Stimulus responsive polymers for the purification of biomolecules

The present invention relates to polymers useful for protein purification. In particular, the present invention relates to stimulus-responsive polymers useful for purifying a target molecule, at least in part, from a sample containing the target molecule and one or more impurities.

Efficient and economical large-scale purification of biomolecules, such as therapeutic proteins, including antibodies, is an increasingly important consideration for the biotechnology and pharmaceutical industries. The purification process is usually quite complex and expensive and involves many different steps. For example, typically in the case of a protein, a cell culture method using a mammalian or bacterial cell line engineered to produce the protein, for example by inserting a recombinant plasmid containing the gene encoding the protein. Is used to produce proteins. Usually, after expressing the target protein, separation from one or more undesired components, including host cell proteins, media by-products and DNA, poses a great challenge. Such separation is especially important if the therapeutic protein is intended for human use and requires Food and Drug Administration (FDA) approval.

Usually, the separation and / or purification process currently used for proteins involves at least the following steps: lysing to recover intracellular proteins or, in the case of secretory proteins, recovering the protein from the medium. Steps; cells and cell debris are removed using fractional centrifugation or filtration to obtain a clarified sample containing the protein; and in a multi-step process, the said from various impurities in the sample using various chromatographic media. Includes steps to separate proteins.

Various types of polymers, including polymeric electrolytes, are used in one or more steps to purify biomolecules, especially proteins. For example, the use of polymeric electrolytes in agglutination to purify proteins is well established (eg, International PCT Patent Application No. WO. See 2008/091740). This can be done with a wide range of polymers, the only required general property is that the polymer must have some level of interaction with the species (eg, target molecule or impurity). The most common method is the use of ionic species-containing polymers (eg, polymer electrolytes). Usually, a polymeric electrolyte is added to the protein mixture and purification is done by selective aggregation of one or more components of the mixture. A significant drawback of this approach is that carefully controlled levels of polymer are used to avoid residual polymer contamination (eg, when polymer levels are too high) or inefficient aggregation (eg, when polymer levels are too low). The electrolyte must be added. Since ion exchange and other charged chromatography media are commonly used in protein purification, residual polymeric electrolytes potentially bind to the media used in the downstream purification step, polluting the process and complicating it. There is a risk of doing.

Recently, technologies have been developed that solve some of the challenges associated with the use of polymers for the purification of biomolecules (see, eg, International PCT Publication No. WO 2008/079302 A2). For example, stimulus-responsive, or "smart" polymers have been developed that can bind to both soluble components (eg, host cell proteins, DNA, cell culture additives) and insoluble components (eg, cells and cell debris). , US Publication Nos. 20080255027 and 20090036651). Although stimulus-responsive polymers are usually very promising, a significant challenge facing the widespread use of said polymers is the presence of simple stimuli that can be performed on a variety of scales, from laboratory scale to large scale production.

<p num="0006"><patcit num="1"><text>International Publication No. 2008/09 1740</text></patcit><patcit num="2"><text>International Publication No. 2008/079302</text></patcit><patcit num="3"><text>U.S. Patent Application Publication No. 2008/0255027</text></patcit><patcit num="4"><text>U.S. Patent Application Publication No. 2009/0036651</text></patcit></p>

The present invention is based on a novel polymeric electrolyte that is easily scalable and can be manipulated over a wide range of pH and conductivity and thus used in purifying a series of biomolecules, including, for example, therapeutic proteins. To provide a stimulus-responsive polymer.

In some embodiments according to the invention, a stimulus-responsive polymer comprising a polymeric electrolyte backbone containing one or more hydrophobic groups is provided, which polymer binds to the biomolecule of interest in the sample after stimulation. , Can be precipitated.

In some embodiments, the polymeric electrolyte backbone of the polymer according to the invention comprises at least two monomer units or at least three monomer units. In some embodiments, at least 50% of the monomeric units contain an electric charge. In other embodiments, each monomer unit of the polymeric electrolyte backbone contains an electric charge.

In some embodiments, the stimulus-responsive polymer according to the invention comprises a polyamine backbone. In some embodiments, the one or more hydrophobic groups are phenyl groups.

Stimulus-responsive polymers according to the present invention are useful for purifying the desired target molecule and separate the desired target molecule from one or more unwanted entities present in the sample with the desired target molecule. Purify by

Thus, in some embodiments, the stimulus-responsive polymer according to the invention is itself bound by the stimulus-responsive polymer and binds to and precipitates the target biomolecule, which is the desired target molecule to precipitate. In other embodiments, the stimulus-responsive polymer binds to and precipitates the desired biomolecule of interest, which is an unwanted real entity present in the sample along with the desired target molecule.

In some embodiments, the biomolecule of interest is a therapeutic polypeptide (ie, the desired target molecule). In some embodiments, the therapeutic polypeptide is an antibody (eg, a monoclonal antibody).

In other embodiments, the biomolecule of interest is selected from the group consisting of host cell proteins, DNA, RNA, lipids, viruses, endotoxins, cell culture additives, whole cells and cell debris.

In some embodiments, the polymers according to the invention are responsive to stimuli, which are complex-forming salts.

The methods of using the polymers described herein are also included in the present invention. The stimulus-responsive polymer replaces or improves during one or more steps in the purification process, thereby substantially increasing the total purity of the desired target molecule desired to be purified or separated from one or more unwanted entities. In that respect, it is unique and creative compared to the polymers described in the prior art.

Thus, in some embodiments, (a) preparing a sample containing the target molecule and one or more impurities; (b) high containing one or more hydrophobic groups attached to the polymeric electrolyte skeleton. The sample is contacted with the stimulus-responsive polymer under a first set of conditions suitable for the stimulus-responsive polymer containing the molecular electrolyte skeleton to bind to the target molecule in solution to form a complex of the polymer with the target molecule. Step; and (c) stimulating the sample under a second set of conditions suitable for precipitating the complex from solution; precipitating the complex separates the target molecule from one or more impurities. However, this provides a method for improving the purity of the target molecule.

In some embodiments that follow the method of the invention, the method further comprises the step of recovering the target molecule from the complex.

In another embodiment, the stimulus-responsive polymer according to the invention binds to and precipitates one or more impurities rather than the target molecule, and precipitates a complex of the polymer and one or more impurities and one or more impurities. The target molecule is separated from the target molecule, which improves the purity of the target molecule. Therefore, the method is (a) Steps to prepare a sample containing the target molecule and one or more impurities; (b) A stimulus-responsive polymer containing a polymeric electrolyte skeleton containing one or more hydrophobic groups attached to the polymeric electrolyte skeleton. The step of contacting the sample with the stimulus-responsive polymer under the first set of conditions suitable for binding to one or more impurities to form a complex of the polymer with one or more impurities; and (c. ) Including the step of stimulating the sample under a second set of conditions suitable for precipitating the complex; precipitating the complex separates the target molecule from one or more impurities, which results in the target molecule of the target molecule. Improve purity.

In some embodiments, the stimulus-responsive polymer according to the invention has the following structure:

<chemistry num="1"><img id="000002" he="41" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, x and y represent the monomer units of the polymer; R₁And R₂Is a charged group that forms part of the polymeric electrolyte backbone (B); R₃Is a hydrophobic group attached to a charged group in the skeleton) including. The ratio of y monomer units (ie, having hydrophobic groups attached to the skeleton) to the total number of monomer units (ie, the sum of x and y monomer units) corresponds to the "% of hydrophobic modification" of the polymer.

In some embodiments, the stimulus-responsive polymer according to the invention has the following structure:

<chemistry num="2"><img id="000003" he="38" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, x, y and z represent monomer units in the polymer; R₁, R₂And R₃Is a charged group that forms part of the polymeric electrolyte backbone (B); R₄Is a hydrophobic group attached to a charged group in the skeleton; R₅Is a functional group attached to a charged group in the skeleton) including. The ratio of y monomer units (ie, having hydrophobic groups attached to the skeleton) to the total number of monomer units in the polymer (ie, the sum of x, y and z monomer units) is the "% of hydrophobic modification" of the polymer. Corresponds to. In addition, the ratio of z-monomer units (ie, having functional groups attached to charged groups on the backbone) to the total number of monomer units (ie, the sum of x, y and z monomer units) is the "functional group modification" of the polymer. Corresponds to "%".

Generally, the polymers included in the present invention may have n (where n is greater than or equal to 2) monomer units x, y or z described herein.

In yet other embodiments, the stimulus-responsive polymer according to the invention has the following structure:

<chemistry num="3"><img id="000004" he="42" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, x and y represent the monomer units of the polymer; R₁, R₂Are aliphatic amine groups (eg, primary or secondary and / or aromatic amines) that form part of the carbon-containing backbone of the polymeric electrolyte; R₃Is an amine group R₂It is a hydrophobic group bonded to and contains 4 or more carbon atoms (for example, an alkyl group, an alkenyl group, an alalkenyl group or a fluorocarbon group). including. In some embodiments, the ratio of y (ie, a monomer unit with a hydrophobic group attached to a charged group in the polymeric electrolyte skeleton) to x (ie, an unmodified charged group in the polymeric electrolyte skeleton) is It is 0.01 to 0.75 or 0.05 to 0.75. Therefore, the% hydrophobic group modification is 1% to 75% or 5% to 75% of the total polymer electrolyte monomer unit (ie, x + y).

In yet another embodiment, the stimulus-responsive polymer according to the invention has the following structure:

<chemistry num="4"><img id="000005" he="38" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>(In the formula, R₁, R₂And R₃Are aliphatic amine groups (eg, primary or secondary amines and / or aromatic amines) that form part of the carbon-containing polymeric electrolyte skeleton; R₄Is a hydrophobic group that contains 4 or more carbon atoms and is selected from alkenyl, alalkyl and alalkenyl groups; R₅Is a hydrophobic group that contains 4 or more carbon atoms and is selected from alkyl or fluorocarbon groups) including. The ratio of y monomer units to the total number of polymer electrolyte monomer units is 0.01 to 0.75. The ratio of z-monomer units to the total number of polymer electrolyte monomer units is 0.05-0.5 or 0.01-0.5. Therefore, the hydrophobic group modification% is 1% to 75% or 5% to 75%, and the functional group modification% is 1% to 50% or 5% to 50%.

