RU2794431C1 - Improving immunoglobulin affinity chromatography by the application of pre-capture floculation - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: method for carrying out affinity chromatography with the capture of immunoglobulin from the culture liquid includes the following steps: adding a water-soluble organic polymer that causes flocculation to the culture liquid, depth filtration and affinity chromatography of the filtrate of the resulting mixture, while the immunoglobulin is associated with the medium for affinity chromatography, then the medium for affinity chromatography is washed using a wash buffer of pH 5–9 and an ionic strength of 0.1–5 mol/l and eluting the immunoglobulin from the affinity chromatography resin using an elution buffer of pH 2.5–4.5, with a water-soluble organic polymer being a poly(diallyldimethylammonium chloride) with a molecular weight of 10-10,000 kDa, which is added at a final concentration of 0.01–1.0 w/v.%.

EFFECT: elimination of formation of a precipitate in affinity chromatography.

24 cl, 5 dwg, 19 tbl, 16 ex

Description

Technical field

The present invention relates to methods for preventing precipitation during affinity chromatography of antibodies from a cell culture derived composition using flocculation and filtration steps prior to capture affinity chromatography and additional polishing steps after the capture step to purify antibodies.

State of the art

The selection of efficient and economical 3' sequences for the purification of polypeptides produced by recombinant DNA technology is a critical step in the development of every new biopharmaceutical for therapeutic use. In the recent past, the need for large-scale purification processes for monoclonal antibodies (mAbs), due to their exceptionally high therapeutic doses in medical use, has further increased with the use of improved cell culture techniques, resulting in higher cell density and higher expression rates, and hence , to a higher titer, eg >10 g/L, of the recombinant antibody of interest. Increasing concentrations of product and contaminants in culture fluids place greater demands on capture chromatography, upstream sample clarification steps, and subsequent polishing. The entire subsequent process must: (i) manage increased product weight, (ii) effectively remove increased process and product-related impurities below certain acceptance criteria, (iii) maintain economic yield, and (iv) provide sufficient mAb quality. Typically, the downstream process accounts for most of the total cost of producing therapeutic antibodies.

Mabs in crude fractions are typically associated with impurities such as host cell proteins (HCP), host cell DNA (HCDNA), endotoxins, insulin, viruses, aggregates, other unwanted product variants, and various leachates from process materials. The presence of these impurities poses a potential risk to patient health and therefore their absence from the final product is a regulatory requirement. Only very low residual quantities are allowed.

The classical procedure for purifying polypeptides derived from cell culture follows a sequence of capture-intermediate-polish chromatographs followed by filtration, concentration or dialysis steps at various positions in the downstream process sequence. In recent years, platform approaches have been successfully introduced in the field of purification of monoclonal antibodies. Since mAbs are a well-defined class of glycoproteins with common physicochemical properties, the use of a universal platform process is reasonable (Kelly B, 2009). Such a versatile process, with more or less product-specific adaptations, can be applied to many monoclonal antibodies, especially to immunoglobulins of the same class or subclass, such as IgG1 or IgG2.

One of the most common capture steps used to purify monoclonal antibodies is Protein A affinity chromatography. This capture provides exceptional selectivity for Fc-bearing molecules, thereby removing over 99.5% of contaminants in a single step. However, in addition to the advantages, two more disadvantages should be noted. One disadvantage is the unwanted leaching of Protein A or Protein A fragments known to be toxic (Gagnon P, 1996). Another drawback is the high cost of this type of resin, especially on an industrial scale, required to purify sufficient amounts of therapeutic antibodies. Protein A resin is about 30 times more expensive than ion exchange resin. It has been estimated that for the post-treatment of 10 ^m3 of cell culture, the cost of Protein A affinity chromatography is about US$4-5 million (Farid SS 2009).

In many cases, the capture steps are performed with raw feed materials (loading) that can cause contamination (impurity buildup) of the affinity column resin. In the absence of a proper regeneration step, this can prevent successful reuse of the grip resin. Contaminants in crude culture fluids such as lipids, oxidizing agents, host cell proteins (HCP), host cell DNA (HCDNA), aggregates or particles, metal ions, and other substances contribute to resin contamination and can cause precipitation and haze during elution at low pH values. (Shukla AA, 2005). In addition to the direct effect on the binding fragments, the matrix can also be irreversibly contaminated. The consequence of this is an increase in back pressure and a decrease in productivity and flow from one cycle to the next. This problem is not limited to Protein A resins: fouling of chromatographic resins during their lifetime is a common serious problem for commercial bioseparations. Hydrophobic ligands used for hydrophobic interaction chromatography and mixed chromatography, when used as capture steps for immunoglobulins derived from cell cultures, are particularly sensitive to the capture of lipophilic contaminants from culture fluids. Despite complex protocols for post-run cleanup steps, the lifetime of the capture column is limited and depends on the number of runs, operating and cleanup operating conditions, and sample composition and purity.

To clarify and pre-purify highly contaminated culture fluids, mechanical separation steps were applied that remove most of the cell debris and aggregates. Centrifugation and filtration are the most common pre-treatment steps performed prior to loading the sample onto the trapping resin. For larger volumes, continuous centrifugation is performed using cell separators followed by filtration steps using depth filters and/or microfilters. The resulting culture fluid is then referred to as "clarified cell culture supernatant" (Liu HF 2010). Although direct loading of harvested culture fluid onto Protein A resin is a frequent method of choice (Fahrner RL, 2001), other platform technologies use different clarification steps, i.e. centrifugation, depth filtration and/or microfiltration (Liu HF 2010, WO9522389, WO2001150110) to protect the capture column. This reduces solid and particle contamination of the resin and improves the purity of the antibodies in the capture eluate.

Pre-capture chromatography performed in flow mode, in addition to centrifugation/filtration steps, has been successfully demonstrated for large-scale purification of immunoglobulins (WO2015135884). It has been found that by including an additional anion exchange chromatography step prior to capture chromatography, the overall cost of the purification process can be significantly reduced. The pre-capture anion exchange flow chromatography step reduced the load of impurities with which the expensive capture chromatography material was exposed.

Turbid elution pools and high column backpressure are common during the elution of monoclonal antibodies at acidic pH in Protein A chromatography. Studies have attributed this phenomenon to liquid-liquid phase separation due to self-association of proteins. Several factors influence this effect, including pH, temperature, ionic strength, and protein concentration. Careful selection of process parameters during Protein A elution, including temperature, flow rate, buffer, and salt, can reduce both turbidity and column back pressure (Luo H, 2017). Similar strategies have been taken to avoid the problems of aggregation and particle formation that manifest as haze in the protein A elution pool (Shukla, AA 2005). Stabilizers have been proposed, such as salts, urea and amino acids added to the elution buffer, lower temperature, pH adjustment of wash and elution buffers, or pretreatment of harvested cell cultures to remove certain impurities that may precipitate at low pH. Pretreatment methods include anion exchange chromatography, filtration and acid precipitation of contaminants (Shukla AA, 2005).

Flocculation is a process that is widely used in the chemical and food industries, as well as in wastewater treatment. Both precipitation and flocculation have been successfully used to clarify cell culture liquids. While settling depends on reducing the solubility of the target solutes to create solid particles, flocculation is the agglomeration of particles (cells, cell debris, or colloids in mammalian cell culture) caused by a binding effect caused by a flocculating agent. The flocculant causes destabilization of the biocolloidal suspension, causing the dispersed particles to adhere into larger clusters, which leads to an increase in the average particle size distribution. (Felo M, 2015, Singh N, 2016). Flocculation methods can be divided into anionic, cationic and mixed (Singh N, 2016).

In particular, flocculants such as simple acids, divalent cations, polycationic polymers, caprylic acid, and stimulus-responsive polymers have been evaluated for their ability to improve cell culture clarification and reduce levels of DNA, host cell proteins (HCP), and viruses ( Felo M, 2015). For about a decade, flocculation techniques have been intensively researched for their usefulness in the large-scale production of monoclonal antibodies.

Flocculation with combinations of different cations and anions in the presence of cells, with and without pH adjustment, has been successfully used to purify monoclonal antibodies (WO2007035283). Most preferred was a combination of Ca ²⁺ and phosphate. The marked reduction in HCP and turbidity at the peak of the Protein A elution compared to non-flocculated controls was highlighted.

Other methods use polyelectrolytes such as polyvinyl sulfonic acid, polyvinyl sulfonate, polystyrene sulfonic acid, polyacrylic acid, polylysine, or polyarginine to precipitate impurities in harvested cell culture fluids (WO2008091740). A similar method using caprylic acid as a flocculant has been developed for the precipitation of contaminants (WO2010151632).

Precipitation or flocculation of impurities often requires additional pH manipulation. This has been disclosed for precipitation with divalent cations such as Co ²⁺ or Ni ²⁺ (WO2008127305) and precipitation with dextran (WO2016153983).

Another flocculation process using benzyl poly(allylamine), referred to as a stimulus-responsive polymer, was developed to purify monoclonal IgG1 from CHO cell culture. The stimulus was the addition of sodium phosphate (Kang YK, 2013). Other compounds are known to act as stimulus-responsive polymers in the clarification of antibodies harvested from cell culture (WO2011146394).

The flocculation step by adding allantoin to harvested cells followed by the addition of ethacridine has been described as a conditioning method before subsequent adsorption and chromatography steps (WO2014133459).

Another flocculation method uses a C ₇ -C ₁₀ fatty acid in combination with other functional substrates to clarify harvested cell cultures (WO2014196926). A similar process was applied based on the combination of C ₇ -C ₁₀ fatty acid and allantoin (WO2015130222).

A more sophisticated flocculation method has been disclosed by combining allantoin, a cationic polymer and a fatty acid to remove contaminants from harvested cells. A mixture of three flocculants, allantoin, chitosan and caprylic acid, proved to be the most successful (WO2017217930).

A complete process for the purification of antibodies or other target molecules has been described, including (i) a precipitation step to remove impurities, (ii) Protein A chromatography followed by (iii) one or two flow chromatographies (WO2014004281). The pellet is added to the harvested cell culture and also contains flocculants with and without additional stimulus responsive elements.

The use of poly(diallyldimethylammonium chloride), a cationic water-soluble polymer, has been investigated for clarification of a harvested cell culture of mAb-producing CHO cells (McNerney T, 2015). The authors added the flocculant directly to the culture broth. Centrifugation was omitted in favor of a filtration system. The toxic poly(diallyldimethylammonium chloride) was shown to be reduced to an undetectable level after subsequent capture chromatography on Protein A.

A large-scale cell culture harvesting method has been developed, consisting of an initial flocculation step in the presence of cells and based on poly(diallylmethylammonium chloride) (WO2013090820). The preferred flocculation method uses additional flocculation enhancers, i.e. polyethylene glycol and Triton X-100.

The essence of the invention

The present invention relates to the prevention of precipitation during low pH elution of immunoglobulins from cell cultures from affinity capture media. The invention also relates to methods for purifying immunoglobulin in an efficient and cost-effective manner, with satisfactory purity and yield. In particular, the present invention relates to improving the purity of an immunoglobulin solution eluted from a capture chromatography resin, and hence resulting in an improved quality of the final product.

In addition, the present invention addresses the aspect of recycling rather expensive chromatographic materials, in particular the lifetime of the affinity chromatography materials used in the capture step of the downstream process, and how the lifetime can be increased while reducing the technical complexity of the purification process.

Standard post-chromatography processes for the purification of immunoglobulins from cell culture fluids typically begin with a capture affinity chromatography step in which the immunoglobulin must be captured from a sample containing the immunoglobulin along with impurities. Immunoglobulin is separated from impurities mainly due to the selective binding of immunoglobulin to the affinity ligands of the capture resin, while impurities do not bind to the resin and thus end up in the flow fraction, while the immunoglobulin ends up in the eluate.

Capture affinity chromatography is usually the most expensive step in the purification of immunoglobulins, accounting for 40 to 50% of the total downstream costs when protein A affinity chromatography is used. The same applies to alternative affinity chromatography columns that can be used as a chromatography step. with capture during purification of immunoglobulins.

There is a continuing need for cost-effective purification of immunoglobulins from large volumes of culture fluid and fermentation broth, as well as from clarified samples obtained from such fluid or broth. In particular, there is a need for simple purification methods that are cost effective, yet efficient and satisfactory in terms of purity and yield.

It has been found that by including an additional chromatography step prior to capture chromatography, the overall cost of the purification process can be significantly reduced. This additional chromatography step reduces the amount of impurities with which expensive capture chromatography material is exposed. This so-called "pre-purification" step is performed using an anion exchange or mixed chromatographic material that is less expensive and more durable than the chromatographic material used in the subsequent capture step and is easy to regenerate (WO2015135884). In order to keep the purification process as simple as possible, the prepurification chromatography is carried out in flow mode, i.e. the immunoglobulin to be purified does not bind to the resin and thus is obtained in the flow fraction, while impurities are largely retained on the resin and thus separated from immunoglobulin. The direct connection of the anion exchange pretreatment column to the protein A capture column, so that the run-off fraction of the pretreatment step is not temporarily stored in the collector but is immediately transferred to the capture resin further simplifies this method (WO2015135884).

However, such an anion exchange flow chromatography process used as a pre-capture clarification step, although successfully applied in large scale production, is still subject to further improvements. Anion exchange chromatography depletes mainly negatively charged substances such as HCDNA and HCP along with non-specifically adsorbed cellular debris, aggregates, lipids, etc., which are retained by the column. However, most positively charged HCP and uncharged molecules can pass through the pre-column. This can lead to undesirable clouding of the Protein A elution peak. Moreover, due to the increasing demands caused by high cell density, complex media supply strategies, and high expression rates of current mammalian cell cultures, a significant prepurification column is required to act as an effective Protein A column shield. This affects the price. Finally, the anion exchange resin must undergo rigorous and lengthy cleaning and regeneration procedures. This is undesirable in routine production, which is preferably performed in serial batch operations.

The present invention explores the possibility of using a flocculation step as a pre-capture step to overcome the aforementioned chromatographic pre-purification problems. To achieve the desired high purity of the immunoglobulin intended for therapeutic use, the flocculation step and the capture chromatography step are followed by two or more chromatographic polishing steps.

The problem underlying the present invention is solved by providing a method for purifying immunoglobulin from culture fluid, which includes the following steps in the following order: (a) adding a flocculating compound to the culture fluid;

(b) depth filtration of the mixture of step (a);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the affinity chromatography medium using a wash buffer with a pH of 5-9 and an ionic strength of 0.1-5 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5-4.5.

In some embodiments, step (a) includes the following steps

- centrifugation and/or filtration of the culture fluid;

- adding a flocculating compound to the supernatant or filtrate.

Flocculation can be performed by adding a cationic compound as a flocculant. The cationic compound may be selected from the group consisting of a divalent metal ion salt, a water-soluble organic polymer, and a water-insoluble organic polymer.

In some embodiments, the divalent metal ion salt is CaCl ₂ .

In some embodiments, the water-soluble organic polymer is poly(diallyldimethylammonium) chloride. In other embodiments, the water-insoluble organic polymer is chitosan.

In specific embodiments, the implementation of the flocculation is carried out with poly(diallyldimethylammonium chloride) as a flocculant. More specifically, poly(diallyldimethylammonium chloride) has a molecular weight of 10 kDa to 10,000 kDa.

Typically, poly(diallyldimethylammonium chloride) is added at a final concentration of 0.01 to 1.0% (w/v), preferably a final concentration of 0.02 to 0.1% (w/v), more preferably in a final concentration of 0.03 to 0.08% (w/v).

In some embodiments, no substances other than poly(diallyldimethylammonium chloride) are added to perform flocculation.

In some embodiments, the poly(diallyldimethylammonium chloride) flocculation is carried out without additional pH or conductivity adjustment.

In specific embodiments, the implementation of the flocculation is carried out with stirring at room temperature for at least 5 minutes, preferably at least 15 minutes.

