with equilibration buffer and finally eluted with a buffer step containing 100 mM sodium chloride. A further reduction in HCP content (78%) was obtained with this step.

Eluted antibodies can be further purified by a combination of chromatography and filtration techniques until the required purity specifications are met.

The overall reduction in host cell protein from cell culture harvest through the CEX step is summarized below:

Claims (9)

A method of purifying cell broth containing cells by the following steps:
a. Contacting the cell broth with particulate anion exchange material having an intrinsic particle density of 1.4 to 3 g / ml,
b. Allowing sufficient incubation time to form cell pellet and supernatant layers, and
c. Separating the resulting cell pellet from the supernatant layer. A method of recovering a desired secreted biological material from a cell broth containing cells that produce the desired secreted biological material by the following steps:
a. Contacting the cell broth with particulate anion exchange material having an intrinsic particle density of 1.4 to 3 g / ml,
b. Allowing sufficient incubation time to form the cell pellet and supernatant layer,
c. Separating the resulting cell pellet from the supernatant layer, and
d. Extracting the desired secreted biological material from the supernatant layer. The method of claim 2, wherein the resulting cell pellet is further processed by the following steps:
e. Resuspending the resulting cell pellet,
f. Allowing sufficient incubation time to form the cell pellet and supernatant layer,
g. Separating the resulting cell pellet from the supernatant layer, and
h. Extracting the desired secreted biological material from the supernatant layer. 4. The method of claim 3, wherein steps e through h are repeated. The method according to claim 3 or 4, wherein the resulting cell pellet is resuspended in an aqueous salt solution. The method of claim 5 wherein the aqueous salt solution is PBS. The method of claim 3 or 4, wherein each supernatant layer is harvested prior to extracting the desired biological material. 8. The method of any one of claims 3 to 7, wherein the desired biological material is extracted using cation exchange chromatography. The method according to claim 1, wherein the desired biological substance is an immunoglobulin or part thereof.

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