Additional methods using stimulus-responsive polymers according to the present invention include methods that allow the target molecule or product (eg, antibody) to be purified while reducing the amount of residual polymer in the sample.

In some embodiments, a method of separating a target molecule (eg, an antibody) from one or more impurities using a stimulus-responsive polymer according to the invention is provided while minimizing the residual amount of the stimulus-responsive polymer. The method is to (a) prepare a sample containing the target molecule and one or more impurities; (b) adjust the pH or salt concentration of the sample before, during or after the addition of the stimulus-responsive polymer. Contact the sample with a stimulus-responsive polymer under a first set of conditions suitable for the polymer to bind to one or more impurities to form a first complex of the polymer with one or more impurities. Steps; (c) Precipitate the first complex from the sample under the conditions of the second set; (d) Contact the sample with polyvalent ions to form a second complex of residual polymer and polyvalent ions. One in the sample, including (e) the step of precipitating the second complex; and (f) the step of recovering the target molecule from the sample; thereby reducing the amount of residual polymer in the sample. The target polymer is separated from the above impurities.

In some embodiments, the target molecule is an antibody. In certain embodiments, the antibody is a monoclonal antibody.

In certain embodiments, recovery of the target molecule in the various methods according to the invention comprises a chromatography step. In another embodiment, recovery of the target molecule comprises a filtration step.

In some embodiments, the method according to the invention may include two or more steps using a stimulus-responsive polymer according to the invention. For example, a stimulus-responsive polymer can be used to precipitate one or more impurities in one step of the purification process, for the same or different polymers to precipitate the target molecule or desired product in different steps of the process. Can be used.

In some embodiments, one or more impurities are selected from the group consisting of host cell proteins, DNA, RNA, antibody aggregates, viruses, endotoxins, whole cells, cell debris and cell culture additives.

<figref num="1">It is a figure which shows the reaction of a polyallylamine polymer with benzyl chloride.</figref><figref num="2">The reaction scheme for caproic acid and tert-butyl modified polyallylamine (HC-t-BuMPAA) is shown.</figref><figref num="3">It is a graph which shows the influence of sodium chloride on the polyvalent ion stimulation and pH responsiveness of a caproic acid and a tert-butyl modified polyallylamine (HC-t-BuMPAA). The X-axis represents pH and the Y-axis represents the turbidity of the centrifuge (ie, centrifuge output).</figref><figref num="4">The synthesis of polyvinylamine described in Example 29 is shown.</figref><figref num="5">The deprotection reaction of polyamine after polymerization is shown.</figref><figref num="6">The reaction of polyvinylamine with benzyl chloride is shown.</figref><figref num="7">The polymerization and reaction schemes for forming a multivalent ion stimulus responsive copolymer are shown.</figref><figref num="8">The NMR spectrum of the modified polyvinylamine (PVA) from Example 35 is shown.¹1 H NMR integration shows a benzyl modification level of about 18%.</figref><figref num="9">The NMR spectrum of the modified polyallylamine from Example 36 is shown.¹1 H NMR integration shows a benzyl modification level of about 33%.</figref><figref num="10">FIG. 5 shows a graph showing the effect of polymer addition on centrifugation turbidity for non-stimulation responsive polymers (eg chitosan) and stimulus responsive polymers (eg benzyl modified polyallylamine). As described in Example 37, the X-axis is the amount of polymer added (wt%) and the Y-axis is the centrifugal turbidity (NTU).</figref><figref num="11">The graph which shows the influence of the polymer addition amount in the presence and absence of a stimulus for a benzylpolyallylamine stimulus responsive polymer is shown. As described in Example 38, the X-axis is the amount of polymer added (wt%) and the Y-axis is the centrifugal turbidity (NTU).</figref><figref num="12">A typical scheme used for purification of biomolecules is shown.</figref><figref num="13">A purification scheme containing a stimulus-responsive polymer used to improve cell culture clarification is shown. The stimulus-responsive polymer removes one or more impurities, but the polymer does not bind to the desired target molecule.</figref><figref num="14">Represents a purification scheme that includes a stimulus-responsive polymer used to improve cell culture clarification. The stimulus-responsive polymer removes one or more impurities by aggregation, but the polymer does not bind to the desired target molecule and the residual polymer is removed by applying post-clarification stimulus.</figref><figref num="15">Represents a purification scheme that includes a stimulus-responsive polymer used to improve cell culture clarification. The stimulus-responsive polymer removes one or more impurities by aggregation, but the polymer does not bind to the desired target molecule and the residual polymer is removed by an additional adsorption filtration step after clarification.</figref>

The present invention provides a novel and improved stimulus-responsive polymer containing a polymeric electrolyte backbone modified with one or more hydrophobic groups, at least in part, and the polymer solubility can be altered by stimulating.

The stimulus-responsive polymers and methods of using the polymers described herein are for purifying desired target molecules, including, for example, proteins and for impurities (eg, host cell proteins, DNA, RNA, lipids, endotoxins). , Cell culture additives, cells and cell debris) are more efficient than those described in the prior art in providing an improved pH range for removing unwanted real estate. In some embodiments, the polymers described herein are responsive to low concentrations of monopolyvalent salts, which is an improvement over existing salt responsive polymers. Scalability and low conductivity are obtained. In various embodiments, the polymer according to the invention can effectively remove whole cells, cell debris and other soluble impurities from the cell culture medium. The polymer can also effectively remove impurities from the protein and the protein mixture containing one or more impurities. In addition, the various polymers described herein are capable of effectively capturing the target molecule and the protein / product in the sample, whereby one or more of them are present in the sample. It can be separated from impurities to improve the purity of the target molecule.

The use of polymers described herein for purifying a target molecule (eg, a Therapeutic protein) using a wide range of conditions is also included in the invention.

Without wishing to be bound by theory, the stimulus-responsive polymers described herein are a desired target molecule (eg, a therapeutic protein) or a desired product, or an undesired entity (eg, an undesired substance). , Host cell proteins, DNA, RNA, lipids, endotoxins, cell culture additives, one or more impurities including whole cells and cell debris), could be used to bind and precipitate. Generally, a molecule bound by a polymer according to the present invention, whether it is a desired target molecule or an undesired real substance, is referred to as a target biomolecule.

The choice of specific stimulus-responsive polymer to use as described herein is determined by what the polymer binds to. For example, in the case of a target biomolecule having a net negative charge at a pH higher than its pI (eg, whole cells, cell debris, DNA, endotoxins and proteins), it is cationic (ie, positively charged). It is desirable to use a stimulus-responsive polymer containing a polymeric electrolyte skeleton. On the other hand, in the case of a target biomolecule (eg, a protein) that has a net positive charge at a pH lower than its pI, a stimulus containing an anionic (ie, negatively charged) polymeric electrolyte backbone. It is desirable to use a responsive polymer.

The positive charge can be unique to the polymer under the conditions used during the purification process, or the positive charge can occur in response to changes in the pH at which the stimulus-responsive polymer is charged.

An important parameter affecting the total recovery of biomolecules is the ratio of hydrophobic modifying groups in the polymeric electrolyte backbone to other unmodified charged groups. For example, an increase in% hydrophobic group results in the loss of biomolecules due to non-specific interactions. Thus, for a given biomolecule, a particular ratio of charged groups to hydrophobic groups may be used to maximize biomolecule recovery. In addition, a high percentage of hydrophobic groups can limit the solubility of the polymer and the effectiveness of the charged groups on the polymer electrolyte backbone.

Furthermore, modification of the charged amine group in the backbone of the polyelectrolyte polyallylamine with benzyl chloride yields a secondary amine, which is charged under a wide range of pH conditions. However, benzyl modification adds steric bulk to amine groups that can affect charge-charge interactions. In addition, modifying the charged groups in the polymeric electrolyte backbone with hydrophobic groups can reduce the number of charged groups. For example, modification of the amine group of polyallylamine with benzyl chloride forms an amide bond that is not a charged group, which reduces the number of charged groups in the backbone. Therefore, a reduction in the number of charged groups can also affect the solubility of the polymer and the ability of the polymer to bind through charge-charge interactions.

Some polymers according to the present invention are cationic and others are anionic, but hybrid polymers containing a polymeric electrolyte skeleton modified with one or more hydrophobic groups and anionic groups can also be synthesized. .. In the case of the hybrid polymer, the unmodified group on the cationic polymer electrolyte is responsive to the complex-forming salt, and the anionic modifying group on the skeleton binds to the target biomolecule with a net positive charge. obtain. Therefore, the ratio of unmodified cationic groups to anionic hydrophobic groups in the polymer electrolyte backbone is important to determine the solubility and irritation required for the polymer to form and capture the complex with the biomolecule of interest. .. For example, too few unmodified cationic groups in the polymeric electrolyte backbone can limit or eliminate the response to stimuli. On the other hand, too few anionic groups can limit the ability to capture the biomolecule of interest.

The required level of modification of the polymeric electrolyte backbone with hydrophobic groups, as well as the type and amount of irritation used, will depend on the biomolecule to be purified using the polymer and the conditions under which it will be used, as well as the inherent solubility and molecular weight of the polymeric backbone. Can be determined on the basis. For example, in order to minimize the amount of irritation (eg, polyvalent salt) used, it is desirable to have more hydrophobic groups attached to the polymeric electrolyte backbone. Alternatively, increasing the amount of polyvalent ion stimulation reduces the degree of hydrophobic modification or the required% hydrophobic modification. In some cases, for example, if the multivalent ion stimulation is at a very high concentration, the need for hydrophobic modification can be completely eliminated. Alternatively, increasing the molecular weight of the polymer can reduce the intrinsic solubility of the polymer electrolyte skeleton and reduce or eliminate the hydrophobic modification of the polymer electrolyte skeleton (5% or less). However, eliminating the hydrophobic modification can increase the amount of residual polymer in the case of lower molecular weight or more soluble polymer backbones at higher polymer additions.