In preferred embodiments, the affinity chromatography is Protein A chromatography. The Protein A chromatography medium may contain an alkali-stable derivative of Protein A as a ligand, preferably an alkali-stabilized tetrameric variant of the B domain of Protein A, coupled to a cross-linked agarose matrix.

In some embodiments, microfiltration is performed after depth filtration.

Usually affinity chromatography is carried out at most 8 hours after depth filtration, preferably at least 3 hours after depth filtration.

In some embodiments, the culture fluid is obtained from recombinant CHO cells expressing immunoglobulin. Preferably the immunoglobulin is IgG1 or IgG2. More preferably, the Fc portion of IgG1 or IgG2 is human.

The method described herein may include the steps defined in any of the previous embodiments and one or more additional steps following affinity chromatography selected from viral inactivation, ion exchange chromatography, mixed chromatography, nanofiltration, and ultrafiltration/diafiltration.

Preferably, the method described above further comprises the following steps: (f) incubating the eluate obtained in step (e) for viral inactivation at a low pH of 2.5 to 4.5 for at least 10 minutes;

(g) performing cation exchange chromatography;

(h) performing mixed chromatography or anion exchange chromatography;

(i) nanofiltration of the eluate from step (h) or the composition obtained from it and obtained after one or more additional processing steps performed after step (h); And

(j) ultrafiltration/diafiltration of the filtrate of step (i) or the composition obtained from it and obtained after one or more additional processing steps performed after step (i).

Another preferred method includes the following steps in the following order: (a) adding a flocculating compound to the culture fluid;

(b) depth filtration of the mixture of step (a);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the medium for affinity chromatography using a wash buffer with a pH value of 5-9 and an ionic strength of 0.1-5 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5-4.5.

(f) incubating the eluate from step (e) for viral inactivation at a low pH of 2.5 to 4.5 for at least 10 minutes;

(g) performing cation exchange chromatography;

(h) performing mixed chromatography or anion exchange chromatography;

(i) nanofiltration of the eluate from step (h) or the composition obtained from it and obtained after one or more additional processing steps performed after step (h); And

(j) ultrafiltration/diafiltration of the filtrate of step (i) or the composition obtained from it and obtained after one or more additional processing steps performed after step (i);

where stage g) is performed in the binding-elution mode, and stage h) - in the binding-elution mode or in flow mode.

The invention also solves the problem of increasing viral safety during the production of immunoglobulin.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1: Flow charts of conventional immunoglobulin purification methods

Figure 1A shows a generic process flow for purifying immunoglobulins from large volume cell cultures. The process, which started with a clarified mass of material that is obtained after centrifugation and/or filtration of the collected culture liquid, consists of a capture step on Protein A and two subsequent polishing steps. This scheme also includes two typical stages of virus protection. A viral inactivation step is performed by keeping the protein A eluate at a low pH, and a nanofiltration step to remove the virus is performed after the last polishing step. The final step is usually tangential flow ultrafiltration and/or diafiltration (UF/DF-TFF) to obtain the desired concentrations of immunoglobulin and formulation ingredients.

FIG. 1B shows a classic three-chromatography process for purifying immunoglobulins from large volume cell cultures (eg, according to Fahrner R.L., 2001 or Kelly B, 2009). This is the same process as in Fig. 1A, except that the polishing steps are described as cation exchange chromatography (polish step 1) followed by anion exchange chromatography (polish step 2). It should be emphasized that cation exchange chromatography is carried out in binding mode, while anion exchange chromatography is carried out in flow mode. It should be noted that a commonly used equivalent to this classic scheme is simply to change the order of polishing steps 1 and 2.

Fig 2: Examples of process schemes according to the invention using pre-cleaning steps.

FIG. 2A shows a schematic of a large scale process with a pre-capture flocculation step directly during collection in the presence of cells. The flocculated material is then removed by centrifugation followed by depth filtration and microfiltration. The flocculation step is performed by adding a cationic compound. The clarified culture liquid is loaded onto a capture chromatography column, which is Protein A chromatography. The two polishing steps are cation exchange chromatography (polish step 1) followed by mixed chromatography (polish step 2). Cation exchange chromatography is carried out in the mode of binding and elution. The mixed mode resin has positively charged ligands and can be run in bind and elution mode or in flow mode (FT). The virus safety intermediates and final UF/DF-TFF are described in FIG. 1A.

FIG. 2B shows an alternative large-scale process scheme that is similar to the process in FIG. 2A, except that the flocculation step is performed after cell separation in the absence of cells. All other steps are described in Fig. 2A.

On FIG. 2C shows another alternative large scale process scheme that is similar to the process of FIG. 2B, except that the final polish chromatography is anion exchange flow chromatography (FT) mode. All other steps are described in Fig. 2B and 2A.

DETAILED DESCRIPTION OF THE INVENTION

Used in the present description, the term "culture fluid" refers to the collected culture fluid, cell culture supernatant or pre-treated cell culture supernatant. The culture fluid can be obtained directly from the host cell or immunoglobulin-producing organism. The culture fluid may be partially clarified or purified by centrifugation and/or filtration, such as microfiltration, diafiltration, ultrafiltration and depth filtration.

As used herein, the term "pretreated" refers, for example, to a cell culture supernatant that has been prepared for the chromatography step used in the method of the invention, for example by subjecting the sample to one or more adjustments consisting of buffer change, dilution, addition of salts, detergents, chaotropic substances or organic compounds, pH titration or filtration to adjust the pH range and/or conductivity and/or buffer capacity to achieve the desired chromatography characteristics and stabilization of the immunoglobulin. Since immunoglobulins expressed from mammalian cells are usually secreted into the culture fluid during the culture process, the collection of the product at the end of the culture process occurs by separating the culture fluid from the cells. The cell separation method must be gentle to minimize cell disruption to avoid an increase in cellular debris and the release of proteases and other molecules that may affect the quality of the immunoglobulin product. Typically, the harvest from mammalian cell cultures is subjected to centrifugation followed by filtration. Expanded bed adsorption chromatography is an alternative method to avoid centrifugation/filtration methods. Another pre-purification sample treatment with chromatographic steps may include concentrating and/or diafiltering the cell culture supernatant to a specific immunoglobulin concentration, pH range, conductivity, and buffer concentration.

As used herein, the term "flocculation" (synonyms "coagulation" and "agglomeration") includes a clustering reaction whereby suspended solids in a liquid react with a flocculating agent to form larger aggregates or floes. The terms "flocculating agents" and "flocculating agents", which may be used interchangeably herein, may be cationic or anionic compounds. Preferably the flocculants are cationic compounds. More preferably, the flocculants are cationic water-soluble polymers. The "flocculation step" used in the present invention functions as a pre-purification step prior to capture chromatography. Flocculation can be performed either in the presence of cells, which means that the flocculant is directly added to the culture fluid at the end of the culture and the flocculated material, including cells, is removed from the culture fluid by centrifugation and/or filtration, or the flocculant is added to the culture fluid after the cells are removed by centrifugation and then the flocculated material is removed by filtration.

Used in the present description, the term "cationic compound" includes metal ions and cationic polymers (polycations). "Cationic polymers" may be water-soluble polymers or water-insoluble polymers. "Water-soluble polymers" include organic substances that dissolve in water and thus change the physical properties of aqueous systems. "Water insoluble polymers" have limited solubility in water. They disperse or swell in water and preferably contain hydrophilic polymers which act as flocculants in aqueous suspension. Water insoluble polymers can become water soluble at very low or very high pH. Cationic compounds used as a flocculant perform many functions in aqueous media. Water-soluble or water-insoluble organic polymers include a wide range of substances derived from natural sources or through chemical synthesis. Examples of natural water-soluble or water-insoluble organic polymers are polysaccharides (eg pectin, dextran, starch, chitosan and hyaluronic acid), xanthan gum, carrageenan and many others. Examples of chemically synthesized organic polymers are polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyacrylic acid (PPA), polyacrylamide, and many others (see Kadajii VG 2011 for a review). In the course of the invention, water-soluble polymers act as the most preferred flocculants.

The terms "impurity" and "contaminant" are used interchangeably herein and refer to any material that differs from the immunoglobulin of interest. Examples include components of cell culture media, cell debris, host cell proteins, endotoxins, viruses, lipids, DNA, RNA, leachates from process materials, and aggregates or fragments thereof. Also contemplated as impurities are product-related substances such as aggregates, charge variants, misfolded molecules, or fragments of the immunoglobulin of interest to be purified.

The term "precipitation" refers to impurities that are insoluble and/or in an insoluble state. Precipitation may cause the sample to become cloudy.

As used herein, the term "chromatographic media" or "chromatographic medium" should be understood as a chromatographic material or medium in the form of granules, plates, crystals, monoliths, membranes, fibers, a network of fibers, or any other solid phase. The "environment" carries functional groups, called "ligands", associated with the main backbone, directly or through a spacer, called the "matrix". An exception is size exclusion chromatography resins, which typically do not contain any attached ligand. The term "medium" does not limit the methods of the invention to only column chromatography using chromatographic resins, but also includes other types of chromatography, such as membrane chromatography using membrane adsorbers. Particularly in ion exchange chromatography, the invention includes an ion exchange resin or membrane adsorber for ion exchange chromatography.

"Resin" means any chromatographic material or medium in the form of granules containing a matrix with an associated functional group (ligand) that can interact with a protein or at least one contaminant. An exception is size exclusion chromatography resins, which typically do not contain any attached ligand. Resins can be supplied in the form of granules of different sizes and packed in columns. Alternatively, pre-filled columns can be purchased.

By "matrix" or "solid phase" is meant a non-aqueous matrix to which a ligand can be attached. The matrix of interest herein is typically a matrix that includes glass, ceramic, silica, cellulose, agarose, methacrylate polymer, or polystyrene.

By "ligand" is meant any functional group that interacts with a protein or at least one contaminant and that is covalently linked to a "matrix".

The term "binding mode" or "binding and elution mode" refers to chromatography conditions in which a sample containing the immunoglobulin to be purified is applied to a chromatographic medium, where the immunoglobulin binds to the chromatographic medium. Thus, the immunoglobulin remains on the chromatographic medium, while sample contaminants may be present in the unbound fraction, also referred to as the flow through fraction. When the chromatography step is conducted in the binding mode, one or more washing steps may be performed after the immunoglobulin has bound to the chromatographic medium and before the immunoglobulin has been eluted from the medium. To obtain the immunoglobulin, it is then eluted and obtained in an eluate which can then be further purified in the next chromatography step, if desired. The elution of the immunoglobulin can be performed using selective conditions to allow the contaminants to remain bound to the medium during the elution of the immunoglobulin.

Running the chromatography step in "binding mode" does not necessarily mean that 100% of the immunoglobulin of interest is bound. In the context of the present invention, "bound to a chromatographic resin" or "bound to a chromatographic medium" means that at least 50% of the immunoglobulin is bound, preferably at least 75% of the immunoglobulin is bound, more preferably at least 85% of the immunoglobulin is bound, and most preferably more than 95% of the immunoglobulin is bound to the resin or medium.

In the context of the present invention, it should be understood that the capture chromatography step and the intermediate cation exchange chromatography step are performed in binding mode, with the capture step considered to be the first chromatography step that is performed in binding mode. The final polishing step in the mixed mode chromatography of the present invention is performed either in the binding and elution mode or in the flow mode. In addition, it is understood that instead of mixed chromatography, anion exchange flow chromatography can be used as the final polishing step.

The term "flow mode" refers to chromatography conditions in which a sample containing an immunoglobulin of interest is applied to a chromatographic resin or medium, where the immunoglobulin does not bind to the chromatographic resin, but is predominantly present in a fraction that is not bound to the resin or medium and, thus contained in the eluate. The intermediate chromatographies developed according to the present invention do not use the flow mode. However, the methods of the present invention can be supplemented by additional flow chromatographic polishing techniques. In this mode, impurities can bind to the resin or medium.

A "wash step" is a step performed in binding mode chromatography after the sample is loaded onto the chromatographic column but before the protein is eluted from the column. The wash step further removes contaminants less tightly or non-specifically bound to the matrix, to the immunoglobulin and/or ligand, without significantly eluting the immunoglobulin of interest from the resin. In the wash step, the resin is washed with the desired wash buffer (eg, the wash buffer is passed through a chromatographic column until the UV absorbance measured downstream of the column returns to baseline).

The term "elution" is understood as the process that desorbs the immunoglobulin of interest from the chromatographic resin by changing the solution conditions such that buffer components compete with the molecule of interest for the ligand site on the chromatographic resin. Another way of elution takes place in affinity chromatography, for example using Protein A. In this case, the elution buffer can change the conformation of the ligand or immunoglobulin, thereby weakening the binding. An immunoglobulin of interest can be eluted from ion exchange resins by changing the ionic strength of the buffer surrounding the ion exchange material such that buffer ions in the mobile phase compete with the molecule for the charged ion sites of the ion exchange resin. Alternatively, changing the pH affects the amphoteric protein and increasing the pH above the pI of the protein now prevents it from binding to the cation exchange resin and the protein is eluted. The same effect is observed on anion exchange chromatography resin when the pH is lowered below the pI of the protein. As used herein, the term "elution" includes isocratic elution, one-step elution, and gradient elution with or without preceding washing steps. Elution of the immunoglobulin of interest can be accomplished by increasing the ionic strength or conductivity of the mobile phase, which is affected by increasing the salt concentration in the buffer solution. Alternatively, it may be appropriate to increase or decrease the pH value. Discontinuous step gradients, linear gradients, non-linear gradients, or a suitable combination of such gradients may be used.

Buffers suitable for washing and elution may be selected from acetate, citrate, succinate, maleate, malonate, tris-HCl, tris-acetate, tris-glycine, phosphate, succinate, malonate, MES, MOPS, PIPES, PHEPES, bistris, glycine and other suitable buffers with the addition of salts such as phosphates, sulfates or chlorides such as NaCl or KCl. The ionic strength and salt concentration by which elution is achieved depend on the pH of the buffer solution and the pI of the protein. The wash buffer may further contain a detergent (eg polysorbate), a solvent (eg hexylene glycol, isopropanol or ethanol) or a polymer (eg polyethylene glycol). In addition, the wash buffer may include chaotropic reagents (eg urea or arginine) and/or protease inhibitors (eg EDTA).

Used in the present description, the term "buffer" refers to a solution that resists changes in pH under the action of the components of the acid-base conjugate.

The terms "immunoglobulin" and "antibody" are used interchangeably herein. An immunoglobulin can be a monoclonal antibody, a polyclonal antibody, a polyspecific antibody (eg, a bispecific antibody), and fragments thereof that exhibit the desired antigen-binding activity. Naturally occurring antibodies are molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N- to the C-terminus, each heavy chain has a variable domain (VH), also called a heavy chain variable domain, followed by three or four constant domains (CH1, CH2, CH3 and optionally CH4). Similarly, from the N- to the C-terminus, each light chain has a variable domain (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (), based on the amino acid sequence of its λ constant domain.

"Antibody fragments" include a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; single chain antibody molecules; diantibodies; linear antibodies; and polyspecific antibodies formed from antibody fragments.

Preferably the immunoglobulin is a monoclonal antibody. The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. individual antibodies that make up a population that are identical except for possible natural mutations that may be present in minor amounts. Unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinant. The modifier "monoclonal" indicates the nature of the antibody derived from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.

The immunoglobulin may be of the murine IgG1, IgG2a, IgG2b, IgM, IgA, IgD or IgE class, the human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE class, or combinations or fragments thereof.

An immunoglobulin can recognize one or a combination of proteins, including but not limited to the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 ( B7.1), CD79b, CD86 (B7.2), CD147, CD152, IL-la, IL-1β, IL-1, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-12, IL-13, IL-23, IL23a, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-12 receptor, IL-13 receptor, IL receptor subunits -18, PDGF-β and its analogues, PLGF, VEGF, TGF, TGF-β2, TGF-p1, EGF receptor, PLGF receptor, VEGF receptor, gpIIb/IIIa platelet receptor, thrombopoietin receptor, PD-1 apoptosis receptor, growth factor hepatocytes, osteoprotegerin ligand, interferon alfa, interferon beta, interferon gamma, B-lymphocyte stimulator BLyS, T-cell activation regulator CTLA-4, complement factor C5, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, ErbB2/HER- 2, tumor-associated epitopes that are present at elevated levels in the serum of patients, tumor-associated epitopes or proteins expressed on breast, colon, squamous cell, prostate, pancreas, lung and/or kidney cancer cells and/or on melanoma, glioma or neuroblastoma cells, necrotic tumor nucleus, integrin alpha 4 beta 7, integrin VLA-4, integrins B2, integrin α4β1 and α4β7, TRAIL 1,2,3 and 4 receptors, RANK, RANK ligand (RANKL), TNF -α, VAP-1 adhesion molecule, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrin-adhesin, platelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain, parathyroid hormone, sclerostin, MHC I, carcinoembryonic antigen (CEA), calcitonin (CGRP gene-related peptide), alpha-fetoprotein (AFP), tumor necrosis factor (TNF), Fc-y-1 receptor, HLA-DR beta 10, HLA-DR antigen, L -selectin, Kallikrein, Kirin and IFN-γ.

The immunoglobulin may be, for example, afelimomab, abciximab, adalimumab, ado-trastuzumab, aducanumab, alemtuzumab, alirocumab, anifrolumab, arcitumomab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, brazucumumab, bazitumabumab, blinitumab rentuximab, brodalumab , brolucizumab, canakinumab, caplacizumab, capromab, cemiplimab, certolizumab, cetuximab, clenoliximab, claudiximab, chrizalizumab, daclizumab, daratumumab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, edrecolomab, elotuzumab, evinacizumab, emtinizumab b, evolocumab, fremanezumab , foravirumab, galcanezumab, galiximab, gemtuzumab, golimumab, guselcumab, ibalizumab, ibritumomab, imciromab, idarucizumab, inebilizumab, infliximab, inotuzumab, ipilimumab, ixekizumab, keliximab, lanadelumab, lebrikizumab, lexatumumab, mirikimumab, mauriquizumab mogamulizumab, mosunetuzumab, muromonab -CD3, natalizumab, necitumumab, nivolumab, nofetumumab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, ramucirumab, ranibizumab, ravulizumab, restuzumab, resankisimumab, romosuksimumab, romotuzumab b, sacytuzumab, sarilumab , satralizumab, secukinumab, sirukumab, tanekumab, tezepelumab, tezolizumab, tocilizumab, tositumomab, tralokinumab, trastuzumab, tremelimumab, ublituximab, ustekinumab, and vedolizumab.

The immunoglobulin of the invention is preferably an IgG molecule, such as an IgG1, IgG2, IgG3 or IgG4 molecule. More preferably, the immunoglobulin is IgG1 or IgG2. Even more preferably, the immunoglobulin is an IgG1 or IgG2, wherein at least the Fc portion is human. The immunoglobulin may be a chimeric mouse/human IgG1, wherein the Fc portion of the IgG1 is human. Most preferably, the chimeric immunoglobulin is rituximab or infliximab.

Rituximab is a chimeric anti-CD20 antibody which is described in detail in, for example, WO9411026.

Infliximab is a chimeric anti-TNFα antibody which is described in detail in, for example, WO9216553.

The immunoglobulin may be a humanized IgG1 from a mouse precursor. Most preferably, the humanized antibody is trastuzumab or bevacizumab.

Trastuzumab is a humanized anti-HER2 antibody which is described in detail in, for example, WO9222653.

Bevacizumab is a humanized anti-VEGF antibody which is described in detail in, for example, WO9845331.

The immunoglobulin may be a fully human IgG1 or IgG2 antibody. Most preferably, the human antibody is adalimumab (IgG1) or denosumab (IgG2).

Adalimumab is a human anti-TNFα antibody which is described in detail in, for example, WO9729131.

Denosumab is a human anti-RANKL antibody which is described in detail in, for example, WO03002713.

In one embodiment, the immunoglobulin is bevacizumab, trastuzumab, or denosumab.

Monoclonal antibodies as used herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from a different species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity.

In addition, monoclonal antibodies here also include "humanized" antibodies. Such antibodies are produced by "humanization" of non-human (eg murine) antibodies and contain only minimal sequences derived from animal immunoglobulin. Most of the molecule belongs to the human sequence. Residues from the hypervariable region of the human recipient antibody are replaced by residues from the hypervariable region of a non-human donor antibody having the desired binding properties.

Finally, the monoclonal antibodies of the present disclosure also include fully human antibodies that can be obtained by screening a library of human antibody genes.

In a preferred embodiment, the sample is obtained from the culture fluid, which is obtained from a culture of recombinant CHO cells. Preferably, the culture fluid is obtained from a culture of recombinant cells in the growth phase.

The method of the present invention can be used to purify immunoglobulins on a small and large scale. Preferably, the method is carried out on a large scale.

"Small scale", also referred to as "laboratory scale", refers to the purification of samples containing less than 50 g of immunoglobulin, less than 10 g of immunoglobulin, or less than 1 g of immunoglobulin. "Small scale" also refers to purification processes in which the protein eluted from the capture step column is less than 50 g of immunoglobulin, less than 10 g of immunoglobulin, or less than 1 g of immunoglobulin.

"Large scale", also referred to as "manufacturing scale" or "commercial scale", refers to the purification of samples containing more than 50 g of immunoglobulin, more than 100 g of immunoglobulin, more than 200 g of immunoglobulin, or more than 300 g of immunoglobulin. "Large scale" also refers to purification processes in which the protein eluted from the capture step column is greater than 50 μg immunoglobulin, greater than 100 μg immunoglobulin, greater than 200 μg immunoglobulin, or greater than 300 μg immunoglobulin.

The term "further processing step" refers to any step that is commonly used in protein purification protocols, such as filtration, dialysis, viral inactivation, dilution, concentration, pH adjustment, conductivity adjustment, intermediate chromatography step, or retention step for any purpose. An additional processing step may be applied between all the chromatography steps of the invention. An intermediate chromatography step may be used between any of the chromatography steps. In particular, the term "additional processing step" refers to an intermediate chromatography step used between capture chromatography and cation exchange chromatography. The intermediate stage of chromatography can be carried out with any chromatographic medium in any mode. Any type of chromatography can be used in the intermediate chromatography step, including column chromatography and membrane chromatography.

Other types of chromatography may also be used as an additional intermediate or polishing step. For example, an anion exchange column chromatography and an anion exchange membrane chromatography can be used as a polishing step, flow mode is most preferred. Another possibility is to use hydroxyapatite chromatography, in particular ceramic hydroxyapatite, in coupling mode.

The first aspect of the invention relates to a method for preventing precipitation during affinity chromatography with the capture of immunoglobulin from culture fluid, and the method includes the following steps in the following order:

(a) adding a flocculating compound to the culture fluid;

(b) depth filtration of the mixture of step (a);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the medium for affinity chromatography using a wash buffer with a pH value of 5-9 and an ionic strength of 0.1-5 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5-4.5.

Pretreatment stage: Flocculation

In the experiments that led to the present invention, it was found that the cell-free harvest material (clarified supernatant) still contains several substances that, together with the purified immunoglobulin, bind strongly to the capture resin. This affects the reuse of the resin and the quality of the immunoglobulin after elution from the capture resin. This can be improved with additional prepurification chromatography based on anion exchange flow chromatography (WO2015135884). Despite a marked decline in HCDNA and HCP levels, there is still a need for further improvements. It has been observed that the Protein A eluate still tends to be cloudy, which is caused by impurities and not by the immunoglobulin itself. The low pH required for elution, the relative amount of impurities, and high protein concentrations contribute to haze. In addition to cloudy elution pools, increased column back pressure was also observed. This is best explained by the increased viscosity. In addition, the pre-purification anion exchange chromatography column is an undesirable cost factor due to the relatively large size required for a very high contaminant load. The latter is enhanced by increasing the fermentation time required for a higher titer of monoclonal antibodies and therefore for economical production. Since the filtering effect of the anion exchange resin is limited mainly to retaining negatively charged molecules, and some of the haze-causing impurities still slip through, a need arose for a simpler and more efficient pre-trapping purification strategy. According to the results of the present invention, the introduction of a suitable flocculation pre-treatment method is surprisingly a good solution for a quick and easy purification of the collected culture fluid. By removing additional contaminants, the developed flocculation method improves sample purity, reduces critical contaminants that contribute to turbidity during low pH capture medium elution, and protects the costly affinity column. The flocculation process of the present invention is simple, fast, no loss of product, and reasonably priced. Moreover, it turned out that the flocculation method can be performed either (i) before the cells are removed from the culture fluid, which means in the presence of cells, or (ii) after the cells have been removed from the culture fluid, which means no cells. The purity of the immunoglobulin before and after the stage with Protein A is comparable for both methods. In the first method, the flocculated material, which also includes cells, is separated by centrifugation and the supernatant is further purified using a filtration system. In the latter method, the cells are pelleted by centrifugation and the supernatant is flocculated. The filtration system then removes the flocculated material. In the simplest case, the filtration system consists of a depth filter. However, a series depth filter and microfilter system is preferred. During large scale and full scale production, flocculation after cell separation has proven to be the preferred method. When flocculation was carried out directly in cell culture, the removal of flocculation containing the entire cell mass from the fermenter and separator took a very long time.

A large number of different flocculation methods using various flocculants have been known and experimentally proven to be effective in clarifying mammalian cell cultures expressing monoclonal antibodies. Most methods require a combination of substances and/or further manipulation of the culture fluid, such as pH adjustment. In addition, many published methods use flocculation instead of a centrifugation step. On the contrary, the present invention combines three methods for optimal clarification of the culture liquid in the following order: 1) separation of cells by centrifugation or filtration, 2) flocculation and 3) depth filtration.

When the pH of the culture fluid is less than the pI of the antibody being purified, the antibody carries a net positive charge. Under these conditions, the cationic electrolyte may flocculate or precipitate impurities, residual cells and cellular debris and leave the antibody in solution. The flocs can then be removed by centrifugation and/or depth filtration, optionally followed by microfiltration.

In the present invention, a single cationic compound such as a divalent metal ion, a water-soluble organic polymer, or a water-insoluble organic polymer is used to induce flocculation. No additional substances, such as stimulus-responsive polymers or salts, are required to perform the flocculation step of the present invention. Likewise, the flocculation process of the present invention is performed without further adjustment of pH or conductivity.

The term "in the following order" should be understood to mean that the said process steps are performed in the order indicated. Additional process steps may be included before, after, and between the listed process steps.

The chromatographic medium may be disposable or reusable. In one embodiment, the chromatographic resin is reusable. In a particular embodiment, the chromatographic resins of step (b), (c) and (d) can be reused.

Reusable chromatographic media are more economical than chromatographic media configured as single use. In particular, capture chromatography uses large volumes of chromatography medium. Therefore, it is particularly advantageous to use a reusable chromatographic medium, such as affinity chromatography resin, which is often reused for the capture step. The use of the pre-treatment steps of the present invention allows the costly affinity capture column to be scaled down and the service life to be increased and thus the total cost of goods to be reduced.

The term "reusable" as used herein means that the media or resin is configured to be reused in more than one cleaning cycle, i.e. at least 2, 5, 10, 50, 100, 200, 300, 400, 500 or more cleaning cycles. Between each cycle, the chromatographic medium or resin can be washed and/or regenerated and/or sterilized and/or stored.

In a preferred embodiment, the cationic compound used as the flocculant is selected from a divalent metal ion salt, a water-soluble organic polymer, and a water-insoluble organic polymer. Water-soluble organic polymers are more preferred.

The salt of the divalent metal ion may be, for example, CaCl ₂ . The water-soluble organic polymer may be, for example, poly(diallyldimethylammonium chloride). The water-insoluble organic polymer may be, for example, chitosan. Even more preferably, the flocculant is poly(diallyldimethylammonium chloride).

In one embodiment, the water-soluble organic polymer is used at a final concentration of 0.01 to 1.0% (w/v), preferably 0.02 to 0.15% (w/v), more preferably 0.025 up to 0.090% (w/v). In a preferred embodiment, the water-soluble organic polymer is added at a final concentration of 0.0375 to 0.075% (w/v). In another preferred embodiment, the water-soluble organic polymer is added at a final concentration of 0.015 to 0.05% (w/v).

In a preferred embodiment, the water-soluble organic polymer used to induce flocculation is poly(diallyldimethylammonium chloride) with a molecular weight of 10 kDa to 10,000 kDa. In another preferred embodiment, the flocculation step is carried out with stirring at room temperature for at least 10 minutes, at least 15 minutes or at least 20 minutes, preferably at least 30 minutes. Room temperature in the present invention means 15-25°C, 20-25°C, preferably 18-22°C. Agitation means continuous agitation and/or agitation using suitable mechanical devices.

In another preferred embodiment, the poly(diallyldimethylammonium chloride) stock solution added to the culture liquid to induce flocculation has a concentration of 5-15% (w/v), preferably 9-11% (w/v), more preferably 10% (w/v). Most preferred is a neutral pH solution containing less than 0.1% diallylammonium chloride, having a microbial count of less than 10 cfu/ml, and containing no sodium chloride.

In one embodiment of the present invention, a flocculation step is performed prior to removing the cells from the culture fluid by adding a flocculant directly to the fermenter at the end of the culture period. In other words, in such embodiments, no further centrifugation and/or filtration takes place before the flocculation step. The flocs, including cells, are then removed by centrifugal settling, preferably a plate separator suitable for large volumes. The resulting supernatant can be further clarified by depth filtration, optionally followed by microfiltration.

In other embodiments, step (a) includes the following steps:

(a1) centrifuging and/or filtering the culture fluid; And

(a2) adding a flocculating compound to the supernatant or filtrate obtained in step (a1).

In other words, the flocculation step is performed after removing the cells from the culture fluid by adding a flocculant to the supernatant after performing the centrifugation and/or filtration step.

Thus, in specific embodiments, the implementation of the method according to the invention includes the following steps in the following order:

(a1) centrifuging and/or filtering the culture fluid;

(a2) adding a flocculating compound to the supernatant or filtrate obtained in step (a1);

(b) depth filtration of the mixture of step (a2);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the affinity chromatography medium using a wash buffer with a pH of 5-9 and an ionic strength of 0.1-5 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5-4.5.

Preferably, the flocs from step (a2) are removed by depth filtration, optionally followed by microfiltration.

Preferably, with the exception of poly(diallyldimethylammonium chloride), no additional flocculation agents are added. In other words, no other flocculating compounds are added in step (a) other than poly(diallyldimethylammonium chloride).

Preferably, the flocculation with poly(diallyldimethylammonium chloride) is carried out without further adjustment of pH or conductivity. This means that in the flocculation step and the next filtration step(s) before affinity chromatography, in particular the flocculation step (a2), the pH or conductivity is not adjusted.

Thus, in a specific embodiment, the method of the invention comprises the following steps in the following order:

(a1) centrifuging and/or filtering the culture fluid;

(a2) adding a flocculating compound to the supernatant or filtrate obtained in step (a1);

(b) depth filtration of the mixture of step (a2);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the affinity chromatography medium using a wash buffer with a pH of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5.

where, with the exception of poly(diallyldimethylammonium chloride), no other substances are added to perform flocculation; And

where the flocculation of poly(diallyldimethylammonium chloride) is carried out without additional regulation of pH or conductivity.

Capture Affinity Chromatography Step: Protein A Chromatography

The term "capture step" refers to the first step of chromatography carried out in the binding mode. The capture step for purifying immunoglobulin from culture fluids is typically performed as an affinity chromatography step. Protein A or its derivatives or analogs are primarily used for affinity capture. According to the present invention, affinity chromatography has been successfully used to capture immunoglobulins. The term "affinity chromatography" for the present invention means a method for the selective binding of immunoglobulin from culture fluid based on a highly specific interaction between the ligand and the immunoglobulin.