In various embodiments, the degree of hydrophobic modification on the polymeric electrolyte backbone ranges from 1% to 85% or 5% to 0%. Therefore, the percentage of hydrophobic modification is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, depending on the target biomolecule to be bound by the stimulus-responsive polymer. , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%.

In some embodiments, complex-forming salts are used as stimuli. In various embodiments, the concentration of complex-forming salts is in the range of 2 mM to 500 mM or 25 mM to 100 mM. Examples of complex-forming salts include polyvalent ions (eg, citrates, phosphates, sulfates and EDTA) and ion-associated salts (eg, perchlorates, sodium dodecyl sulfate, dodecylbenzene sulfate, etc.) Fe (II) -4-chloro-2-nitrophenol anion, sodium tetraphenylborate and hexanitrodiphenolamine), but not limited to these (eg ANALYTICAL SCIENCES, DECEMBER 1987, VOL.3, See p.479). Generally, one of ordinary skill in the art is familiar with various complex-forming salts known in the art that can be used as stimuli to the polymers described herein.

The amount of complex-forming salt required to induce precipitation depends on multiple factors, such as pH, polymer concentration and concentration of the biomolecule of interest in the sample. For example, some polymeric electrolytes, such as polyallylamine, have a charge density that varies with pH (the level of amine protonation). As the pH increases, the level of charge density decreases, so the degree of irritation required to induce precipitation is different than when the pH is lower or the charge density state is higher.

Generally, a stimulus-responsive polymer according to the present invention may be added in solid or liquid form to a feedstock containing the target molecule or a sample containing the target molecule. The final polymer concentration is usually 0.01% to 2%. In some of the methods described herein, a stimulus, such as a complex-forming salt (eg, a polyvalent anion), is added after forming a mixture of polymer and biomolecule of interest. The amount of irritation may depend on the polymer concentration. For example, a polymer concentration of 2% requires more stimulation needed to induce polymer precipitation. It is important to apply the correct amount or slightly excessive amount of stimulation in order for the polymer to completely precipitate in response to the stimulation. This is in contrast to polymer agglomeration, which results in problematic residual polymers due to over-addition.

The present invention can be used in various purification schemes. Although stimulus-responsive polymers can be useful at any step in the process, they are preferably used during clarification or capture of the target molecule at the beginning of the process. One stimulus-responsive polymer or polymer mixture can be added in one or more steps and then precipitated using one or more stimuli. Stimulation may be applied before, during, or after the polymer associates with the biomolecule of interest (ie, one or more impurities or the desired target molecule). Stimulation may also be applied before, during or after removal of the precipitate, which is usually in solid form. Precipitation is then one or more techniques known in the art in simultaneous, parallel or series of separation schemes and / or techniques described herein, such as filtration, sedimentation, centrifugation or other solid / liquid separation. It can be removed using a method or a combination of methods.

The addition of the stimulus-responsive polymer can be done in several ways. The cell culture medium can be adjusted to the desired conditions prior to the addition of the stimulus-responsive polymer, eg, pH and / or conductivity can be adjusted (eg, reduced). The stimulus-responsive polymer can then be added to the cell culture medium and mixed. The stimulus-responsive polymer can be added in liquid or solid format. The polymer-containing solution itself can be prepared just to adjust the pH of the cell culture medium to the desired conditions. For example, the stimulus-responsive polymer can be dissolved in a concentrated acetic acid solution. The concentration of this acetic acid solution can be varied based on volume, fermentation solution conditions and protein concentration to give the necessary pH adjustment upon addition of the stimulus-responsive polymer, thereby producing the desired polymer concentration and solution pH. The stimulus-responsive polymer is typically 0.01-0.1% wt polymer or 0.01-0.5% wt at a concentration that causes natural aggregation, depending on the type and% solid, so that the solution begins to fog and form a precipitate. It can be added in the range of polymers. Alternatively, the stimulus-responsive polymer can be added at a concentration where polymer-biomolecular association occurs, eg, typically in the range of 0.5% to 2% wt polymer, without spontaneous aggregation, and the solution is the original solution. It may be more transparent, slightly cloudy, or more cloudy. In addition, the stimulus-responsive polymer may be added at a concentration at which both spontaneous aggregation and polymer-biomolecular association occur.

Although it is more desirable to use stimuli in purification schemes as it alleviates problems associated with polymer overdose, such as in the case of agglomeration processes, the polymers described herein are used as flocculants. It is also possible to do it.

In an exemplary purification scheme, a stimulus-responsive polymer according to the invention is added to the cell culture after fermentation is complete and the polymer is prepared to bind to a target biomolecule that is not the desired target molecule. In this method, the stimulus-responsive polymer is added to the cell culture under the conditions of the first set, eg, conditions that can be adjusted before, during or after the addition of the polymer that binds the biomolecule of interest. After adding the stimulus-responsive polymer under the first set of conditions, the stimulus is added under the second set of conditions, thereby causing the biomolecule of interest (eg, cells, cell debris, host cell proteins, DNA, endotoxins and viruses). Precipitation containing one or more impurities) such as. Subsequent removal of the solid precipitate by centrifugation and / or filtration yields a clarified cell culture medium. The resulting clarified cell culture can then be subjected to a capture step using a chromatographic medium to bind to the desired target molecule. The target molecule can then be eluted from the capture step. Thus, in some cases, the number of additional steps can be reduced, removed or altered by using a stimulus-responsive polymer according to the invention that can remove one or more impurities in the clarification step.

In some embodiments, the stimulus-responsive polymer is added to the cell culture under conditions where the stimulus-responsive polymer binds to a target biomolecule that is not the target molecule. After the stimulus-responsive polymer is thoroughly mixed under the desired solution conditions, aggregates containing the biomolecule of interest are formed. The solid containing the target biomolecule is removed by primary clarification. The resulting cell culture medium is collected and stimulated to precipitate the residual polymer. The precipitated residual polymer is then removed by secondary clarification. The resulting clarified cell culture solution is subjected to a capture step using a chromatography medium to bind to the target molecule. Residual polymers can also be removed by stimulating in the purification step after primary clarification. Stimulation may also be applied to remove the residual polymer at any step, or one or more stimuli may be applied multiple times throughout the process.

In another purification scheme, the stimulus-responsive polymer is added under conditions suitable for the polymer to bind to one or more impurities so that the polymer does not bind the target molecule to the cell culture medium after fermentation is complete. To do. After the stimulus-responsive polymer is thoroughly mixed under the desired solution conditions, a stimulus is applied to form a solid precipitate with one or more impurities. The solid precipitate is removed by centrifugation and / or filtration. The resulting clarified cell culture solution is passed through a filter to which the stimulus-responsive polymer can be attached. One of ordinary skill in the art can easily select / identify a filter to which a stimulus-responsive polymer can be attached. For example, a filter having properties similar to those of the target biomolecule to be bound by the polymer can be prepared. Alternatively, a filter having a charge group having the same charge as the target biomolecule and a charge opposite to the charge of the stimulus-responsive polymer may be used.

Membranes, packed beds or filters can also be used to remove the polymer from the solution. For example, an anionic membrane containing a sulfonic acid group can be used to remove the polyamine stimulus responsive polymer. Filters with similar binding properties can also be used for the stimuli used to precipitate the polymer. If the stimulus is a multivalent anion, a filter with a surface containing the multivalent anion can remove the polymer from the solution. For example, a phosphate-modified membrane can bind to polyamines in much the same way that phosphate ions form a complex with polyamines, thereby inducing precipitation. In addition, beads modified with polyvinyl phosphate (for example, polymethacrylate) may be used. In some embodiments, the solution is filtered through a filter capable of removing the stimulus-responsive polymer, and then the resulting solution is subjected to a capture step using a chromatographic medium to bind the target biomolecule. The polymer can be removed using a chromatographic medium, a depth filter, or other porous material to which the stimulus-responsive polymer can be attached. The polymer can also be removed using adsorption means in a single step or in two or more steps. In addition, the polymer can be removed using adsorption means at any step after adding the polymer to the mixture.

It is believed that the present invention can be used in many modifications of the purification schemes described herein. Stimulus-responsive polymers can be used to replace or enhance both the clarification and capture steps. For example, two separate stimulus-responsive polymers may be used: a first polymer that binds to one or more impurities but not to the target molecule, and a second polymer that binds to the target molecule. These two polymers can be applied in separate steps or in a single step. Similarly, a single polymer having a functional group capable of binding to the target molecule and a polymeric electrolyte backbone capable of binding to one or more impurities can be used. A single polymer can bind to a target molecule and one or more impurities in a single step or multiple steps. After several different precipitation / stimulation additions or washing steps, elution of the target molecule from the polymer may be followed. A stimulus-responsive polymer may be added and used after the capture step to clarify suspended solids or impurities resulting from virus inactivation after the capture step or other steps. A stimulus-responsive polymer may be used in place of or to enhance the polishing step.

Some embodiments include a virus inactivating step (ie, exposing the solution to low pH, detergent or heat). The solution conditions may be adjusted and the sample may be subjected to a series of polishing steps (ie, one or more ion exchanges, hydrophobic interactions, mixing modes and others). The solution may then be subjected to a series of filtration steps including virus filtration and ultrafiltration, or diafiltration.

Some terms are first defined so that the disclosure of the present invention can be more easily understood. Additional definitions will be given through a detailed description.