As used herein, the term “immunoglobulin-binding protein/peptide affinity chromatography” refers to affinity chromatography that uses recombinant proteins of microbial origin (e.g., Staphylococcus aureus, Streptococcus, Peptostreptococcus magnus) as ligands, or variants thereof, or synthetic peptides that can be of microbial origin with the ability to bind to immunoglobulins. Exemplary immunoglobulin binding proteins can be Protein A, Protein G, Protein L, or Protein A/G. In addition, binding proteins from proteins of microbial origin or peptides derived from the Fc gamma A receptor (FcR-IIIA) are also examples of affinity ligands. Preferably, the immunoglobulin-binding protein or peptide is Protein A. The ligands may include one or more of the E, D, A, B, and C domains of Protein A. More preferably, the ligands include the B domain of Protein A or engineered protein Z. An example of a resin using as The 14 kDa ligand peptide recombinantly produced with Saccharomyces cerevisiae is IgSelect (GE Healthcare). This ligand, for which no further information is available, was specifically designed to provide high affinity for all human IgG-Fc subclasses.

Using the Protein A affinity chromatography step as a capture step after the flocculation prepurification step, the method of the present invention provides a cost-effective method for purifying immunoglobulins while exploiting the significant binding specificity of Protein A affinity chromatography in purifying immunoglobulins.

In a preferred embodiment, the invention relates to a method for purifying immunoglobulin from a sample containing immunoglobulin and at least one contaminant, the method comprising the following steps in the following order:

(a) adding a flocculating compound to the culture fluid;

(b) depth filtration of the mixture of step (a);

(c) Protein A chromatography of the filtrate obtained in step (b), c, wherein the immunoglobulin is coupled to Protein A chromatography medium;

(d) washing the Protein A chromatography medium using a wash buffer with a pH of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5-4.5.

In a preferred embodiment, the Protein A chromatography resin used in the capture step contains an alkali-stable Protein A derivative as a highly cross-linked agarose ligand. In a more preferred embodiment, the alkali-stable protein A derivative is an alkali-stabilized tetrameric variant of the B domain of Protein A.

In order to make the Protein A affinity resin more resistant to harsh purification conditions and provide protection from the effects of cross-contamination between runs, improved Protein A affinity resins containing ligands specially designed to provide alkali resistance, high binding capacity and low ligand leaching. However, one of the main disadvantages of these improved resins is that they are significantly more expensive than conventional Protein A resins. An important advantage of the process of the present invention is that both conventional Protein A resins and newer generation Protein A resin products can be used. . Because Protein A resins are exposed to less impurities, conventional and cheaper Protein A resins become acceptable despite being limited by rather mild regeneration conditions. However, as a result of the pre-purification step of the present invention, and regardless of the selected resin for Protein A, both conventional resins and new generation resins can be used for a longer service life. In addition, because the purification of the Protein A column becomes easier, the process also becomes more economical.

Examples of conventional Protein A resins that can be used for the purposes of the invention may include, but are not limited to, Unosphere SUPrA (Bio-Rad), Protein A Ceramic HyperD F (Pall Corporation), Poros MabCapture A (Applied Biosystems), ProSep HC , ProSep Ultra and ProSep Ultra Plus (EMD Millipore), Protein A-Sepharose FF, pProtein A-Sepharose FF, rmp Protein A-Sepharose FF, IgSelect, MAbSelect, MAbSelect SuRe, MAbSelect SuRe LX, MabSelect Xtra, mAb GESelect PrismA (Healthcare ), Praesto A, Praesto AP, Praesto APc, Praesto Jetted A50 (Purolite Life Sciences), and Toyopearl pProtein A (Tosoh Bioscience).

As used herein, the term "Protein A" includes Protein A isolated from its natural source, Protein A obtained synthetically or biosynthetically (for example, by peptide synthesis or recombinant methods), and its variants that retain the ability to bind proteins that contain CH2/CH3 and/or Fc regions. Preferably, resins with high binding capacity and/or alkaline stability can be used. For example, Protein A derived from Protein A or an alkali-stabilized affinity medium derived from Protein A can be used. Preferably, alkali-stabilized ligands based on Protein A (E. coli, Saccharomyces cerevisiae) can be used. The alkali-stabilized ligand derived from Protein A can be attached to a highly cross-linked agarose matrix, preferably immobilized with a chemically stable thioether bond. One example is the MabSelect SuRef from GE Healthcare Life Sciences, which can be quickly and efficiently purified after a run with up to 0.5 M NaOH. The alkali-stabilized MabSelect SuRe ligand is derived from the B-domain of Protein A and essentially lacks the VH3 binding domain, resulting in a higher elution pH. The preferred product is MabSelect SuRe LX which has a higher binding capacity than MabSelect SuRe.

One or more wash steps between loading the sample onto the Protein A affinity column and eluting the immunoglobulin from the Protein A column can be included using dedicated wash buffer(s). The wash buffer is a buffer used to remove impurities from the protein A resin without removing significant amounts of protein A-bound immunoglobulin of interest. The wash buffer may contain salt and detergent (eg, polysorbate); salt and solvent (eg hexylene glycol); high concentration salt (for example, high molarity Tris buffer); or a salt and a polymer (eg polyethylene glycol). In addition, the wash buffer may include chaotropic reagents (eg urea or arginine) and/or protease inhibitors (eg EDTA). Finally, the wash buffer may have a lower pH as loading buffer and/or a higher pH as elution buffer.

An elution buffer is used to elute the immunoglobulin of interest from the Protein A column. Preferably, the elution buffer has a low pH and thereby disrupts the interactions between Protein A and the immunoglobulin of interest by changing the conformation of the protein. Preferably, the low pH elution buffer has a pH in the range of about 2 to about 5, most preferably in the range of about 3 to about 4. Examples of buffers that will control the pH in this range include phosphate, acetate, citrate, glycine, and ammonium buffers. , as well as their combinations.

Such preferred buffers are citrate and acetate buffers, most preferably sodium citrate or sodium acetate buffers. Other elution buffers are contemplated, including high pH buffers (eg, having a pH of 9 or greater) or buffers containing a compound or composition such as MgCl ₂ (2 mM) to elute the immunoglobulin of interest.

The Protein A affinity resin can be regenerated with 0.1-0.5 NaOH, preferably inside the column (CIP).

Intermediate/Polishing Chromatography Step: Cation Exchange Chromatography

The method described herein may further include a cation exchange chromatography step.

Cation exchange chromatography is based on the charge-charge interaction between the proteins in the sample and the charges immobilized on the resin. In cation exchange chromatography, the bound molecules are positively charged, while the immobilized functional groups (ligands) are negatively charged. Commonly used cation exchange resins are S (sulfonate) resins, SP (sulfopropyl) resins, SiB (sulfoisobutyl) resins, SE (sulfoethyl) resins and CM (carboxymethyl) resins.

However, in general, the cation exchange chromatography step can be carried out with all common commercially available cation exchange resins or membranes. Cation exchange resins can be used in the form of pre-packed columns or membranes on which a functional group, such as sulfonic acid, is fixed. Alternatively, the resins can be purchased as a bulk material and the columns can be packed by the user. There are no special restrictions regarding the capacity and size of columns, except for the usual ones. One skilled in the art knows the amount of cation exchange resin and the size of the column to be used. It depends on the overall scale of the process.

Typical commercially available products include, for example, Macro-Prep High S, Macro-Prep CM, Unosphere Rapid S, Unosphere Rapid S40, Nuvia S and Nuvia HR-S (Bio-Rad, CA, USA), Toyopearl CM, Toyopearl SP, Toyopearl Sulfate 650 F and Toyopearl GigaCap S (Tosoh Bioscience, Germany), Millipore ProRes S, Fractogel EMD COO ^- , Fractogel EMD SO ₃ ^- , Fractogel EMD SE Hicap, Eshmuno CPX (Merck KGaA, Germany), Biosepra CM Ceramic, Biosepra S Ceramic HyperD, S HyperCel (Pall Corperation, New York, USA), Poros HS, Poros XS (Applied Biosystems, Germany), BioPro IEX SmartSep S, BioPro IEX S75 (YMC Europe), Praesto SP, Praesto Jetted SP35 (Purolite Life Sciences, Europe), CM-Sepharose FF, SP-Sepharose FF, S-Sepharose FF, SP-Sepharose HP, SP-Sepharose XL, SP-Sepharose Large Beads, CM-Sephadex, Capto S, Capto SP ImpRes and Source S ( all GE Healthcare, Germany).

Typically, cation exchange chromatography is carried out using buffers at pH values from 4 to 7.

Preferred cation exchange resins of the present invention are strong cation exchangers using sulfonate, sulfopropyl or sulfoisobutyl ligands. Most preferred are sulfonate or sulfopropyl ligands bound to rigid matrices such as highly cross-linked agarose eg Nuvia HR-S or poly(styrene vinylbenzene) eg Poros 50 HS or polymethacrylate eg Fraktogel EMD SO ₃ ^- . The most preferred cation exchange resin is Poros HS 50 with sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ ^- ) ligands bound to a cross-linked poly(styrenedivinylbenzene) matrix.

The cation exchange chromatography can be balanced with a buffer having a pH of about pH 4 to about pH 8. The buffer concentration can be from 10 to 100 mM, preferably from 20 to 50 mM.

Examples of buffers used for cation exchange chromatography are citric acid, lactic acid, succinic acid, formic acid, butanedioic acid, acetic acid, malonic acid, glycine, MES, PIPES, phosphate, bistris, or mixtures thereof.

The cation exchange chromatography step can separate charged immunoglobulin variants and can deplete residual host cell proteins, DNA, aggregates, fragments, viruses, endotoxins, flocculants, and leached Protein A.

Immunoglobulin can bind to the resin at a pH value below the isoelectric point (pI) of the immunoglobulin and at low conductivity.

The isoelectric point or pI of a protein refers to the pH at which the protein has an overall net charge of zero, i.e. the pH at which the protein has an equal number of positive and negative charges. The determination of pI can be performed according to techniques developed in the prior art, such as isoelectric focusing.

Low conductivity means less than 2 mS/cm.

For elution, an increase in the ionic strength of the elution buffer, provided by either a single step or a gradient, can be used. Examples of salts used in the elution of cation exchange chromatography are chloride salts, sulfate salts, phosphate salts, citrate salts, formate salts or acetate salts. Preferably NaCl or KCl is used. The ionic strength can be increased up to 1M.

As an alternative, an increase in the pH of the elution buffer, either in a single step or via a gradient, can be used.

A preferred embodiment of cation exchange chromatography is an operating pH range of 4 to 6, more preferably a pH range of 4.5 to 5.5. As buffer substances, either carboxylic acids or amino acids can be used, most preferably citric acid or glycine.

In another preferred embodiment, the elution of the immunoglobulin associated with the cation exchange resin is carried out by changing the pH value, ie increasing the pH. This can be achieved with a low pH to high pH gradient provided by mixing two different buffer solutions. Citrate buffers for low pH and phosphate buffers for high pH are preferred. In a preferred embodiment, a pH gradient is formed by mixing a citrate buffer at a pH of about 5 to 6 with a phosphate buffer at a pH of about 7 to 9. Buffers can be prepared using sodium salts of acids at a concentration of 10 to 50 mM. The preferred pH gradient range is from 5 to 7.5. In another preferred embodiment, the pH gradient is formed by mixing glycine buffers of pH 8.7 and pH 10.5. Optionally, the pH of the elution pool is adjusted, for example using acetic acid, before loading onto the last chromatographic column.

Alternatively, an increase in both the pH and the ionic strength of the elution buffer, either in a single step or in a gradient, can be used for elution.

In one embodiment, the Protein A chromatography eluate is subjected to cation exchange chromatography.

Typically, the protein A chromatography eluate is subjected to a viral inactivation step followed by cation exchange chromatography.

Thus, in some embodiments, the implementation of the method according to the invention may include the following steps in the following order:

(a) adding a flocculating compound to the culture fluid;

(b) depth filtration of the mixture of step (a);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the affinity chromatography medium using a wash buffer with a pH of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5.

(f) incubating the eluate from step (e) for viral inactivation at a low pH of 2.5 to 4.5 for at least 10 minutes; And

(g) performing cation exchange chromatography.

Resin for cation exchange chromatography can be regenerated with 1M NaCl for 3-5 column volumes. In addition, a clean-in-place procedure can be applied, including the following steps: (a) washing with 1-5 column volumes of 1 M NaOH, 1 M NaCl, (b) washing with 1-5 column volumes of 1 M acetic acid or TFA, (c ) rebalancing.

Polishing stage: Mixed chromatography

The method of the invention may further include a mixed chromatography step as a polishing step, i.e. it is carried out in sequence (as one or more steps) after the affinity chromatography.

The media, referred to as mixed media or resins, are chromatographic media containing functional groups composed of either charged hydrophobic ion-exchange ligands or crystalline minerals such as hydroxyapatite or fluorapatite. Instead of "mixed chromatography", the term "multimodal chromatography" was sometimes used, or in connection with a particular procedure, "hydrophobic charge induction chromatography". Mixed chromatography is the interaction of at least two principles: hydrophobic interaction and ion exchange or metal affinity interaction and ion exchange. Mixed chromatography provides less predictable selectivity that cannot be replicated with a single mode chromatography method such as ion exchange chromatography or hydrophobic interaction chromatography, respectively. Positively charged hydrophobic ligands belong to the mixed mode anion exchanger group (eg CaptoAdhere) and negatively charged ligands belong to the mixed mode cation exchanger group (eg Capto MMC). Some mixed media are zwitterionic (eg Bakerbond ABx). Other mixed mode media contain hydrophobic ligands that ionize and change from uncharged to positively charged by lowering the pH (eg MEP HyperCel). Finally, hydroxyapatite and fluorapatite media have more complex mixed mode functions as they contain positively charged calcium ions and negatively charged phosphate groups.

In a preferred embodiment, the mixed chromatography used as the polishing step uses a resin having hydrophobic and anion exchange functions. More preferred are mixed type resins containing positively charged N-benzyl-N-methylethanolamine ligands that are bound to a highly cross-linked agarose matrix.

In some embodiments, the method includes the following steps in the following order: (a) adding a flocculating compound to the culture fluid;

(b) depth filtration of the mixture of step (a);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the affinity chromatography medium using a wash buffer with a pH of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5.

(f) incubating the eluate from step (e) for viral inactivation at a low pH of 2.5 to 4.5 for at least 10 minutes;

(g) performing cation exchange chromatography; And

(h) performing mixed chromatography.

Preferably, the mixed chromatography step follows the cation exchange chromatography. More preferably, the mixed chromatography step after the cation exchange chromatography is performed with a medium containing positively charged ligands. More preferably, the positively charged ligand is N-benzyl-N-methylethanolamine and the ligand is linked to highly cross-linked agarose.

The following conditions may apply when loading a positively charged mixed coupling and elution chromatography resin: pH 6 to pH 9, preferably pH 7.0 to 8.5; conductivity 0.5 to 10 mS/cm, preferably 1 to 4 mS/cm. One or more washing steps may be used. The conditions depend on the pI of the immunoglobulin and can be specifically adjusted according to the desired separation.

The most preferred mixed mode resin used in polishing step (d) is CaptoAdhere (GE Healthcare Life Science).

Preferred loading conditions for CaptoAdhere binding mode chromatography may be as follows: the resin is equilibrated with 0.5 M Na-phosphate, pH 8.2, followed by 20 mM Na-phosphate, pH 8.2. The sample (cation exchange pool) is adjusted to pH 8-8.5 and conductivity 1-4 mS/cm and loaded onto the column. After washing with equilibration buffer 20 mM Na-phosphate, pH 8.2, the immunoglobulin of interest can be eluted from the CaptoAdhere resin with, for example, 20 mM Na-phosphate, pH 5-7, preferably pH 5.5-6.5.

Alternatively, CaptoAdhere chromatography can be run in flow mode. Thus, pH and ionic strength must be adjusted so that the immunoglobulin does not bind to the mixed mode ligand while residual contaminants to be removed (DNA, aggregates, leached protein A, host cell proteins) remain bound. The conditions depend on the pI of the immunoglobulin. Preferably phosphate or Tris buffers are used in the pH range of 6.5 to 8.5, more preferably pH 7 to 8. The electrical conductivity is controlled by salt, eg NaCl, or buffer concentration. Most preferred is Na-phosphate buffer in the concentration range from 10 to 50 mm with the addition of NaCl in the concentration range from 50 to 200 mm. It should be taken into account that high salt concentrations, although desorbing the ionic interaction, promote hydrophobic interaction with the ligand.

Regeneration (clean-in-place) of the mixed mode resin can be performed at low pH, high salt and high pH, for example using 10 to 200 mM citric acid, 0.5 to 2 M NaCl and 10 to 1 M NaOH.

The preferred regeneration procedure is performed by successive washing with solutions A-D: solution A: 100 mM citric acid, 2 M NaCl; Solution B: 2M NaCl; Solution C: 1M NaOH; Solution D: 10 mM NaOH. Resin storage can be done in Solution D.

In another preferred embodiment, the method of the invention comprises the following steps in the following order:

- adding a flocculating compound to the culture liquid;

- depth filtration;

- affinity chromatography, where the immunoglobulin binds to the medium for affinity chromatography;

- washing the medium for affinity chromatography using a wash buffer with a pH value of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l;

- eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5; And

- strong cation exchange chromatography, in which the immunoglobulin binds to the resin for strong cation exchange chromatography, and obtaining immunoglobulin in the eluate by desorption of the immunoglobulin from the resin for strong cation exchange chromatography; And

- positively charged mixed chromatography; where the resin is equilibrated with a phosphate buffer pH 8 to 8.5, the immunoglobulin is bound to a positively charged mixed chromatography resin and the immunoglobulin is eluted with a phosphate buffer pH 5 to 7.

Polishing Stage: Anion Exchange Chromatography

The method of the invention may further include an anion exchange chromatography step as a polishing step, i.e. it is carried out in sequence (as one or as one or more steps) after the affinity chromatography.

The media, referred to as anion exchange media or resins, are chromatographic media containing functional groups consisting of positively charged ion exchange ligands. The anion exchange chromatography medium can be a strong or weak anion exchange chromatography medium, including anion exchange membranes.

In anion exchange chromatography, the bound molecules are negatively charged, while the immobilized functional groups (ligands) are positively charged. Commonly used anion exchange chromatography media are Q-medium (quaternary amine ligands), TMAE resins (trimethylaminoethyl ligands), and DEAE resins (diethylaminoethyl ligands). However, in general, the anion exchange chromatography step can be carried out using all the usual commercially available anion exchange media. The anion exchange medium can be used in the form of prepacked columns or membranes. Alternatively, the resins can be purchased as a bulk material and the columns can be packed by the user. There are no special restrictions on the capacity and size of columns, except for the usual ones. One skilled in the art will know the amount of anion exchange chromatography medium and the size of the column to be used. It depends on the overall scale of the process.

In particular, the strong anion exchange chromatography medium has been found to be effective in removing residual impurities.

It is preferred to use a strong anion exchange chromatography medium containing a ligand selected from the group consisting of quaternary aminoethyl (QAE) moieties, quaternary ammonium moieties, and trimethylammonium moieties.

Typical strong anion exchange chromatography media that can be used for the purpose of the invention contain functional groups such as quaternary aminoethyl (QAE) moieties, resins include e.g. Toyopearl QAE (available from Tosoh Bioscience, Germany), Selectacel QAE available from Polysciences Inc., Pennsylvania, USA), QAE Sephadex (available from GE Healthcare, Germany) and others; quaternary ammonium (Q) fragments, resins include, for example, Q Sepharose XL, Q Sepharose FF, Q Sepharose HP, Q Sepharose CL-4B, Q Sepharose Big Beads, Source Q, Resource Q, Capto Q, Capto Q ImPres (all available from GE Healthcare, Germany), Poros HQ (Applied Biosystems, Germany), Q HyperCel, Biosepra Q Ceramic HyperD (available from Pall, New York, USA) Macro Prep High Q (Bio-Rad, California, USA), Toyopearl Super Q (available from Tosoh Bioscience, Germany), UNOsphere Q (available from Bio-Rad, CA, USA), BioPro IEX SmartSep Q (YMC Europe), Praesto Q, Praesto Jetted Q35 (Purolite Life Sciences, Europe), trimethylammonium methyl (TMAE ) include, for example, Fractogel EMD TMAE (Merck KgaA, Germany) and trimethylammonium resins include, for example, Nuvia Q (available from Bio-Rad, CA, USA).

More preferably, the anion exchange chromatography may be high anion exchange chromatography, which is performed using a strong anion exchange resin containing -N(CH ₃ ) ₃ ⁺ (trimethylammonium) bound to highly cross-linked agarose (for example, Capto Q, available from GE Health Care, Germany), or media with similar characteristics.

In some embodiments, the method includes the following steps in the following order: (a) adding a flocculating compound to the culture fluid;

(b) depth filtration of the mixture of step (a);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the affinity chromatography medium using a wash buffer with a pH of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5;

(f) incubating the eluate from step (e) for viral inactivation at a low pH of 2.5 to 4.5 for at least 10 minutes;

(g) performing cation exchange chromatography; And

(h) performing anion exchange chromatography.

Another preferred embodiment involves anion exchange chromatography in flow mode after the step of cation exchange chromatography in binding mode.

Thus, in a preferred embodiment of the present invention, a method is provided comprising the following steps in the following order:

- adding a flocculating compound to the culture liquid;

- depth filtration;

- affinity chromatography, where the immunoglobulin binds to the medium for affinity chromatography;

- washing the medium for affinity chromatography using a wash buffer with a pH value of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l;

- eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5; And

- strong cation exchange chromatography, where the immunoglobulin binds to the resin for strong cation exchange chromatography, and obtaining immunoglobulin in the eluate by desorption of the immunoglobulin from the resin for strong cation exchange chromatography; And

- strong anion exchange chromatography; where the immunoglobulin does not bind to the resin for strong anion exchange chromatography, and obtaining immunoglobulin in the eluate.

The most preferred anion exchange chromatography resin is Capto Q (GE Health Care, Germany). Preferred loading conditions for Capto Q flow chromatography may be as follows: the resin is equilibrated with 25 mM Tris buffer, pH 8, and the chromatography is carried out with the same buffer.

The preferred regeneration procedure is carried out by sequential washing with solutions A-C: solution A: 2M NaCl; Solution B: 1M NaOH; Solution C: 10 mM NaOH. The resin can be stored in solution C.

Depth Filtration

In addition, the method of the present invention may include one or more stages of depth filtration. Unlike membrane filters, which separate by holding particles on the membrane surface, depth filters consist of a matrix of fibers or granules, with separation occurring throughout the matrix rather than on its surface.

Examples of depth filters include, but are not limited to, the Pall SXL series (e.g., SXLP700416 and SXLPDE2408SP filter capsules, the Pall HP series (e.g., PDD1, PDE2, PDH4, PDK5, PEKM) (Pall Corporation), the Millistak series (e.g., XOHC, FOHC), DOHC, AlHC and BlHC Pod), HC-Pro series, Clarisolve series (e.g. 40 MS) (EMD Millipore) or Zeta Plus series (90ZB08, 30ZA/60ZA, 60ZN90ZA, delipid, VR07 and VR05 filter pods) (3M).

Preferably the depth filter consists of a pre-extracted inorganic filter aid, cellulose and a resin system which imparts a strong positive charge to the filter matrix, such as Zeta Plus from 3M, UK.

Another preferred filter module is the Emphaze AEX (3M) Hybrid Purifier, which is an anion exchange depth filter.

The most preferred depth filters used in the present invention are PDE2, PDH4 and P700 series filter capsules from Pall Corporation.

The process parameters for performing depth filtration include a volume load from 100 to 2000 l/m ² , preferably from 200 to 1500 l/m ² , more preferably below 1200 l/m ² and most preferably from 400 to 1000 l/m ² , pressure from 0.2 to 2 bar, preferably 0.4 to 0.6 bar and room temperature.

In another embodiment, the purification may include one or more filtration steps preceding the first chromatography step. In a preferred embodiment, the purification may include one centrifugation step and one or more filtration steps. In another preferred embodiment, the first chromatography step is preceded by depth filtration and the microfiltration step is performed after flocculation. In a more preferred embodiment, the first chromatography step is preceded by a cell separation step, a flocculation step, a depth filtration step, and a microfiltration step. Additional filtration steps using positively and/or negatively charged membranes can be included in the filtration series clarifying the sample after flocculation and before the capture chromatography step. Filter modules combining different materials, charged and uncharged, are also included in the preferred embodiment.

Preferably the depth filter contains more than one layer. For example, a two-layer filter can be used for depth filtration.

Ultrafiltration, Viral Filtration, Microfiltration

In addition, the method according to the invention may include one or more stages of microfiltration, ultrafiltration and/or nanofiltration. Ultrafiltration is a form of membrane filtration in which pressure presses a liquid against a semi-permeable membrane. Suspended solids and high molecular weight solutes are retained while water and low molecular weight solutes pass through the membrane. Ultrafiltration is a widely used method for separating, purifying and concentrating macromolecular solutions, especially protein solutions. Ultrafiltration can be combined with diafiltration. This mode is suitable for buffer exchange, removal of salts and other microparticles from the solution by multiple or continuous dilution and re-concentration. Ultrafiltration can be performed with stacked membranes in a tangential or cross flow filtration system (TFF or TF-UF), especially for processing large sample volumes. As an alternative to ultrafiltration, hollow fiber systems are commonly used. The cutoff size of the membrane is from 1 to 300 kDa. For immunoglobulins, typical cutoff values for ultrafiltration membranes are 10-100 kDa. Within the scope of the present invention, a molecular weight cutoff of 30 or 50 kDa is preferred for UV membranes.

Microfiltration is a particle filtration method using membranes with a pore size of approximately 0.1 to 10 µm. For sterile filtration, which places special demands on the environment, sterilized microfilters with a pore size of approximately 0.2 µm are used. The use of additional pre-filters with large pore sizes (0.45 µm, 3 µm) is common. This prevents flow reduction due to fast blocking of filters with small pore sizes.

Finally, in the biopharmaceutical industry, nanofiltration is predominantly used to filter viruses and is essential for the safety of therapeutic proteins produced in mammalian cell cultures. The nanofiltration steps are typically performed at the end of the downstream, closer to filling the bulk of the purified immunoglobulin. The pore size of commonly used nanofilters is 15 to 35 nm (Planova, Asahi Kasei, Japan; or Viresolve, EMD-Millipore, Germany).

Within the scope of the present invention, the terms "nanofiltration" and "viral filtration" are used interchangeably.

The present invention provides an additional method for purifying immunoglobulin from culture fluid containing immunoglobulin and at least one contaminant, wherein the method includes the following steps in the following order:

- centrifugation and/or filtration of the culture fluid;

- adding a flocculating compound to the supernatant or filtrate;

- depth filtration;

- microfiltration;

- affinity chromatography, where the immunoglobulin binds to the medium for affinity chromatography;

- washing the medium for affinity chromatography using a wash buffer with a pH value of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l; And

- elution of the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5.

In a preferred embodiment of the invention, the purification process comprises one or more ultrafiltration/diafiltration and/or nanofiltration steps. These filtration steps can be performed using commercially available filter devices such as those available from Pall Corporation, GE Healthcare, EMD-Millipore, or Sartorius.

In a further embodiment, the method includes an additional step of nanofiltration of the eluate resulting from the affinity chromatography or polishing step, or the composition obtained therefrom and obtained after one or more additional processing steps performed after said chromatography step. Preferably, filters can be used for nanofiltration that are capable of retaining parvoviruses, most preferably with a pore size of 15-35 nm.

In some embodiments, the present invention provides a method for purifying an immunoglobulin from a culture fluid containing an immunoglobulin and at least one contaminant, wherein the method includes the following steps in the following order:

- adding a flocculating compound to the culture liquid;

- depth filtration;

- affinity chromatography, where the immunoglobulin binds to the medium for affinity chromatography;

- washing the medium for affinity chromatography using a wash buffer with a pH value of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l;

- eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5;

- cation exchange chromatography, where the immunoglobulin binds to the cation exchange chromatographic resin, and obtaining immunoglobulin in the eluate by desorption of the immunoglobulin from the cation exchange chromatographic resin;

- mixed chromatography, where the immunoglobulin is either bound to the mixed chromatography resin, and obtaining the immunoglobulin in the eluate by desorption of the protein from the mixed chromatography resin, or without binding to the mixed chromatography resin, and obtaining the immunoglobulin in the eluate; And

- filtering the eluate through a viral filter, optionally followed by an ultrafiltration/diafiltration step.

In further embodiments, the present invention provides a method for purifying immunoglobulin from culture fluid containing immunoglobulin and at least one contaminant, the method comprising the following steps in the following order:

- adding a flocculating compound to the culture liquid;

- depth filtration;

- affinity chromatography, where the immunoglobulin binds to the medium for affinity chromatography;

- washing the medium for affinity chromatography using a wash buffer with a pH value of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l;

- eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5;

- cation exchange chromatography, where the immunoglobulin binds to the cation exchange chromatographic resin, and obtaining immunoglobulin in the eluate by desorption of the immunoglobulin from the cation exchange chromatographic resin;

- anion exchange chromatography, where the immunoglobulin does not bind to the resin for anion exchange chromatography, and obtaining immunoglobulin in the eluate; And

- filtering the eluate through a viral filter, optionally followed by an ultrafiltration/diafiltration step.

In a preferred embodiment, the invention provides a method for purifying immunoglobulin from culture fluid containing immunoglobulin and at least one contaminant, the method comprising the following steps in the following order:

- adding a flocculating compound to the culture liquid;

- deep filtration of the mixture;

- affinity chromatography of the filtrate, where the immunoglobulin binds to the medium for affinity chromatography;

- washing the medium for affinity chromatography using a wash buffer with a pH value of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l;

- eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5;

- cation exchange chromatography, where the immunoglobulin binds to the cation exchange chromatographic resin, and obtaining immunoglobulin in the eluate by desorption of the protein from the cation exchange chromatographic resin;

- mixed mode chromatography, where the immunoglobulin binds to the mixed mode chromatography resin, and obtaining the immunoglobulin in the eluate by stripping the protein from the mixed mode chromatography resin;

- nanofiltration; And

- ultrafiltration/diafiltration.

In further preferred embodiments, the present invention provides a method for purifying immunoglobulin from culture fluid containing immunoglobulin and at least one contaminant, the method comprising the following steps in the following order:

- adding a flocculating compound to the culture liquid;

- depth filtration;

- affinity chromatography, where the immunoglobulin binds to the medium for affinity chromatography;

- washing the medium for affinity chromatography using a wash buffer with a pH value of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l;

- eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5;

- cation exchange chromatography, where the immunoglobulin binds to the cation exchange chromatographic resin, and obtaining immunoglobulin in the eluate by desorption of the immunoglobulin from the cation exchange chromatographic resin;

- anion exchange chromatography, where the immunoglobulin does not bind to the resin for anion exchange chromatography, and obtaining immunoglobulin in the eluate; And

- nanofiltration; And

- ultrafiltration/diafiltration.

Viral inactivation (V10)

Virus removal and/or inactivation is required in a method for producing a recombinant protein drug, such as a cell culture-derived monoclonal antibody, due to the possibility of accidental contamination by viruses from the starting material or production steps. As a result, there is significant regulatory demand for the viral safety of each manufacturing process, resulting in a biological therapeutic protein derived from cell cultures.