I.<u style="single">Definition</u> As used interchangeably herein, the term "stimulus" is meant to refer to a physical or chemical change in an environment that results in a response by a stimulus-responsive polymer according to the present invention. Accordingly, the present invention provides novel polymers that are responsive to stimuli and that stimulate changes in the solubility of the polymer. Examples of stimuli in which one or more polymers described herein are responsive include, but are not limited to, for example, changes in temperature, changes in conductivity and / or changes in pH. In some embodiments, the stimulus involves the addition of a complexing agent or complexing salt to the sample. In various embodiments, the stimulus is usually added after adding the polymer to the sample. However, irritation may be applied during or before the addition of the polymer to the sample.

As used herein, the term "polymer" refers to a molecule formed by covalent bonds of two or more monomer units. The monomer unit may be synthetic or natural. The polymer formed by the repeating unit may be linear or branched. Examples of polymers include polyethylene glycol, polypropylene glycol, polyethylene, polyallylamine, polyvinyl alcohol, polystyrene and copolymers (eg, polystyrene-co-polypyridine, polyacrylic acid-co-methylmethacrylate, pluronic, PF68, etc.) , Not limited to these. In some embodiments according to the present invention, the polymer comprises a polypolymer electrolyte backbone. Also described herein are copolymers that are responsive to stimuli that can be used in methods according to the invention. Generally, in the case of polymers, the monomer units are of the same type, but copolymers are usually understood to have different types of monomer units.

As used herein, the term "stimulus-responsive polymer" is a polymer or copolymer that exhibits a change in physical and / or chemical properties after being stimulated. A typical stimulus response is a change in the solubility of the polymer. For example, polymer poly (N-isopropylacrylamide) is soluble in water at temperatures below about 35 ° C, but becomes insoluble in water at temperatures below about 35 ° C. In certain embodiments, the stimulus-responsive polymer is a polyallylamine or polyvinylamine polymer that is responsive to polyvalent ion stimuli (eg, phosphate stimuli).

As used herein, the term "polymer electrolyte skeleton" refers to a carbon-containing polymer that contains two or more monomer units, at least 50% of the units, at least 55% of the units, at least 60% of the units, At least 65% of units, at least 70% of units, at least 75% of units, at least 80% of units, at least 85% of units, at least 90% of units, or at least 95% of units contain charged functional groups There is. In other words, at least 50% of the monomeric units contain charged groups that form part of the unit. In some embodiments, the polymeric electrolyte scaffolds described herein contain at least two or more monomer units, each of which contains a charged functional group. In the case of a polymeric electrolyte skeleton of a polymer in which each of the monomeric units contains a charged functional group, the polymer may be referred to as a "continuous polymeric electrolyte". Examples of polymeric electrolytes include, but are not limited to, polyallylamine, polyvinylamine, polyacrylic acid, polyethyleneimine, chitosan and polyvinylphosphoric acid. It is also conceivable that one or more real substances different from the monomer unit may be bonded to the polymer electrolyte skeleton.

As used herein, the term "hydrophobic group" refers to a non-polar real or chemical group that has little or no affinity for water. Examples of hydrophobic groups include phenyl groups, tertiary butyl groups, cyclic hydrocarbons, polycyclic aliphatic hydrocarbons, polycyclic aromatic hydrocarbons, and short chain hydrocarbons (eg, hexyl and octyl groups). Included, but not limited to. In certain embodiments, the hydrophobic group is a phenyl group. The hydrophobic group may be a non-hydrocarbon or may contain a heteroatom (eg, nitrogen, oxygen, sulfur, phosphorus, etc.). Various embodiments according to the present invention provide a stimulus-responsive polymer comprising a polymeric electrolyte scaffold having one or more hydrophobic groups attached to a charged group in the scaffold. Although not bound by theory, it is understood that the number of hydrophobic groups attached to the polymer electrolyte backbone is important for altering polymer solubility and improving the stimulus responsiveness of the polymer. To. However, it may be desirable to have a number of hydrophobic groups that make the polymer water insoluble without irritation.

The% of charged groups in the polymer electrolyte backbone modified with hydrophobic groups is commonly referred to as the "hydrophobic modified%" of the polymer. Therefore, in various embodiments, the hydrophobic modification% is important and ranges from 1% to 85% or 5% to 5%. Therefore, the hydrophobic modification% is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%. , At least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least It can be 80%, or at least 85%.

As used herein, the term "hydrophobic modified%" is usually the unmodified high of a hydrophobic group modified polymeric electrolyte charged group as% of the total polymeric electrolyte monomer unit in the polymeric electrolyte polymer backbone. Molecular electrolyte Refers to the ratio of charged groups.

In some embodiments, the hydrophobic group attached to the polymeric electrolyte backbone further has a charged group attached to a real hydrophobic group that is distinct from the charged groups in the backbone.

As used herein, the term "alkyl" usually refers to straight or branched hydrocarbon chains. Linear or branched hydrocarbon chains refer to substituted or unsubstituted acyclic carbon-containing compounds including, for example, alkanes, alkenes and alkynes. Examples of alkyl groups include lower alkyls such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or isohexyl; higher alkyls such as n-heptyl, n-octyl, isooctyl, nonyl, decyl. Etc .; lower alkylenes such as ethylene, propylene, propyrine, butylene, butadiene, pentene, n-hexene or isohexene; and higher alkylenes such as n-heptene, n-octene, isooctene, nonene, decene and the like. Those skilled in the art are familiar with the large number of linear (ie, linear) and branched alkyl groups included in the present invention.

In addition, the alkyl group may also contain various substituents in which one or more hydrogen atoms are substituted with functional groups. Examples of functional groups include, but are not limited to, carboxylic acids, sulfonic acids, phosphonic acid groups and the like. As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain in which at least one of the carbon-carbon bonds is a carbon-carbon double bond.

As used herein, the term "alalkyl" refers to an alkyl group whose end is substituted with at least one aryl group.

As used herein, the term "alalkenyl" refers to an alkenyl group whose terminal is substituted with at least one aryl group.

As used herein, the term "aryl" often refers to a hydrocarbon ring with a conjugated double bond system containing at least 6 n (π) electrons. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anisyl, toluyl and xylenyl.

As used herein, the term "fluorocarbon" refers to a straight or branched carbon chain in which one or more hydrogen atoms are substituted with fluorine groups. A straight or branched fluorocarbon chain usually refers to a substituted or unsubstituted acyclic carbon-containing compound including, for example, alkanes, alkenes and alkynes. Examples of alkyl groups include lower alkyls such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or isohexyl; higher alkyls such as n-heptyl, n-octyl, isooctyl, nonyl, decyl. Etc .; lower alkylenes such as ethylene, propylene, propyrine, butylene, butadiene, pentene, n-hexene or isohexene; and higher alkylenes such as n-heptene, n-octene, isooctene, nonene, decene and the like. Generally, one of ordinary skill in the art is familiar with a number of linear (ie, linear) and branched alkyl groups within the scope of the present invention. In addition, alkyl groups can also contain various substituents in which one or more hydrogen or one or more fluorine atoms are substituted with functional groups. Examples of functional groups include, but are not limited to, carboxylic acids, sulfonic acids, phosphonic acid groups and the like .

As used herein, the term "functional group" is a group that imparts additional functionality to the polymers described herein. In other words, the functional group is different from the hydrophobic group and is a group bonded to a polymer electrolyte skeleton, for example, a charged group in the skeleton. For example, in some embodiments, the functional group can be a ligand for altering the binding properties of the polymer, such as carboxylic acids, sulfonic acids, sulfates, primary amines, quaternary amines and diethylamino groups. Functional groups can alter properties or impart additional desired properties to the polymer, such as altering the stimulus response or making the polymer responsive to a second stimulus. Examples of functional groups for altering stimulus response behavior include, but are not limited to, carboxylic acid groups (pH responsive), pyridine groups (pH responsive) and N-isopropylacrylamide groups (temperature response).

As used herein, the term "ligand" usually refers to an entity that provides specific binding ability to another entity. Examples of "ligants" include ion exchange groups, bioaffinity or biospecific groups, hydrophobic groups, thiophyllic interacting groups, chelates or chelating groups, groups having π-π interactions with so-called target compounds. Includes, but is not limited to, hydrogen bonding groups and hydrophilic groups.

As used herein, the term "aggregation" is a solution of a flocculant (eg, a polymer described herein) to remove one or more suspended insoluble or soluble impurities. Refers to the addition to. The polymer must be added to the solution at a concentration that can naturally form insoluble agglomerates that can be removed from the solution by typical solid-liquid separation methods.

As used herein, the terms "composition," "solution," or "sample" are target molecules that are intended to be purified using one or more stimulus-responsive polymers described herein. Alternatively, it refers to a mixture of a desired product and one or more undesired reals or impurities. In some embodiments, the sample comprises a feed material or cell culture medium from which the target molecule or desired product is secreted. In some embodiments, the sample is a target molecule (eg, a therapeutic protein or antibody) along with one or more impurities (eg, host cell protein, DNA, RNA, lipids, cell culture additives, cells or cell debris). )including. In some embodiments, the sample comprises the target molecule secreted into the cell culture medium. In some embodiments, a sample for which the target molecule is to be purified using one or more of the stimulus-responsive polymers described herein is conditioned before contacting the sample with the stimulus-responsive polymer. "Partially purified". Partial purification can be achieved, for example, by subjecting the sample to one or more purification steps, such as one or more non-affinity chromatography steps. The target molecule can be separated from one or more undesired reals or impurities by precipitating one or more impurities or precipitating the target molecule.

In some embodiments, the stimulus-responsive polymer according to the invention binds to a target biomolecule that is itself a target molecule or product (eg, a target protein or polypeptide) under the conditions of the first set, and the second set. Under the above conditions, for example, the sample is stimulated to precipitate the target molecule. In other embodiments, the biomolecule of interest is a molecule other than the target molecule. In other words, the biomolecule of interest bound by the stimulus-responsive polymer described herein can be a molecule that you do not want to bind to the target molecule in the sample. Although not bound by theory, in some embodiments, stimulus-responsive polymers according to the invention are one or more host cell proteins, DNA, whole cells, cell debris, viruses when stimulated. , Endotoxin and / or cell culture additives are believed to bind and precipitate. Thus, the target molecule (eg, target protein or polypeptide) is present in a sample containing the desired target molecule or precipitating the desired target molecule with the polymers described herein. It can be purified by precipitating one or more unwanted real estates (eg, one or more impurities) that may be present.