The viral inactivation step occurs after capture affinity chromatography, maintaining the eluate at a low pH. The advantage is achieved by elution at low pH from the affinity matrix. In such an aqueous acidic environment, many viruses, especially enveloped viruses, are unstable and disintegrate.

Low pH means pH values from 2 to 5, preferably from 2.5 to 4.5, more preferably from 3 to 4.

In a preferred embodiment, the incubation time for viral inactivation is 10 minutes to 2 hours, more preferably 30 minutes to 90 minutes, most preferably 45 minutes to 75 minutes.

In a preferred embodiment, viral inactivation is carried out at room temperature with stirring, and after incubation the pH is raised.

The present invention provides a method for purifying immunoglobulin from culture fluid containing immunoglobulin and at least one contaminant, the method comprising the following steps in the following order: (a1) separating cells from the culture fluid by sedimentation;

(a) adding a flocculating compound to the culture fluid;

(b) depth filtration of the mixture of step (a);

(c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium;

(d) washing the affinity chromatography medium using a wash buffer with a pH of 5 to 9 and an ionic strength of 0.1 to 5.0 mol/l; And

(e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer with a pH of 2.5 to 4.5.

(f) incubating the eluate from step (e) for viral inactivation at a low pH of 2.5 to 4.5 for at least 10 minutes;

(g) cation exchange chromatography of the eluate after viral inactivation in step (f) or a composition obtained therefrom and obtained after one or more additional processing steps performed after step (f), where the immunoglobulin is associated with a resin for cation exchange chromatography and obtaining immunoglobulin in the eluate by desorption of the protein from the resin for cation exchange chromatography;

(h) mixed chromatography of the eluate obtained in step (g) or a composition obtained therefrom and obtained after one or more additional processing steps performed after step (g), where the immunoglobulin is associated with a mixed mode chromatography resin and obtaining immunoglobulin in eluate by stripping the protein from the mixed chromatography resin;

(i) nanofiltration of the eluate obtained in step (h) or the composition obtained from it and obtained after one or more additional processing steps performed after step (h); And

(j) ultrafiltration/diafiltration of the filtrate of stage (i) or the composition obtained from it and obtained after one or more additional stages of processing performed after stage (i).

In further embodiments, step h) is as follows:

(h) Mixed chromatography of the eluate obtained in step (g), or a composition obtained from it and obtained after one or more additional processing steps performed after step (g), where the immunoglobulin does not bind to the mixed chromatography resin, and obtaining immunoglobulin in mixed chromatography resin eluate.

Or, alternatively, instead of mixed chromatography, anion exchange chromatography can be used, and step h) is as follows:

(h) anion exchange chromatography of the eluate obtained in step (g) or a composition obtained therefrom and obtained after one or more additional processing steps performed after step (g) where the immunoglobulin does not bind to the anion exchange chromatography resin, and obtaining the immunoglobulin in eluate from resin for anion exchange chromatography.

In a preferred embodiment, ultrafiltration/diafiltration is the last step in the purification of the immunoglobulin and no further chromatography is performed prior to steps (c), (g) and (h).

EXAMPLES

The methods for purifying immunoglobulins of the invention are supported and illustrated with reference to the following examples. It should be emphasized that these examples should in no way be construed as limiting the scope of the invention.

Example 1: Immunoglobulins and cell cultures

The methods of the invention are independent of the specific antibodies or specific host cells used to express the immunoglobulins. The same is true for the expression method and the selected culture conditions, which have been optimized to maximize the harvested cell yield. Various monoclonal antibodies have been used in the development of the methods of the present invention. They have been successfully purified on various scales according to the methods of the invention. Most of the selected experiments presented in the tables were performed with bevacizumab, a humanized anti-VEGF IgG1 antibody. In addition, some other experiments were performed with trastuzumab, a humanized anti-HER2, IgG1 antibody, and with denosumab, a fully human anti-RANKL IgG2 antibody. All three antibodies were recombinantly expressed in CHO cells, which were grown in fed-batch cultures of various scales. Most of the flocculation experiments at the development stage were carried out with culture fluids from a laboratory volume of 10 L or a pilot volume of 100 L or 200 L. The production scale and the maximum cultivation volume used in the examples were 5000 L. Some experiments were performed using a smaller production scale - 1000 liters. Unless otherwise noted, scale always refers to culture volume.

Example 2: Comparison of different flocculants (Tables 1-3)

The flocculation experiments shown in Tables 1-3 were performed in different batches using an experimental scale (100 L) culture of CHO cells producing humanized antibodies of the IgG1 subclass (bevacizumab). The antibody titer ranged from 1.16 to 1.52 g/l. The flocculants were added to 20 ml culture broth aliquots at the end of fermentation before cell separation. Three different cationic substances were tested at different concentrations. 1) pDADMAC=poly(diallyldimethylammonium chloride), 2) chitosan, and 3) calcium chloride. pDADMAC was purchased as a 10% (w/v) solution (EMD Millipore). Chitosan was purchased as a solid (Sigma) and dissolved 1% (w/v) in 0.1 M acetic acid. The operating pH range of chitosan is from 4.5 to 5. Calcium chloride was added using the original aqueous solution.

The experiments were carried out in the following sequence of stages:

- Taking a 20 ml aliquot from the fermentation broth

- Adding flocculant

- Incubation 15 min at room temperature with stirring

- Centrifugation at 4600 rpm/10 min (Thermo Scientific benchtop centrifuge)

- Microfiltration (PVDF, 0.2 µm, EMD Millipore)

- Analysis for turbidity, HCDNA and HCP.

In order to evaluate the direct effect of different flocculants at different concentrations, depth filtration was not performed. At the centrifugation stage, flakes, cells and cell debris were removed. Subsequent microfiltration removed the remaining particles. For controls (null values), no flocculant was added and no incubation was performed. However, before analysis, the samples were centrifuged and filtered. Protein A HPLC was used to determine antibody concentrations.

Table 1a: Effect of flocculation with pDADMAC on HCDNA reduction and haze

pDADMAC Conc. [% w/v] 0 0.0250 0.0375 0.0750 0.0900 HCDNA [ng/ml] 62900 59.9 16.0 16.0 16.0 Decrease [%] 0 99.91 99.98 99.98 99.98 Haze [FNU] 19.90 10.30 3.20 2.55 2.46 Decrease [%] 0 48.24 83.92 87.19 87.64

Table 1b: Effect of flocculation with chitosan on HCDNA reduction and haze

Chitosan Conc. [% w/v] 0 0.025 0.050 0.075 0.200 0.400 HCDNA [ng/ml] 62900 32700 24200 20700 6690 5380 Decrease [%] 0 48.01 61.53 67.09 89.36 91.45 Haze [FNU] 19.90 17.60 17.30 17.10 16.1 16.3 Decrease [%] 0 11.56 13.07 14.07 19.10 18.09

Table 1c: Effect of flocculation with CaCl ₂ on HCDNA reduction and haze

Calcium chloride Conc. [% w/v] 0 40 50 60 70 80 100 120 HCDNA [ng/ml] 62900 21000 12000 1330 590 318 130 56.4 Decrease [%] 0 66.61 80.92 97.89 99.06 99.49 99.79 99.91 Haze [FNU] 19.90 11.20 14.60 13.20 11.70 10.50 7.86 7.40 Decrease [%] 0 43.72 26.63 33.67 41.21 47.24 60.50 62.81

Tables 1a-1c show flocculation results for HCDNA reduction and turbidity using three different flocculants. HCDNA was determined using the Quant-iT™ dsDNA High Sensitivity Assay Kit (Thermo Fisher) and qPCR (PrepSEQ™ Residual DNA Sample Preparation Kit and resDNASEQ™ Quantitative CHO DNA Kit, Thermo Fisher). Turbidity measurements were made on a Hach 2100Q turbidimeter and expressed in formazin nephelometric units (FNU). The instrument measured the light scattered by the sample at an angle of 90 degrees to the incident infrared light.

Table 1a shows the effect of pDADMAC up to 0.09% (w/v). Already at the lowest concentration (0.025% w/v), HCDNA was almost completely removed (99.9% reduction, three orders of magnitude). An 87% reduction in turbidity can be achieved using 0.075% (w/v) pDADMAC. Further turbidity reduction could not be achieved with the higher concentration of pDADMAC in this experiment.

The results with chitosan, as shown in Table 1b, showed effective removal of HCDNA, however, at higher concentrations required for pDADMAC. A reduction of about 91.5% was obtained at 0.4% (w/v). With regard to haze reduction, a reduction of no more than 19% was measured.

The results of the experiments with CaCl ₂ are shown in Table 1c. Also a very effective reduction of HCDNA was obtained for this flocculant. A 99% reduction was measured at 70 mM and a 99.9% reduction at 120 mM CaCl ₂ . The haze reduction was greater for chitosan but lower for pDADMAC. A reduction of about 63% can be achieved at 120 mM. Higher concentrations of CaCl ₂ were not tested.

Conclusions (Tables 1a-c): The initial concentration of HCDNA (ie controls without flocculation) was 62.9 μg/ml cell culture supernatant and 51.1 mg/g IgG, respectively. Losses of total protein or antibodies were not detected for all flocculants and concentrations tested. Similarly, no increase in cell rupture caused by flocculation was observed. Surprisingly, it is clear that HCDNA has little, if any, effect on haze. For example, with 0.4% (w/v) chitosan, approximately 91.5% of HCDNA was flocculated and removed, while the turbidity remained almost unchanged at 82% of control (Table 1b). To reduce both HCDNA and turbidity, the flocculant pDADMAC is best suited.

Table 2a: Effect of flocculation with pDADMAC on HCP reduction

pDADMAC Conc. [% w/v] 0 0.0250 0.0375 0.0500 0.0750 0.0900 HCP [µg/ml] 2310 2280 2240 2170 2100 2060 Decrease [%] 0 1.30 3.03 6.06 9.09 10.82

Table 2b: Effect of flocculation with chitosan on HCP reduction

Chitosan Conc. [% w/v] 0 0.025 0.050 0.075 0.100 0.200 0.400 HCP [µg/ml] 1720 1670 1720 1720 1590 1580 1610 Decrease [%] 0 (0) 0 0 7.56 8.14 6.40

Table 2c: Effect of calcium chloride flocculation on HCP reduction

Calcium chloride Conc. [% w/v] 0 10 20 50 60 70 HCP [µg/ml] 1410 1330 1200 1080 1080 1000 Decrease [%] 0 5.67 14.89 23.40 23.40 29.08

Tables 2a-2c show the results of HCP reduction flocculation using three different flocculants. HCP was measured using CHO HCP ELISAKit, 3G (Cygnus technology).

Table 2a shows the effect of pDADMAC on HCP removal. Even at the highest concentration tested (0.09% w/v), the reduction was only about 10.8%.

As shown in Table 2b, the effect of chitosan as a flocculant on the reduction of HCP was negligible. The best reduction was about 8.1% at 0.2% (w/v).

The best reduction in HCP was obtained in experiments with CaCl ₂ as shown in table 2c. A 29.1% reduction was measured at 70 mM. Higher concentrations may result in better recovery but have not been tested.

Conclusions (Tables 2a-c): Initial concentrations of HCP ranged from 1.4 to 2.3 mg/ml. By mass, this is about 20-40 times more than the HCDNA load. Again, it is clear that HCP also has little effect on haze. For example, 0.075% (w/v) pDADMAC reduced haze by about 87% (Table 1a), while the reduction in HCP was only about 9% (Table 2a). For HCP reduction by flocculation, CaCl ₂ was the best. Tables 3a-3c below summarize the favorable concentrations of the three flocculants tested for reduction of HCDNA, turbidity and HCP.

Table 3a: Favorable concentrations of various flocculants for reducing HCDNA

flocculant Concentration decline pDADMAC 0.025% w/v 99.9% Chitosan 0.400% w/v 91.5% _CaCl2 70 mm 99.1%

Table 3b: Favorable Concentrations of Various Flocculants for Turbidity Reduction

flocculant Concentration decline pDADMAC 0.075% w/v 87.2% Chitosan 0.200% w/v 19.1% _CaCl2 120 mm 62.8%

Table 3c: Favorable concentrations of various flocculants for reducing HCP

flocculant Concentration decline pDADMAC 0.090% w/v 10.8% Chitosan 0.200% w/v 8.1% _CaCl2 70 mm 29.1%

All three flocculants were able to effectively remove HCDNA due to their cationic nature. Since the original problem of the present invention was mainly related to the turbidity observed in the Protein A elution pools, pDADMAC was chosen as the most suitable flocculant. This polymer can be used at lower concentrations and surprisingly results in almost complete removal of HCDNA already at the lowest concentration tested.

Example 3: Further flocculation experiments with pDADMAC (Tables 4-6)

In subsequent larger series of trials, the effect of pDADMAC on HCDNA reduction and turbidity was investigated in parallel experiments using samples from six different cell cultures expressing bevacizumab. The test methods were the same as described in Example 2. The non-flocculated supernatant (control) ranges were 44.3 to 62.9 µg/ml HCDNA, 19.9 to 29.1 FNU turbidity, and 1.24 to 2. 14 mg/ml HCP. The antibody titer ranged from 1.16 to 1.52 mg/ml. Five different concentrations of pDADMAC were tested and mean % reductions were calculated.

Table 4a: Effect of pDADMAC concentration on HCDNA reduction in different cell culture lots

Host cell DNA [ng/ml]
% reduction pDADMAC
[% w/v] 0 0.0250 0.0375 0.0500 0.0750 0.0900 Cell culture 1 46500
0 3.9
99.99 3.1
99.99 0.2
100 0.2
100 0.2
100 Cell culture 2 62900
0 59.9
99.91 16.0
99.98 Not
done 16.0
99.98 16.0
99.98 Cell culture 3 45400
0 16.1
99.96 5.7
99.99 5.7
99.99 5.7
99.99 5.7
99.99 Cell culture 4 44300
0 4.0
99.99 0.4
100 0
100 0.3
100 0.9
100 Cell culture 5 51500
0 0.7
100 0.1
100 0.1
100 0.1
100 0.1
100 Cell culture 6 50200
0 18.9
99.96 0.9
100 2.5
100 0.5
100 0.6
100 Average % Decrease 0 99.97 99.97 100 100 100

The results shown in Table 4a further confirm the high potential of the cationic polymer pDADMAC to flocculate HCDNA. Already at the lowest concentration tested (0.025% w/v), almost 100% reduction was obtained in all six cell cultures. In practice, the quantitative removal of HCDNA at an early stage of subsequent processing is very beneficial and facilitates subsequent filtration and capture chromatography.

Turbidity reduction is the most important requirement when designing pre-treatment steps. In the case of several cell cultures expressing IgG1 or IgG2 antibodies (eg, bevacizumab, trastuzumab, and denosumab), turbidity occurs in the concentrated solutions eluting from the capture column. In the experiments of the present invention, it was found that the reduction of turbidity in the pre-purification step also prevents haze after capture chromatography if the process steps are performed without interruption. No reappearance of turbidity was observed in the protein A elution pools.

Table 4b: Effect of pDADMAC concentration on turbidity reduction in different batches of cell cultures

Haze [FNU]
% reduction pDADMAC [%w/v] 0 0.0250 0.0375 0.0500 0.0750 0.0900 Cell culture 1 28.10
0 22.80
18.86 8.53
69.64 5.57
80.18 2.92
89.61 2.85
89.86 Cell culture 2 19.90
0 10.30
48.24 3.20
83.92 Didn't do 2.55
87.19 2.46
87.64 Cell culture 3 29.10
0 8.95
69.24 8.50
70.79 3.50
87.97 3.25
88.83 3.40
88.32 Cell culture 4 24.90
0 9.68
61.12 6.74
72.93 2.30
90.76 2.18
91.25 2.38
90.44 Cell culture 5 20.80
0 17.10
17.79 15.20
26.92 5.01
75.91 3.18
84.71 3.10
85.10 Average % Decrease 0 48.93 64.84 83.70 88.32 88.27

The results presented in Table 4b confirm the use of pDADMAC as a flocculant. At 0.05% (w/v), the average reduction in cell cultures was above 80%. At a concentration of 0.075% (w/v), the average reduction was about 88%. Higher concentrations (0.09% w/v) did not give a further increase.