As used herein, the terms "precipitate," "precipitate," or "precipitate" are used in an aqueous and / or soluble state of a bound (eg, complex with a biomolecule of interest) or unbound polymer. Refers to the change from to non-aqueous and / or insoluble state.

As used herein, the term "target biomolecule" refers to a molecule that binds and precipitates with the stimulus-responsive polymers described herein. For example, the biomolecule of interest can be the desired target molecule, eg, the desired product or polypeptide (eg, antibody) of interest, or is an unwanted entity that must be removed from a sample containing the desired target molecule. possible. Unwanted entities include, for example, one or more impurities selected from host cell proteins, DNA, RNA, protein aggregates, cell culture additives, viruses, endotoxins, whole cells and cell debris. Not limited.

The terms "target molecule", "target biomolecule", "desired target biomolecule" and "desired target biomolecule" used interchangeably herein generally contain the polypeptide or product. Refers to the polypeptide or product that you want to purify or separate from one or more unwanted entities (eg, one or more impurities) that may be present in the sample. The terms "protein in question", "target polypeptide", "polypeptide" and "target protein" used interchangeably herein generally refer to a Therapeutic protein or polypeptide, which are referred to herein. Antibodies to be purified using stimulus-responsive polymers according to the invention are included, but not limited to.

As used interchangeably herein, the term "polypeptide" or "protein" usually refers to peptides and proteins having about 10 or more amino acids. In some embodiments, the stimulus-responsive polymers described herein are proteins or polypeptides from one or more unwanted entities present in the sample with the protein or polypeptide. Used to separate. In some embodiments, the one or more entity is one or more impurities that may be present in the sample along with the protein or polypeptide to be purified. As discussed previously, in some embodiments, the stimulus-responsive polymers described herein specifically bind to and precipitate the protein or polypeptide upon stimulation of the sample. In other embodiments, the stimulus-responsive polymers described herein are real substances other than the protein or polypeptide, such as host cell proteins, DNA, viruses, whole cells, cells, when the sample is stimulated. It binds to debris and cell culture additives and precipitates.

In some embodiments, the protein or polypeptide to be purified using the stimulus-responsive polymers described herein is a mammalian protein, such as a Therapeutic protein or a protein that can be used in therapy. Examples of proteins include, for example, renin; growth factors including human growth hormone and bovine growth hormone; growth hormone release factors; parathyroid hormone; thyroid stimulating hormone; lipoprotein; α-1-antitrypsin; insulin A chain; insulin Chain B; proinsulin; follicular stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factors and von Wilbrandt factors; anticoagulant factors such as protein C; atrial sodium diuresis Peptide factors; Pulmonary surfactants; Plasminogen activators such as urokinase or human urine or tissue type Plasminogen activators (t-PA); Bombesin; Trombin; Hematopoietic growth factors; Tumor necrosis factors-α and -β; Enke Farinase; Lantes (activator of normal T cell expression and secretion); Human macrophage inflammatory protein (MIP-1-α); Serum albumin, such as human serum albumin; Muller's tube suppressor; Rilaxin A chain; Rilaxin B Chains; proliluxins; mouse gonadotropin-related peptides; microbial proteins such as β-lactamase; Dnase; IgE; cytotoxic T lymphocyte-related antigens (CTLA) such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF) ); Receptors for hormones or growth factors; Protein A or D; Rheumatology factor; Neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3) , NT-4, NT-5 or NT-6), or nerve growth factors such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors such as α-FGF and β-FGF; epithelial growth factor (EGF);

Moreover, in some embodiments, the protein or polypeptide purified using a smart polymer according to the invention is an antibody, a functional fragment or variant thereof. In some embodiments, the protein is a recombinant protein containing the Fc region of an immunoglobulin.

The terms "immunoglobulin," "Ig," or "antibody" (used interchangeably herein) are two heavy chains and two that have the ability to specifically bind to an antigen. Refers to a protein having a basic 4-polypeptide chain structure composed of the light chain of the above, and the chain is stabilized by, for example, an interchain disulfide bond. The terms "single-chain immunoglobulin" or "single-chain antibody" (used interchangeably herein) are from heavy and light chains that have the ability to specifically bind to an antigen. It refers to a protein having a two-polypeptide chain structure that is composed, and the chain is stabilized by, for example, an interchain peptide linker. The term "domain" refers to the spherical region of a heavy or light chain polypeptide containing, for example, a β-pleated sheet and / or a peptide loop stabilized by an interchain disulfide bond (including, for example, 3-4 peptide loops). .. Domains are further herein based on the relative deficiency of sequence variation within the domains of various class members in the case of "stationary" domains, and within the domains of various class members in the case of "variable" domains. It is called "steady" or "variable" based on large fluctuations. An antibody or polypeptide "domain" is often referred to in the art as compatible with an antibody or polypeptide "region". The "constant" domain of an antibody light chain is referred to interchangeably with the "light chain constant region", "light chain constant domain", "CL" region or "CL" domain. The "constant" domain of an antibody heavy chain is referred to interchangeably with the "heavy chain constant region", "heavy chain constant domain", "CH" region or "CH" domain. The "variable" domain of an antibody light chain is referred to as compatible with the "light chain variable region", "light chain variable domain", "VL" region or "VL" domain. The "variable" domain of the antibody heavy chain is referred to as compatible with the "heavy chain variable region", "heavy chain variable domain", "VH" region or "VH" domain.

The immunoglobulin or antibody may be monoclonal or polyclonal and may be present in monomeric or polymeric form. For example, IgM antibodies are present in pentamer form and / or IgA antibodies are present in monomeric, dimer or multimer form. Immunoglobulins or antibodies also include multispecific antibodies (eg, bispecific antibodies) and antibody fragments, as long as they retain or are modified to contain a ligand-specific binding domain. It can be. The term "fragment" refers to an intact, i.e., a complete antibody, or part or portion of an antibody or antibody chain that contains fewer amino acid residues than the antibody chain. Fragments can be obtained by treating the intact, ie complete antibody, or antibody chain, chemically or enzymatically. Fragments can also be obtained by recombinant means. When recombinantly produced, the fragments can be expressed alone or as part of a large protein called a fusion protein. Examples of fragments include Fab, Fab', F (ab') 2, Fc and / or Fv fragments. Examples of fusion proteins include Fc fusion proteins.

Usually, immunoglobulins or antibodies are suitable for the "antigen". Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal suffering from a disease or disorder can produce a therapeutic effect in that mammal. However, antibodies to non-polypeptides (eg, tumor-related glycolipid antigens; see US Pat. No. 5,091,178) are also conceivable. If the antigen is a polypeptide, it can be a transmembrane molecule (eg, a receptor) or a ligand (eg, a growth factor).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. it is believed that the individual antibodies that make up the population may be present in trace amounts. It is identical except for naturally occurring mutations. Monoclonal antibodies are highly specific and are suitable for single antigen sites. Moreover, each monoclonal antibody is oriented towards a single determinant on the antigen, in contrast to typical (polyclonal) antibody formulations that typically contain different antibodies to different determinants (epitopes). There is. The modifier "monoclonal" indicates that the properties of an antibody are derived from a substantially homogeneous population of antibodies, but it is not considered that the antibody must be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or recombinant DNA methods (eg, US Pat. No. 4,816,567). Can be created by). A "monoclonal antibody" is a phage antibody using the techniques described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). It can also be isolated from the library.

Monoclonal antibodies are further identical to or identical to the corresponding sequence in an antibody in which a portion of the heavy chain and / or light chain is derived from a particular species or belongs to a particular antibody class or subclass, as long as it exhibits the desired biological activity. A "chimeric" antibody (immunoglobulin) that is homologous and the rest of the chain is identical or homologous to the corresponding sequence in an antibody that is derived from another species or belongs to another antibody class or subclass, as well as fragments of said antibody. (US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

As used herein, the term "hypervariable region" refers to an amino acid residue of an antibody involved in antigen binding. Hypervariable regions are amino acid residues from the "complementarity determining regions" or "CDRs" (ie, residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain. ), And residues 31-35 (HI), 50-65 (H2) and 95-102 (H3) in heavy chain variable domains; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, Amino acid residues from the National Institutes of Health, Bethesda, Md. (1991)) and / or "hypervariable loops" (ie, residues 26-32 (L1), 50-52 (L2) in the light chain variable domains. ) And 91-96 (L3), and residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) in heavy chain variable domains; Chothia and Includes Lesk, J. Mol. Biol., 196: 901-917 (1987)). A "framework" or "FR" residue is a variable domain residue other than the hypervariable region residues defined herein.

The "humanized" form of a non-human (eg, mouse) antibody is a chimeric antibody that contains the smallest sequence from a non-human immunoglobulin. Often, humanized antibodies are non-human species (donor antibodies) such as mice, rats, households or non-human primates in which the recipient's hypervariable region residues have the desired specificity, affinity and competence. It is a human immunoglobulin (recipient antibody) substituted with a hypervariable region residue of origin. In some cases, the Fv framework region (FR) residue of human immunoglobulin has been replaced with the corresponding non-human residue. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or donor antibody. These modifications further purify antibody performance. Generally, a humanized antibody corresponds to at least one in which all or substantially all of the hypervariable loop corresponds to that of a non-human immunoglobulin and all or substantially all of the FR region is of a human immunoglobulin sequence. It typically contains virtually all of the two variable domains. Humanized antibodies optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant immunoglobulin constant region (Fc). For more details, see Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op.Struct.Biol., 2: 593-. See 596 (1992).