Another study evaluated pDADMAC flocculation (without subsequent depth filtration) for primary clarification of nutrient fluxes for high density cell cultures. In contrast to previous experiments, cell culture samples were 1000 L aliquots of the selected fermentation scale (cell density 9.3×10 ⁶ cells/mL) expressing bevacizumab.

The experiments were carried out in the following sequence of stages:

- Taking a 40 ml aliquot from the fermentation broth (50 ml tubes),

- Adding flocculant in different concentrations,

- Incubation 15 min at room temperature with gentle stirring,

- Centrifugation at 3000 rpm/2000g/5 min,

- Microfiltration (Millex filter, 0.2 µm, EMD Millipore),

- Immediate turbidity analysis,

- Subsequent analysis of the concentration of monoclonal antibodies, HCDNA and HCP.

Table 5 summarizes the results of this study. Turbidity was measured on a Hach 2100Q turbidimeter and, unlike previous experiments, was expressed in nephelometric turbidity units (NTU). The instrument measures the scattered light from the sample at a 90 degree angle to the incident white light.

Table 5: Dosing of pDADMAC

pDADMAC [%w/v] 0 0.0250 0.0375 0.0500 0.0750 0.1000 pDADMAC [pg/cell] 0 27 40.5 54 81 108 mab conc. [mg/ml] 1.14 1.14 1.15 1.15 1.12 1.20 HCDNA [ng/mg]
% reduction 39500
0 3.61
99.99 1.54
100 0.58
100 0.49
100 1.76
100 Turbidity [NTU]
% reduction 43.70
0 12.96
70.34 3.30
92.45 1.35
96.91 1.63
96.27 1.94
95.56 HCP [µg/mg]
% reduction 2140
0 1960
8.41 1990
7.01 1970
7.94 1990
7.01 1820
14.95

The results presented in Table 5 confirm previous results for the reduction of HCDNA, turbidity and HCP (see Tables 1a, 2a, 4a and 4b). The optimal dose of pDADMAC is defined as the minimum polymer concentration that results in a significant reduction in supernatant turbidity. Adding higher concentrations of pDADMAC may not be practical. Higher concentrations may only result in a slightly lower level of turbidity (see Table 5, 0.1% w/v pDADMAC) or may potentially result in a slight decrease in the concentration of the product of interest in the supernatant (not observed to 0.1% w/v pDADMAC) or promote faster haze return (see Table 6). Higher concentrations can also lead to the formation of a population of smaller flocculants that can break through the depth filters. The optimal concentration of pDADMAC is in the range of 0.0375 to 0.075% (w/v). This refers to approximately 40-80 pg/cell under the conditions of this experiment. In this range, the reduction in HCDNA was 100%, the reduction in turbidity was about 92-97%, and the reduction in HCP was 7-8%. It should be emphasized that flocculation did not cause a noticeable loss of mAbs.

A further experiment was performed with the same starting material used for the experiments in Table 5, which monitored turbidity stability after performing flocculation, centrifugation and microfiltration. The same methods were used as described in Example 1. After flocculation with three concentrations of pDADMAC (0.0375% w/v, 0.075% w/v, and 0.09% w/v), the turbidity of the sterile filtered supernatant measured over 24 hours at room temperature. Through parallel studies, it was found that centrifugation and microfiltration do not remove residual free pDADMAC. On the contrary, in the chromatographic stages (for example, with Protein A), the polymer was quantitatively removed. Table 6 shows the results of experiments on the stability and kinetics of the reappearance of turbidity.

Table 6: Stability of filtered culture fluids after pDADMAC flocculation

Haze [FNU]/% increase Time
[h] pDADMAC concentration [% w/v] 0.0375 0.0750 0.0900 0 5.48/0 3.09/0 3.65/0 1.5 6.09/11.1 3.16/2.3 4.64/27.1 3 6.85/25.0 3.91/26.5 6.49/77.8 8 6.95/26.8 7.41/139.8 11.9/126.0 24 8.12/48.2 14.7/375.7 21.5/489.0

Surprisingly, the turbidity level increased after three hours, indicating continued settling. The effect is highly dependent on the concentration of pDADMAC. The increase in turbidity after 3 hours is 25% for 0.0375% (w/v), but about 78% for 0.09% (w/v). After 24 hours, the turbidity at the highest concentration of pDADMAC reached non-flocculated (control) values (see Table 4b). The original turbidity reduction by flocculation was cancelled.

Based on these results, it was concluded that 1) flocculated and filtered supernatants require rapid further processing (depth filtration, Protein A chromatography) and 2) the lowest effective amount of pDADMAC should be selected. Compared to 0.075% (w/v), the stability of 0.0375% (w/v) supernatants is much better with a slight increase up to 8 hours. This is an acceptable time window for standard production.

Example 4: Depth Filtration Experiments with and without Flocculation.

When flocculating with 0.0375-0.075% (w/v) pDADMAC in high volume fermentation liquors (5000 L), the cell separator, which then removes flocs and cells, was heavily loaded and post-run cleanup became quite cumbersome. This is also true for the fermenter, which must be cleared of residual flakes. Therefore, in further experiments, alternative pretreatment strategies were tested to overcome this shortcoming.

First, it was investigated whether the flocculation step could be skipped and replaced with different depth filter installations, including two-stage depth filtration. Although several well-established filter systems, including prefilters, were tested in many combinations, the results were not satisfactory, i.e. turbidity after cell separation and depth filtration remained at about 10 FNU, and subsequent precipitation in the Protein A elution pools occurred. with a cloudiness of approximately 30 FNU for better filter tuning. Moreover, depth filters did not reduce HCDNA and HCP.

Secondly, it was investigated whether it is possible to dispense with the separator after flocculation when only depth filters are used. Indeed, some of the filter settings clarified the flocculated culture fluid sufficiently with an acceptable low eluate turbidity (1-2 FNU). Although this strategy offers promising cleaning, the required filter sizes and processing times are quite high and are a significant economic factor. This strategy has not been implemented.

Example 5 Flocculation Experiments with Cell Free Culture Supernatant

In this example, one preferred pretreatment strategy has been developed. The order of the pre-cleaning steps was as follows (see also Figure 2B):

- Collection of culture fluid,

- Cell separation/centrifugation,

- Flocculation with pDADMAC (0.0375% w/v - 0.075% w/v),

- Depth filtration,

- Microfiltration (0.2 microns),

- Chromatography with Protein A (MabSelect SuRe LX).

Compared to Examples 2 and 3, flocculation was performed after cell separation/centrifugation followed by depth filtration. This method was performed with 1L, 10L, 100L and 5000L culture fluid (bevacizumab) and several depth filtration parameters were compared and optimized. The turbidity of the supernatants before flocculation was in the range of 30-100 FNU for the 5000 L scale.

The following types of depth filters were studied:

- Pall Supercap 50 PDH4

- Pall Supercap 50 PDE2

- Pall Supercap 50 PDD1

- Pall Supercap 50 PEKM

- Pall Supercap 50 PEKX

- 3M BC25 90ZB08A

- Hybrid cleaner 3M BV8 Emphaze AEX.

The concentrations of HCDNA, turbidity, HCP and mAbs were again analyzed (see methods in previous Examples) and based on these results PEKM, PDD1 and PDE2 are the most suitable for clarification of flocculated supernatants to an acceptable degree. Due to their dual layer configuration, PDD1 and PDE2 showed more reliable processing than PEKM (single layer) and were therefore the preferred depth filters for floc removal. The turbidity of the eluate behind the depth filters was 1-2 FNU. The filtration time ranged from 80 to 240 minutes at a load of 250-500 l/m ² . Both PDD1 and PDE2 filtrates showed only a slight increase in turbidity over 24 hours, and both filtrates are considered stable enough for further processing on Protein A.

Conclusion. Flocculation with cell-free supernatant (after separation/centrifugation) has proven to be the preferred pre-purification strategy and is well suited for large scale production of immunoglobulins. Flocculated supernatants with a turbidity of 30-100 FNU can be successfully processed with two layer depth filters such as PDD1 and PDE2. At least 500 l/m ² can be loaded into filters of this type. In the case of supernatants with higher turbidity (>100 FNU), the effective filtration area can be increased to avoid long processing times. The final concentration of pDADMAC was 0.0375% (w/v) and is in complete agreement with small scale flocculation experiments in the presence of cells and without depth filtration (Examples 1-3).

Example 6: Flocculation of cell culture supernatants with a higher concentration of IgG using various concentrations of pDADMAC (Table 7)

In this experiment, two different small scale cell cultures were compared by accumulating different amounts of IgG2 antibody (denosumab). Culture volumes were 10 L and two 10 L cultures were pooled for starting material. The culture was harvested after 8 and 10 days, respectively. The pooled 8 day old culture contained 3.86 mg/ml IgG, 42.4 µg/ml HCDNA and 0.454 mg/ml HCP. The 10-day culture contained 5.79 mg/ml IgG (+50%), 85 µg/ml HCDNA (+102%) and 0.506 mg/ml HCP (+11%). Cell culture supernatants were centrifuged (10 min at 4600 rpm) and flocculated (15 min incubation) as described in Example 1.

Table 7: Comparison of various starting materials for impurity reduction by pDADMAC flocculation

pDADMAC
[% w/v] Culture 1/8 d/3.86 mg/ml IgG2 Culture 2/10 d/5.79 mg/ml IgG2 HCDNA
[ng/ml]
(% down) HCP
[µg/ml]
(% down) Turbidity
[FNU]
(% down) HCDNA
[ng/ml]
(% down) HCP
[µg/ml]
(% down) Turbidity
[FNU]
(% down) 0.0000 40100
(0.00) 424
(0.00) 55.90
(0.00) 80700
(0.00) 525
(0.00) 101.00
(0.00) 0.0100 6150
(84.66) 492
(-15.22) 148.00
(-164.76) 18600
(76.95) 511
(2.67) 238.00
(-135.64) 0.0250 0.495
(100.00) 427
(-0.71) 2.02
(96.39) 3,250
(100.00) 469
(10.67) 3.93
(96.11) 0.0375 0.015
(100.00) 365
(13.92) 1.51
(97.30) 0.091
(100.00) 406
(22.67) 2.29
(97.73) 0.0750 0.011
(100.00) 330
(22.17) 1.54
(97.25) 0.012
(100.00) 376
(28.38) 1.88
(98.14) 0.0900 0.023
(100.00) 354
(16.51) 1.84
(96.71) 0.028
(100.00) 374
(28.76) 1.98
(98.04) 0.1000 0.035
(100.00) 322
(24.06) 1.89
(96.62) 0.040
(100.00) 378
(28.00) 2.03
(97.99)

Surprisingly, two very different cultures behaved quite similarly in flocculation experiments. Again, the high potential of pDADMAC to remove HCDNA was confirmed. Already at 0.025% pDADMAC, the reduction in HCDNA was 100% (about 5 orders of magnitude). Looking at turbidity, the reduction is as high as 97-98% for 0.0375% and 0.075% pDADMAC. In this range, HCPs decreased by 22% and 28%, respectively. Surprisingly, the pre-flocculation strategy proved to be quite reliable in terms of antibody type, cell density and monoclonal antibody titer. Stoichiometric dosing for specific crops is not required.

Example 7: Effect of flocculation on impurities after affinity capture with Protein A (Tables 8-10)

This example demonstrates the effect of flocculation on residual contaminants in the Protein A elution pool. Starting materials were cell cultures expressing trastuzumab (IgG1) at experimental scale (200 µl, Table 8) and bevacizumab at full volume (5000 µl, Table 9). Protein A chromatography was performed as described in Example 8.2. The pDADMAC concentration was 0.0375% (w/v) and the depth filter was a Pall Supercap 50 PDE2.

Table 8: Reduction of impurities in Protein A eluates by prior flocculation of pDADMAC

Pre-cleaning steps HCDNA [pg/mg]
(% Decrease) HCP [ng/mg]
(%Reduction) Haze [FNU]
(%Reduction) centrifugation
+ Depth filtering
(control) 889
(0) 394
(0) 42.9
(0) centrifugation
+ Flocculation
+ Depth filtering 76
(91.5) 204
(48.2) 8.8
(79.5)

The IgG concentrations in the protein A eluates were 20.36 mg/ml for the non-flocculated control and 23.32 mg/ml for the flocculation process. No flocculation was performed during the control, and the supernatants after centrifugation were directly subjected to depth filtration and chromatographed over Protein A. Protein A elution pools showed significant precipitation in terms of turbidity of about 43 FNU in the control. In contrast, during the flocculation of pDADMAC, the turbidity was greatly reduced by about 80%. HCDNA dropped over 90% to a very low level of 0.076 ppm which is difficult to detect. Residual HCP was more prominent, ie 204 ppm, reflecting 2-3 orders of magnitude higher loading in the starting material. Its concentration was approximately double that of the control. These results clearly demonstrate the remarkable beneficial effect of pDADMAC flocculation on product purity in Protein A eluates.

The following Tables 9a and 9b show standard production control data from two production lots (5000 L of bevacizumab), which additionally demonstrate a surprisingly high impurity reduction before and after Protein A chromatography. Protein A chromatography was performed in two consecutive runs with 50% of the total volume to reduce the column volume of expensive resin. Subsequent incubations for viral inactivation were performed as described in Example 9.3.

Table 9a: Large scale pDADMAC flocculation process: HCDNA concentrations

Party 1 Party 2 Average value Stage IgG
[mg/ml] HCDNA
[ng/mg] IgG
[mg/ml] HCDNA
[ng/mg] HCDNA
[%] Collection 1.5 87000 1.6 105250 100 centrifugation 1.6 56500 1.6 57000 59 flocculation 1.6 n.d. 1.6 150 0.16 Filtration 1.5 n.d. 1.5 n.d. 0.00 Protein A (1)* 16.9 n.d. 18.4 n.d. 0.00 Protein A (2)* 17 n.d. 18.4 n.d. Inactivation (1) 14.6 n.d. 14.5 n.d. 0.00 Inactivation (2) 15 n.d. 15.1 n.d.

*Chromatography with Protein A was performed in two consecutive runs with a share of 50% in each of the total volume;

n.d.: = not detectable, below test method detection limit.

Table 9b: Large scale pDADMAC flocculation process: HCP concentrations

Party 1 Party 2 Average value Stage IgG
[mg/ml] HCP
[µg/mg] IgG
[mg/ml] HCP
[µg/mg] HCP
[%] Collection 1.5 778.1 1.6 871.4 100.0 centrifugation 1.6 839.0 1.6 921.9 106.8 flocculation 1.6 740.4 1.6 811.2 94.1 Filtration 1.5 662.2 1.5 744.9 85.3 Protein A (1)* 16.9 0.269 18.4 0.252 0.028 Protein A (2)* 17.0 0.255 18.4 0.160 Inactivation (1) 14.6 0.259 14.5 0.225 0.027 Inactivation (2) 15.0 0.270 15.1 0.152

HCDNA was practically not detected after flocculation and below the detection limit in subsequent stages (Table 9a). Flocculation and depth filtration reduced the HCP by only about 15% (Table 9b). However, Protein A chromatography, due to its high selectivity for IgG-Fc, reduced the amount of HCP impurities by more than 99.9%. However, residual concentrations of HCP 160-270 ppm after chromatography on Protein A indicate the need for further polishing steps, at least in the case of a therapeutic antibody for human use.