In some embodiments, the antibody separated or purified using the stimulus-responsive polymer according to the invention is a Therapeutic antibody. Examples of therapeutic antibodies include, for example, trastuzumab (HERCEPTIN ; Genentech, Inc., Carter et al. (1992), Proc. Natl. Acad. Sci. USA, 89: 4285-4289; US Pat. No. 5,725,856); Anti-CD20 antibody, eg, chimeric anti-CD20 C2B8; US patent No. 5,736,137); chimeric or humanized variant of rituximab (RITUXAN ), oclerismab, 2H7 antibody (US patent No. 5,721,108; WO 04/056312) Or tositumomab (BEXXAR ); anti-IL-8 (St John et al. (1993), Chest, 103: 932 and WO 95/23865); humanized and / or affinity matured anti-VEGF antibodies (eg, humanized anti-VEGF) Antibody huA4.6.1 Bevasizumab (AVASTIN , Genentech, Inc .; Kim et al. (1992), Growth Factors, 7: 53-64; WO Anti-VEGF antibody including 96/30046; WO 98/45331)); Anti-PSCA antibody (WO 01/40309); Anti-CD40 antibody including S2C6 and its humanized variant (WO 00/75348); Anti-CD11a ( US Pat. No. 5,622,700; WO 98/23761; Steppe et al. (1991), Transplant Intl., 4: 3-7; Hourmant et al. (1994), Transplantation, 58: 377-380); Anti-IgE (Presta et al. (1993)) , J.Immunol., 151: 2623-2632; WO 95/19181); Anti-CD18 (US Patent No.5,622,700; WO 97/26912); Anti-IgE including E25, E26 and E27 (US Patent No.5,714,338; US Pat. No. 5,091,313; WO 93/04173; US Pat. No. 5,714,338); Anti-Apo-2 Receptor Antibody (WO) 98/51793); anti-TNF-α antibodies including cA2 (REMICADE ), CDP571 and MAK-195 (US Patent No. 5,672,347; Lorenz et al. (1996), J.Immunol., 156 (4): 1646) -1653; Dhainaut et al. (1995), Crit. Care Med., 23 (9): 1461-1469); Anti-tissue factor (TF) (EP 0 420 937 B1); Anti-human α4β7 integrin (WO 98/06248); Anti-EGFR chimerized or humanized 225 antibody (WO 96/40210); anti-CD3 antibody, eg OKT3 (US Pat. No. 4,515,893); anti-CD25 or anti-tac antibody, eg CHI-621 SIMULECT and ZENAPAX . (US Pat. No. 5,693,762); Anti-CD4 antibody, eg cM-7412 antibody (Choy et al. (1996), Arthritis) Rheum., 39 (1): 52-56); Anti-CD52 antibody, eg CAMPATH-1H (Riechmann et al. (1988), Nature, 332: 323-337); Anti-Fc receptor antibody, eg M22 antibody against FcγRI (Graziano) Et al. (1995), J. Immunol., 155 (10): 4996-5002); Anti-cancer fetal antigen (CEA) antibodies such as hMN-14 (Sharkey et al. (1995), Cancer Res., 55 (23 Suppl): 5935s-5945s; Antibodies to breast epithelial cells including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al. (1995), Cancer Res., 55 (23): 5852s-5856s; and Richman et al. (1995), Cancer Res., 55 (23Supp): 5916s-5920s); Antibodies that bind to colon cancer cells, such as C242 (Litton et al. (1996), Eur.J.Immunol., 26 (1): 1-9); Anti-CD38 antibody , For example AT 13/5 (Ellis et al. (1995), J.Immunol., 155 (2): 925-937); Anti-CD33 antibodies such as Hu M195 (Jurcie et al. (1995), Cancer Res., 55 (23Suppl): 5908s- 5910s, and CMA-676 or CDP771; anti-CD22 antibody, eg LL2 or phosphoside (Juweid et al. (1995), Cancer Res., 55 (23Suppl): 5899s-5907s); anti-EpCAM antibody, eg 17-1A (PANOREX®). )); Anti-GpIIb / IIIa antibody, eg abciximab or c7E3 Fab (REOPRO ); Anti-RSV antibody, eg MEDI-493 (SYNAGIS ); Anti-CMV antibody, eg PROTOVIR ); Anti-HIV antibody , For example PRO542; anti-hepatitis antibody, for example anti-Hep B antibody OSTAVIR ); anti-CA 125 Antibodies OvaRex; Anti-idiotype GD3 epitope antibody BEC2; Anti-αvβ3 antibody VITAXIN ; Anti-human renal cell carcinoma antibody, eg ch-G250; ING-1; Anti-human 17-1A antibody (3622W94); Anti-human rectal colon Tumor antibody (A33); anti-human melanoma antibody against GD3 ganglioside R24; anti-human squamous cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA) antibody, such as Smart ID 10 and anti-HLA DR antibody Oncolym (Lym-1). Is included.

The terms "contaminant", "impurity" and "fragment" used interchangeably herein will be separated from one or more foreign substances or improper substances using stimulus-responsive polymers according to the present invention. Biological macromolecules (eg, DNA, RNA, one or more host cell proteins (HCP or CHOP)) that may be present in a sample containing the protein or polypeptide (eg, antibody), Refers to foreign substances or inappropriate substances including endotoxins, viruses, lipids and one or more additives). In some embodiments, the stimulus-responsive polymers described herein bind to and precipitate the protein or polypeptide from a sample containing the protein or polypeptide and one or more impurities. Let me. In other embodiments, the stimulus-responsive polymers described herein bind to and precipitate one or more impurities, thereby separating the polypeptide or protein from the one or more impurities.

The terms "Chinese hamster ovary cell protein" and "CHOP" used interchangeably herein refer to a mixture of host cell proteins ("HCP") from a Chinese hamster ovary ("CHO") cell culture. Point to. HCP or CHOP is usually present as an impurity in cell culture medium or lysate (eg, collected cell culture medium containing the protein or polypeptide (eg, antibody or immunoadhesin expressed in CHOP cells)). doing. Usually, the amount of CHOP present in the mixture containing the protein gives an indication of the purity of the protein. Typically, the amount of CHOP in the protein mixture is expressed as a percentage of the amount of the protein in the mixture.

If the host cell is another mammalian cell type, Escherichia coli, yeast cell, insect cell or plant cell, it should be understood that HCP refers to a protein other than the target protein present in the host cell's rise.

As used herein, the term "cell culture additive" refers to a molecule (eg, a non-protein additive) that is added to a cell culture process to promote or improve the cell culture or fermentation process. In some embodiments according to the invention, the stimulus-responsive polymers described herein bind to and precipitate one or more cell culture additives. Examples of cell culture additives include antifoaming agents, antibiotics, dyes and nutrients.

The terms "parts per million" or "ppm" used interchangeably herein are the desired target molecules purified using the stimulus-responsive polymers described herein (eg, for example. It refers to a measure of the purity of the target protein or antibody). Therefore, this measure can be used to assess the amount of target molecule present after the purification process or to assess the amount of undesired real substance. In some embodiments, the unit "ppm" is an impurity in solution in nanograms / milliliters of the protein in milligrams / milliliters (eg, HCP or CHOP) (ie, CHOP ppm = (CHOP ng). / ml) / refers to the amount of (the protein mg / ml). If the protein has been dried (eg, by freeze-drying), ppm refers to (CHOP ng) / (the protein mg).

The terms "isolate," "purify," and "separate" refer to a composition or composition containing a target molecule and one or more impurities using the stimulus-responsive polymers described herein. It is used interchangeably herein in the context of purifying a target molecule (eg, the polypeptide or protein) from a sample. In some embodiments, the purity of the target molecule in the sample removes one or more impurities (completely or partially) from the sample by using a stimulus-responsive polymer as described herein. ) Increases by removing. In another embodiment, the purity of the target molecule in the sample is increased by precipitating and removing the target molecule from one or more impurities in the sample.

In some embodiments, the purification process further uses one or more "chromatographic steps". Typically, these steps can be performed as required after separating the target molecule from one or more unwanted entities using a stimulus-responsive polymer according to the invention.

In some embodiments, the "purification step" for isolating, separating or purifying the polypeptide or protein using the stimulus-responsive polymers described herein is "homogeneous" or "homogeneous." It can be part of a total purification process that yields a "pure" composition or sample. The term refers to HCP less than 100 ppm in a composition containing the protein, or less than 90 ppm, less than 80 ppm, less than 70 ppm, less than 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm or Used to refer to compositions or samples that contain less than 3 ppm HCP.

As used herein, the term "clarification" step usually refers to one or more initial steps in the purification of a biomolecule. The clarification step usually involves the removal of cells and / or cell debris using one or more steps, including, for example, centrifugation and debs filtration, precipitation, aggregation and precipitation, or various combinations thereof. The clarification step usually involves the removal of one or more unwanted entities, typically performed prior to the step involving the capture of the desired target molecule. Another important aspect of clarification is to remove soluble and insoluble components in the sample that may later contaminate the sterile filter in the purification process, thereby making the entire purification process more economical. In some embodiments, the present invention is commonly used in various purification schemes, as evidenced by the lower turbidity / impurities described in the examples herein and the higher throughput of downstream filters. It provides improvements beyond the conventional clarification steps that have been made.

As used herein, the term "chromatography" refers to any type of technique that separates the analysis (eg, target molecule) from other molecules present in the mixture. Usually, the analyze is separated from other molecules as a result of the rate at which individual molecules of the mixture move through the stationary medium under the influence of the mobile phase, or differences in binding and elution processes.

The terms "chromatographic resin" or "chromatographic medium" are used interchangeably herein and optionally separate the analyst (eg, target molecule) from other molecules present in the mixture. Refers to a phase of the type (eg, solid phase). Usually, the analysts are separated from other molecules as a result of the rate at which individual molecules of the mixture move through the stationary solid phase under the influence of the mobile phase, or differences in binding and elution processes. Examples of various types of chromatography media include, for example, cation exchange resins, affinity resins, anion exchange resins, anion exchange membranes, hydrophobic interaction resins and ion exchange monoliths.