Table 10: Comparison of leached Protein A for different processes

Process Pre-cleaning stage Leached Protein A in the Protein A Elution Pool Cycle 1 [ng/mg] Cycle 2 [ng/mg] Average value [%] A No 25.1 21.6 100 B Nuvia Q Flow 10.3 7.8 39 C pDADMAC Flocculation 5.8 3.8 21

The experiments in Table 10 compared three different processes for leached Protein A in eluted Protein A pools. Process C is a method according to the present invention that uses pDADMAC flocculation under the same conditions as described in Example 7/Table 8 (200 μl of trastuzumab ). The results of the two runs were compared to the previously established process (Process B) using an anion exchange flow chromatography (Nuvia Q) preceding the Protein A step. Process A is an additional control without a pre-treatment step (other than centrifugation and filtration). Processes A and B are described in WO2015135884. All processes were performed at room temperature. Protein A chromatography was performed as described in Example 8.2.

Process A: cell separation → depth filtration → microfiltration → Protein A

Process B: cell separation → Depth filtration → Microfiltration → Anion exchange chromatography (Nuvia Q, flow through) → Protein A (as described in WO2015135884)

Process C: Cell Separation → Flocculation (pDADMAC) → Depth Filtration → Microfiltration → Protein A

The amount of Protein A leached by Process C was significantly lower than by Process B, which was already a valuable improvement for the lower level of Protein A leached compared to Control Process A. The reduction in Protein A leached by Process C was approximately 48% compared to process B and approximately 79% compared to process A. Thus, in addition to a marked reduction in HCDNA and turbidity, pDADMAC flocculation additionally significantly reduces Protein A leaching.

Example 8: Subsequent Process - Purification of Immunoglobulin from Pretreated Culture Supernatants.

The following describes methods that were developed on a small scale and were first applied to a pilot process for the production of bevacizumab (IgG1) on a 100 L scale. The same process, with only minor modifications, was later successfully scaled up to 5,000 hp. After further minor adaptations, this process was also developed for trastuzumab (IgG1) and densosumab (IgG2).

Example 8.1: Selection of Chromatography Resins (Table 11)

Following an extensive screening of commonly used chromatographic media for the capture, intermediate and polishing steps, which was described in WO2015135884, the following chromatographic resins were selected for the subsequent immunoglobulin process (Table 11):

Table 11: Preferred Chromatography Resins

Process stage Resin Type ligand Provider capture MabSelect SuRe LX affine Protein A derivative stabilized with alkali GE Healthcare Intermediate Poros 50HS CEX Sulfopropyl (SP) Applied Biosystems Polishing Capto Q AEX Trimethylammonium GE Healthcare Polishing CaptoAdhere MMC N-benzyl-N-methylethanolamine GE Healthcare

CEX=cation exchange chromatography; AEX=anion exchange chromatography; MMC=Mixed Chromatography.

Chromatographic runs were performed using an Äkta Purifier System (GE Healthcare) at room temperature.

Example 8.2: Chromatography with Protein A.

Protein A capture chromatography was performed with a MabSelect SuRe LX (Table 11). A sample was taken after deep filtration and microfiltration of the flocculated supernatant as described in Example 5. The column was 20 cm in diameter by a bed height of 10.4 cm (packed volume approx. 3.3 L). The Protein A column was equilibrated with 20 mM tris-acetate buffer, pH 7.2. The product solution (about 49.5 L) was charged with 15.8 to 40.6 protein/L resin. The column was washed with equilibration buffer (2 CV) followed by 40 mM Na-phosphate, 1.5 M NaCl, 2 M urea, 10 mM EDTA, pH 7.4. Elution was performed with 100 mM sodium citrate, pH 3.5. The flow rate was 210 cm/h. MabSelect SuRe LX resin was regenerated by backwashing sequentially with (i) 0.2 M NaOH (2 CV), (ii) WFI (2 CV), 3.5% acetic acid, 100 mM Na-sulfate (2 CV). and (iii) WFI (2 CVs). The column was re-equilibrated for the next run or stored in 20% ethanol. The yield was 94-98%.

Example 8.3: Viral inactivation

As shown in Figures 1 and 2, the viral inactivation step occurs after Protein A affinity chromatography. The advantage is achieved by elution at low pH from the affinity matrix. In such an aqueous acidic environment, many viruses, especially enveloped viruses, are unstable and disintegrate. The Protein A method developed for this invention gives an eluate having a pH of 3.5 (see Example 8.2). Monoclonal antibodies were eluted from the Protein A column in 100 mM Na-citrate buffer, pH 3.5, directly into tank A for viral inactivation. The eluate was diluted approximately 2-fold with WFI directly in tank A. The pH of the solution was monitored and re-adjusted to 3.5 with 100 mM citric acid if necessary, and the solution was subsequently transferred to virus inactivation tank B where it was agitated at 65 rpm at 20-24°C for 60 minutes. The pH of the solution was then adjusted to pH 4.5 with 100 mM NaOH, providing the initial conditions for the subsequent cation exchange chromatography step.

Example 8.4 Cation exchange chromatography on Poros 50 HS

Separation of contaminants such as HCP and leached protein A and product-related substances such as charge variants and aggregates was performed using strong cation exchange chromatography with sulfopropyl (SP) ligands (Table 11) used as an intermediate step. A packed Poros 50 HS resin column (size=14 cm diameter x 41 cm bed height, packing volume about 6.3 L) was equilibrated sequentially with (i) WFI (water for injection, 2 CV) and (ii) 20 mM sodium citrate, pH 4.9 (2CV). The product solution obtained after viral inactivation and sample adjustment (pH 4.5) as described in Example 8.3 was loaded onto a column with about 7.3-17.1 g protein/l resin, thereby passing through a Kleenpak Nova 0 prefilter, 45 µm (Pall Corporation). The column was washed with WFI (1 CV) and equilibration buffer (2 CV) before subsequent gradient elution. The gradient consists of two parts with varying degrees of variation. The gradient was formed by mixing 20 mM Na-citrate, pH 4.9 (buffer A) and 40 mM Na-phosphate, pH 7.2 (buffer B) in the following ratios and sequence: (i) 100% A (0.2 CV), (ii) linear gradient to 40% A+60% B (2 CV); (iii) linear gradient to 100% B (10 CV); (iv) 100% B (2 CVs). The flow rate was 75 cm/h during the gradient, otherwise 150 cm/h. The eluate was divided into fractions to provide specific pooling. Fraction collection during the gradient elution started when A280 exceeded >0.1 AU. The volume of the fraction was about 2 liters. In total, approximately 20 fractions were collected. Poros 50 HS resin was regenerated by sequential washing with (i) 1 M NaCl (2 CV) and (ii) 1 M NaOH (2 CV). Thereafter, the column was stored in 10 mM NaOH. The yield in the selected pool was approximately 62-68% due to the depletion of a significant amount of impurities associated with the product.

Example 8.5: Mixed chromatography with CaptoAdhere

The final polishing step was performed with a CaptoAdhere mixed type resin using N-benzyl-N-methylethanolamine ligand (Table 11). The ligand carries positively charged groups and, therefore, in addition to hydrophobic interactions, also provides the functions of an anion exchanger. Chromatography is able to reduce the remaining traces of contaminants such as HCDNA, HCP and major variants depending on the charge. Residual leached Prothien A, product aggregates and product fragments can also be removed at this stage. The CaptoAdhere polishing step was performed in binding mode, which has the advantage of exchanging the buffer with the chosen final buffer.

The pH of the Poros 50 HS pool obtained after the procedures described in Example 8.4 was adjusted to pH 7.2 by adding 100 mM NaOH. The size of the packed column was 14 cm in diameter by 23 cm of bed height (stuffing volume approx. 3.5 liters). The resin was equilibrated with 50.48 mM Na-phosphate, pH 7.2 (3 CV). The product solution was loaded onto a 6.3-17.8 g protein/l resin) column followed by the addition of equilibration buffer (3 CV). Elution was carried out with 50.48 mm Na-phosphate, pH 5. The flow rate was 240 cm/h. The yield ranged from 87 to 98%. The column was regenerated with 2 M NaCl+100 mM citric acid (2 CV) and 1 M NaOH (2 CV) in reverse flow.

Example 8.6 Anion Exchange Chromatography with Capto Q

Instead of mixed chromatography, Capto Q anion exchange chromatography was performed in this example as the final polishing step. Anion exchange chromatography was carried out in flow mode. Before loading the columns, the pH of the combined cation exchange chromatography fractions was adjusted to pH 8 with acetic acid. The Capto Q column was equilibrated with 25 mM Tris, pH 8 buffer. After sample loading, chromatography was performed with the same buffer. The immunoglobulin did not bind to the chromatography resin and was obtained in the eluate.

Example 8.7: Virus filtering

Nanofiltration represents the final step in virus removal and is the most demanding and most reliable virus removal method based on size exclusion in the nanometer range. Viral filtration was performed immediately after mixed chromatography using the CaptoAdhere elution pool from Example 8.5 or the Capto Q flow pool from Example 8.6 after adjusting the pH to 6.2 with Na-phosphate or to pH 5 with acetic acid. Virus removal filtration was performed at room temperature with a Viresolve Pro Modus 1.1 (EMD Millipore) using a QuattroFlow 150 diaphragm pump. An Opticap XL 150 PES 0.1 µm pore size filter (EMD Millipore) was placed in front of the virus filter to avoid clogging virus filter aggregates. The filters were conditioned with 1.5 L of formulation buffer, eg 50.48 mM Na-phosphate buffer pH 6.2 or 18 mM Na-acetate buffer pH 5.2. The set pressure during the filtration process was 2±0.2 bar. After a loading of about 500-600 L/m ^{2 ,} a volume of about 0.5-1 L of formulation buffer, eg 50.48 mM Na-phosphate buffer, pH 6.2, was used to wash out residual monoclonal antibodies from the filter series. The integrity of the virus filter was checked at a pressure of 3.4 bar and a test duration of 1 min.

Example 8.8: Tangential Flow Ultrafiltration/Diafiltration (TF-UF/DF)

The role of the final ultrafiltration/diafiltration step is to control the concentration of the protein solution and, if required, to perform buffer exchange in the bulk of the drug formulation. In the event that the final polishing step was mixed chromatography in binding mode, the buffer exchange was already performed at the level of mixed chromatography allowed by the binding mode (see Example 8.5). In addition, in addition to the buffer, other ingredients of the final composition, such as surfactants, stabilizers and/or cryopreservatives, can be introduced by diafiltration. Alternatively, a concentrated mAb solution is supplemented directly with these substances.

Nanofiltered product solution from Example 8.7. was collected in a UF/DF block tank and concentrated to about 40±2 g/l using an Omega Centrasette membrane cassette (Pall Corporation, cutoff 30 kDa). The feed pressure was provided by a QuattroFlow 150 diaphragm pump. The transmembrane pressure was set to 1 bar, and the feed and retentate pressures were 1 and 0.6 bar, respectively. At this stage, there was no loss of mAbs. Polysorbate 20 and trehalose or sorbitol were added to the concentrated solution of the product. The final total mAb concentration was adjusted, for example, to 30±2 g/l or to 80±5 g/l. The final microfiltration (sterile filtration) was performed with a Mini Kleenpak 0.2 µm filter capsule (Pall Corporation).

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Claims (37)

1. A method for carrying out affinity chromatography with the capture of immunoglobulin from the culture fluid, comprising the following steps in the following order: (a) adding a water-soluble organic polymer to the culture fluid; (b) depth filtration of the mixture of step (a); (c) affinity chromatography of the filtrate obtained in step (b), wherein the immunoglobulin is bound to the affinity chromatography medium; (d) washing the affinity chromatography medium using a wash buffer with a pH of 5-9 and an ionic strength of 0.1-5 mol/l; And (e) eluting the immunoglobulin from the affinity chromatography resin using an elution buffer of pH 2.5-4.5, where the water-soluble organic polymer is poly(diallyldimethylammonium chloride), where poly(diallyldimethylammonium chloride) has a molecular weight of 10-10,000 kDa, and where poly(diallyldimethylammonium chloride) is added at a final concentration of 0.01-1.0% w/v. 2. The method according to p. 1, where stage (a) includes the following stages: (a1) centrifuging and/or filtering the culture fluid; (a2) adding a water-soluble organic polymer to the supernatant or filtrate obtained in step (a1). 3. The method according to p. 1, where poly(diallyldimethylammonium chloride) is added at a final concentration of 0.02-0.1% wt./about., preferably at a final concentration of 0.03-0.08% wt./about. 4. Process according to any one of the preceding claims, wherein no other flocculating agents are added except for poly(diallyldimethylammonium chloride). 5. The method according to any one of the preceding claims, wherein the flocculation with poly(diallyldimethylammonium chloride) is carried out without further adjustment of pH or conductivity. 6. The method according to any one of the preceding claims, wherein the flocculation is carried out with stirring at room temperature for at least 5 minutes, preferably at least 15 minutes. 7. The method according to any one of the preceding claims, wherein the affinity chromatography is Protein A chromatography. 8. The method of claim 7, wherein the Protein A chromatography medium contains an alkali-stable derivative of Protein A as a ligand, preferably an alkali-stabilized protein A B domain tetramer variant bound to a cross-linked agarose matrix. 9. The method according to any one of the preceding claims, wherein the depth filter in step (b) comprises more than one layer, preferably two layers. 10. The method according to any one of the preceding paragraphs, wherein microfiltration is carried out between steps (b) and (c). 11. The process according to any one of the preceding claims, wherein step (c) is carried out at least 8 hours after step (b), preferably at least 3 hours after step (b). 12. The method according to any one of the preceding claims, wherein the culture fluid is obtained from recombinant CHO cells expressing immunoglobulin. 13. The method according to any one of the preceding claims, wherein the immunoglobulin is IgG1 or IgG2. 14. The method of claim 13 wherein the Fc portion of IgG1 or IgG2 is human. 15. A method for purifying an immunoglobulin, comprising the steps as defined in any of the preceding claims, and comprising one or more additional steps after affinity chromatography selected from viral inactivation, anion exchange chromatography, cation exchange chromatography, mixed chromatography, nanofiltration, and ultrafiltration/diafiltration. 16. The method of claim 15, wherein the one or more additional steps comprise: (f) incubating the eluate of step (e) for viral inactivation at a low pH of 2.5-4.5 for at least 10 minutes. 17. The method according to claim 15, where one or more additional stages include: (f) incubating the eluate of step (e) for viral inactivation at a low pH of 2.5-4.5 for at least 10 minutes; (g) performing cation exchange chromatography; (h) performing mixed chromatography or anion exchange chromatography; (i) nanofiltration of the eluate of stage (h) or the composition obtained from it and obtained after one or more additional stages of processing performed after stage (h); And (j) ultrafiltration/diafiltration of the filtrate of stage (i) or the composition obtained from it and obtained after one or more additional stages of processing performed after stage (i). 18. The method of claim 17, wherein the cation exchange chromatography (g) is carried out in a coupling-elution mode, optionally including one or more washing steps. 19. The method of claim 17, wherein the mixed chromatography in step (h) is performed in a coupling-elution mode, optionally including one or more washing steps. 20. The method according to p. 17, where the chromatography (h) is carried out in flow mode. 21. The method of claim 17 wherein the cation exchange chromatography in step (g) is carried out with a medium containing a strong cation exchange medium, preferably sulfopropyl(-CH ₂ CH ₂ CH ₂ SO ₃ ^- ) as ligand. 22. The method of claim 21 wherein the sulfopropyl(-CH ₂ CH ₂ CH ₂ SO ₃ ^- ) is bonded to a cross-linked poly(styrenedivinylbenzene) matrix. 23. The method of claim 17, wherein the mixed chromatography in step (h) is carried out with a positively charged mixed chromatography medium, preferably containing N-benzyl-N-methylethanolamine as a ligand, bound to a cross-linked agarose matrix. 24. The method of claim 17, wherein the anion exchange chromatography in step (h) is carried out with a medium containing a strong anion exchange medium, preferably trimethylammonium (-N ⁺ (CH ₃ ) ₃ ) as ligand, bound to a cross-linked agarose matrix.

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