As used herein, the term "capture step" typically combines a target molecule with a stimulus-responsive polymer or chromatographic resin to give a solid phase containing the target molecule and a precipitate of the polymer or resin. Refers to the method used for. Typically, the target molecule is then recovered using an elution step that removes the target molecule from the solid phase and separates the target molecule from one or more impurities. In various embodiments, the capture step can be performed using a chromatographic medium (eg, resin, membrane or monolith) or polymer (stimulus responsive polymer, polymer electrolyte, or polymer that binds the target molecule).

As used herein, the term "salt" refers to a compound formed by the interaction of an acid and a base. The various salts that can be used in the various buffers used in the methods described herein include acetates (eg, sodium acetate), citrates (eg, sodium citrate), chlorides (eg, sodium citrate). For example, sodium chloride), sulphate (eg, sodium sulphate) or potassium salt, but is not limited to these.

As used interchangeably herein, the term "multivalent salt" or "multivalent ion" refers to a compound containing two or more charges or charge-containing groups. In some embodiments, the polyvalent salt is used as a stimulus, resulting in a change in the solubility of the polymer that is responsive to salt stimuli, usually precipitating the polymer from solution. Examples of polyvalent salts that can be used include, for example, phosphates and sulfates. Possible counterions, such as citrate, are also included in the invention. When used as a stimulus as described herein, the polyvalent salt can be added as a stand-alone reagent to a sample containing the biomolecule of interest along with a stimulus-responsive polymer. Alternatively, the salt may be attached to a substrate, such as a membrane. In certain embodiments, the membrane is modified with polyvinyl phosphate, which is a polyvalent salt-containing polymer coating. It is believed that the multivalent ions used as stimuli as described herein do not significantly impair protein structure and stability compared to other stimuli (eg, temperature and pH).

In some embodiments, the polyvalent salt can interact with one or more reals to form an associated species or complex. Therefore, the salt may also be referred to as a "complex-forming salt". Non-limiting examples of complex-forming salts and resulting complexes include multivalent cations (eg, Cu).²⁺And Ca²⁺) And its complex with carboxylic acid groups present in ethylenediaminetetraacetate; polyvalent anions (eg, phosphates (PO))₄^3-) And its complex with citrates) and primary amines present in polyallylamine; and in ionic salt (eg, perchlorate, dodecyl sulfate and dodecylbenzene sulfonate) and polyallylamine. It is a complex with the primary amine present in.

An "ion-associative salt" is a monovalent (cationic or anionic) bulky charge-dispersed salt. In some embodiments, the ion-associating salt is used as a stimulus, resulting in a change in the solubility of the polymer that is responsive to salt stimuli, causing the polymer to precipitate from solution. Examples of ion-associative salts that can be used include, for example, perchlorate, dodecyl sulfate, dodecylbenzene sulfonate, tetraphenylborate and hexanitrodiphenolamine.

As used herein, the term "solvent" generally refers to a liquid substance from which one or more other substances can be dissolved or dispersed to obtain a solution. Solvents include aqueous and organic solvents, and useful organic solvents include non-protic solvents, ethanol, methanol, isopropanol, acetonitrile, hexylene glycol, propylene glycol and 2,2-thiodiglycol.

As used interchangeably herein, the term "pI" or "isoelectric point" of a polypeptide refers to the pH at which the positive charge of a polypeptide balances its negative charge. The pI can be calculated from the net charge of the amino acid or sialic acid residues of the conjugated carbohydrate of the polypeptide, or can be measured by isoelectric focusing.

The present invention will be further described by the following examples, which should not be construed as limiting. The contents and drawings of all documents, patents and published patent applications cited throughout this specification are incorporated herein by reference.

<p num="0112">[Example 1]<u style="single">Preparation of unclarified non-expressing cell culture medium (CCF)</u> In a representative experiment, cells from a non-expressed Chinese hamster ovary (CHO) cell line were placed in a 10 L bioreactor (New Brunswick Scientific) at 10 × 10.⁶It was grown to a cell / mL density and collected at 64% viability. IgG was spiked to a concentration of 1.3 g / L. Host cell protein (HCP) levels were found to be 8300 ng / ml using ELISA (Cygnus # CM015). The pH of the unclarified cell culture was 7.2.</p><p num="0113">[Example 2]<u style="single">Synthesis of stimulus-responsive polymers containing a polymeric electrolyte backbone modified with hydrophobic groups</u> In a representative experiment, a stimulus-responsive polymer containing a hydrophobic group-modified polyallylamine (BzMPAA) backbone was synthesized. A mixture of 40% wt linear polyallylamine (NITTOBO, 150kD) (10.3g), lithium hydroxide (2g) and 50% water / methanol (20ml) containing a cationic polymer electrolyte skeleton modified with a hydrophobic group. Was synthesized and stirred until well mixed. A solution containing benzyl chloride (2.1 ml) in methanol (15 ml) was added to the polymer solution. The resulting mixture was heated at 60 ° C. for 14 hours. As a result of thermodynamic incompatibility with the solvent, the benzyl-modified polyallylamine precipitated at the end of the reaction period. The precipitate was washed with acetone (30 mL) and redissolved in 1 M acetic acid (400 mL). The polymer was further purified by precipitating with 50 mM sodium phosphate at pH 7. FIG. 1 shows an outline of the reaction of a polyallylamine polymer electrolyte polymer with a hydrophobic group, that is, benzyl chloride.</p><p num="0114">[Example 3]<u style="single">Preparation of a solution of benzyl-modified polyallylamine (BzMPAA)</u> A 10% BzMPAA solution was prepared by dissolving the polymer (10 g) from Example 2 in 1 M acetic acid (90 g) with continuous stirring at room temperature for 16 hours. The resulting viscous solution was slightly cloudy.</p><p num="0115">[Example 4]<u style="single">Use of different concentrations of benzyl-modified polyallylamine (BzMPAA) in clarification of non-expressing cell culture medium (CCF)</u> To a 5 mL sample of unclarified cell culture (CCF) from Example 1, 0.2 g, 0.3 g, 0.4 g and 0.5 g of BzMPAA from Example 3 were added. Samples were mixed at room temperature for 2 minutes. Since the pH dropped to the pH range of 4.5 to 5.5 due to the addition of the polymer, the pH of the mixture was adjusted to pH 7 using a 2M Tris base. Dipotassium hydrogen phosphate (0.043 g) was added to the resulting solution to precipitate the polymer-target molecule, cell and cell debris complex. The precipitate in the form of a dispersed solid suspension was continuously mixed for 5 minutes. The precipitate was then collected by centrifugation (4000 rpm for 1 minute). The supernatant from each sample was filtered through a 0.2 um Durapore® filter. The resulting purification is detailed in Table 1 below.</p><p num="0116">[Example 5]<u style="single">Use of different pH values in clarification of unexpressed CCF with BzMPAA</u> BzMPAA (0.4 g each) from Example 3 was added to 4 samples containing the unclarified cell culture medium (5 mL) from Example 1. Samples were mixed at room temperature for 2 minutes. Since the addition of the polymer lowered the pH to the pH range of 4.5-5.5, the pH of the mixture was adjusted to pH 5.5, 6.5, 7.5 and 8.5 using 2M Tris bases, respectively. Dipotassium hydrogen phosphate (0.043 g) was added to the resulting solution to precipitate the polymer-target molecule, cell and cell debris complex. The precipitate in the form of a dispersed solid suspension was continuously mixed for 5 minutes. The precipitate was then collected by centrifugation (4000 rpm for 1 minute). The supernatant from each sample was then filtered through a 0.2um Durapore® filter. The resulting purification is detailed in Table 1 below. This table lists the BzMPAA purification of spiked non-expressed CHOCCF.</p><p num="0117"><tables num="1"><img id="000006" he="57" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0118">[Example 6]<u style="single">Assay for purity levels resulting from the use of BzMPAA in CCF clarification</u> Samples from Example 4 and Example 5 were assayed for IgG recovery using an HPLC assay for Affinity Protein A analysis. Alternatively, IgG levels in solution were measured using a protein A column for analysis. Specifically, Poros A / 20 protein A column (Applied Biosystems) was equilibrated with PBS, eluted with 0.1 M lysine (pH 2) and washed with 6 M guanidine HCl. An IgG standard curve was created using a series of polyclonal IgG (Seracare) with different injection volumes. The sample was injected and the IgG concentration was determined from the standard curve.</p><p num="0119"> Samples from Example 4 and Example 5 were assayed for host cell protein (HCP) using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Cygnus Technologies Inc., Southport, NC, Cygnus # CM015). Samples from Example 4 and Example 5 were also assayed for DNA using the standard picogreen assay and herring semen DNA as standard.</p><p num="0120">[Example 7]<u style="single">Preparation of unclarified cell culture medium (CCF)</u> 10 × 10 cells from the expressed Chinese hamster ovary (CHO-DG44) cell line in a 10 L bioreactor (New Brunswick Scientific)⁶It was grown to a cell / ml density and collected at 30% viability. The monoclonal antibody (MAb) titer was measured to be 0.8 g / L. Host cell protein (HCP) levels were found to be 200,000 ng / ml using an ELISA assay (Cygnus # 3G ELISA). The pH of the unclarified cell culture was pH 6.9.</p><p num="0121">[Example 8]<u style="single">Synthesis of 20% benzyl-modified polyallylamine (BzMPAA)</u> Place polyallylamine (PAA) (Nittobo, 150kD; 40% wt./wt.) (10g) in a 100ml round bottom flask and add sodium hydroxide (3.34g) (1.2 eq / monomer) to H.₂The solution contained in O (25 mL) was added little by little with magnetic stirring at room temperature. Benzyl chloride (1.38 g, 1.25 mL) was then added all at once, stirred at room temperature for several minutes and then heated to 60 ° C. for 17 hours overnight. The reaction was then cooled to room temperature and the solvent was removed to precipitate the polymer. The precipitated polymer was washed with water and then stirred in 1 M aqueous AcOH solution (40 mL) until completely dissolved. Then H the solution₂Dilute to the final volume of 400 mL (1% polymer solution) with O and dipotassium hydrogen phosphate (K)₂HPO₄) (3.48 g) was added with stirring to adjust the pH of the solution to about 6.8 to precipitate the modified polymer. The polymer was collected by filtration through a glass filter and finally dried in a vacuum oven at 50-60 ° C. overnight.</p><p num="0122">[Example 9]<u style="single">Synthesis of 40% benzyl-modified polyallylamine (BzMPAA)</u> Place polyallylamine (PAA) (Nittobo, 150kD; 40% wt / wt.) (10g) in a 100ml round bottom flask and add sodium hydroxide (3.34g) (1.2 eq / monomer) to H.₂The solution contained in O (25 mL) was added little by little with magnetic stirring at room temperature. Benzyl chloride (2.30 g, 2.09 mL) was then added, stirred at room temperature for several minutes and then heated to 60 ° C. for 17 hours overnight. The reaction was then cooled to room temperature and the solvent was removed to precipitate the polymer. The precipitated polymer was washed with water and stirred in 1 M aqueous AcOH solution (40 mL) until completely dissolved. Then H the solution₂Dilute to the final volume of 400 mL (1% polymer solution) with O and dipotassium hydrogen phosphate (K)₂HPO₄) (3.48 g) was added with stirring and the pH of the solution was adjusted to pH 6.8 to precipitate the modified polymer. The polymer was collected by filtration through a glass filter and finally dried in a vacuum oven at 50-60 ° C. overnight.</p><p num="0123">[Example 10]<u style="single">Synthesis of 60% benzyl-modified polyallylamine (BzMPAA)</u> Place polyallylamine (PAA) (NITTOBO, 150 kD; 40% wt / wt.) (10 g) in a 100 ml round bottom flask and add sodium hydroxide (3.34 g) (1.2 eq / monomer) to H.₂The solution contained in O (25 mL) was added little by little with magnetic stirring at room temperature. Benzyl chloride (3.23 g, 2.94 mL) was then added, stirred at room temperature for several minutes and then heated to 60 ° C. for 17 hours overnight. The reaction was then cooled to room temperature and the solvent was removed. The precipitated polymer was washed with water and then stirred in 1 M aqueous AcOH solution (40 mL) until completely dissolved. Then H the solution₂Dilute to the final volume of 400 mL (1% polymer solution) with O and dipotassium hydrogen phosphate (K)₂HPO₄) (3.48 g) was added with stirring and the pH of the solution was adjusted to pH 6.8 to precipitate the modified polymer. The polymer was collected by filtration through a glass filter and finally dried in a vacuum oven at 50-60 ° C. overnight.</p><p num="0124">[Example 11]<u style="single">Synthesis of diphenyl-modified polyallylamine (DPhMPAA)</u> In an exemplary experiment, polyallylamine in the polymeric electrolyte polymer backbone was modified with a diphenyl group. Briefly, polyallylamine (PAA) (Nittobo, 150 kD; 40% wt / wt.) (10 g) is placed in a 100 ml round bottom flask and sodium hydroxide (3.34 g) (1.2 eq / monomer) is added to H.₂The solution contained in O (25 mL) was added little by little with magnetic stirring at room temperature. Then, chlorodiphenylmethane (3.68 g, 3.23 mL) was added, stirred at room temperature for several minutes, and then heated to 60 ° C. for 17 hours overnight. The reaction was then cooled to room temperature and the solvent was removed. The precipitated polymer was washed with water and stirred in 1M aqueous AcOH solution (40 mL). The remaining white solid produced by the hydrolysis of diphenylchloromethane was filtered off. Then H the solution₂Dilute to the final volume of 400 mL (1% polymer solution) with O and dipotassium hydrogen phosphate (K)₂HPO₄) (3.48 g) was added with stirring and the pH of the solution was adjusted to pH 6.8 to precipitate the modified polymer. The polymer was collected by filtration through a glass filter and finally dried in a vacuum oven at 50-60 ° C. overnight.</p><p num="0125">[Example 12]<u style="single">6% Dichlorobenzyl Modified Polyallylamine (Synthesis of DClBzMPAA)</u> In another experiment, polyallylamine (PAA) (NITTOBO, 150 kD; 40% wt / wt.) (10 g) was placed in a 100 ml round bottom flask and sodium hydroxide (3.34 g) (1.2 eq / monomer) was added to H.₂The solution contained in O (25 mL) was added little by little with magnetic stirring at room temperature. Then 3,4-dichlorobenzyl chloride (1.71 g, 1.21 mL) was added and the mixture was stirred at room temperature for 17 hours overnight. Then the reaction mixture is H₂After diluting with O (100 ml), the pH was adjusted to neutral (pH 7.0) with phosphoric acid. The precipitated polymer is filtered off and H₂Washed with O and dried overnight at 60 ° C in a vacuum oven. The polymer was collected by filtration through a glass filter and finally dried in a vacuum oven at 50-60 ° C. overnight.</p><p num="0126">[Example 13]<u style="single">Synthesis of 10% dichlorobenzyl-modified polyallylamine (DClBzMPAA)</u> In another representative experiment, 10% dichlorobenzyl-modified polyallylamine was synthesized as follows. Place polyallylamine (PAA) (NITTOBO, 150 kD; 40% wt / wt.) (5 g) in a 100 ml round bottom flask and add sodium hydroxide (1.68 g) (1.2 eq / monomer) 50/50 H.₂The solution contained in O / 1,2-dimethoxyethane (DME) (40 mL) was added little by little with magnetic stirring at room temperature. Then, 3,4-dichlorobenzyl chloride (0.57 g, 0.40 mL) was added, stirred at room temperature for several minutes, and then heated to 60 ° C. for 21 hours overnight. The reaction was then cooled to room temperature, the DME was removed under vacuum at 60-70 ° C, and the remaining solvent was removed. The precipitated polymer was washed with water and then stirred in 1 M aqueous AcOH solution (20 mL) until completely dissolved. Then H the solution₂Dilute with O to a final volume of 200 mL (1% polymer solution) and dipotassium hydrogen phosphate (K)₂HPO₄) (1.74 g) was added with stirring and the pH of the solution was adjusted to pH 6.8 to precipitate the modified polymer. The polymer was collected by filtration through a glass filter and finally dried in a vacuum oven at 50-60 ° C. overnight.</p><p num="0127">[Example 14]<u style="single">Synthesis of 33% chlorobenzyl-modified polyallylamine (DClBzMPAA)</u> In another representative experiment, 33% chlorobenzyl-modified polyallylamine was synthesized as follows. Place polyallylamine (PAA) (NITTOBO, 150 kD; 40% wt / wt.) (5 g) in a 100 ml round bottom flask and add sodium hydroxide (3.34 g) (1.2 eq / monomer) 50/50 H.₂The solution contained in O / 1,2-dimethoxyethane (DME) (40 mL) was added little by little with magnetic stirring at room temperature. 4-Chlorobenzyl chloride (1.48 g) was then added, stirred at room temperature for several minutes and then heated to 60 ° C. for 21 hours overnight. The next day, the DME was evaporated under vacuum at 60-70 ° C to separate the remaining solvent from the precipitated polymer. The latter was washed with water and then stirred in 1 M aqueous AcOH solution (20 mL) until completely dissolved. Then H the solution₂Dilute with O to a final volume of 200 mL (1% polymer solution) and dipotassium hydrogen phosphate (K)₂HPO₄) (1.74 g) was added with stirring and the pH of the solution was adjusted to pH 7 to precipitate the purified polymer. The polymer was collected by filtration through a glass filter and finally dried in vacuo at 50-60 ° C. overnight.</p><p num="0128">[Example 15]<u style="single">Synthesis of 13% Phenylbenzyl Modified Polyallylamine (DClBzMPAA)</u> In another experiment, 13% phenylbenzyl-modified polyallylamine was synthesized as follows. Place polyallylamine (PAA) (Nittobo, 150 kD; 40% wt / wt.) (4.7 g) in a 100 ml round bottom flask and add sodium hydroxide (3.34 g) (1.2 eq / monomer) 50/50 H.₂The solution contained in O / 1,2-dimethoxyethane (DME) (40 mL) was added little by little with magnetic stirring at room temperature. 4-Phenylbenzyl chloride (1 g) was then added and the mixture was heated at 55 ° C. for 20 hours overnight. The reaction was then cooled to room temperature, the DME was removed under vacuum at 60-70 ° C. and the remaining solvent was separated from the precipitated polymer. The latter was washed with water and then stirred in 1 M aqueous AcOH solution (40 mL) until completely dissolved. Then H the solution₂Dilute with O to a final volume of 200 mL (1% polymer solution) and dipotassium hydrogen phosphate (K)₂HPO₄) (1.74 g) was added with stirring and the pH of the solution was adjusted to pH 7.0 to precipitate the purified polymer. The polymer was collected by filtration through a glass filter and finally dried in vacuo at 50-60 ° C. overnight.</p><p num="0129">[Example 16]<u style="single">Synthesis of 27% Phenylbenzyl Modified Polyallylamine (DClBzMPAA)</u> In another experiment, 27% phenylbenzyl-modified polyallylamine was synthesized as follows. Place polyallylamine (PAA) (Nittobo, 150 kD; 40% wt / wt.) (2.8 g) in a 100 ml round bottom flask and add sodium hydroxide (3.34 g) (1.2 eq / monomer) 50/50 H.₂The solution contained in O / 1,2-dimethoxyethane (DME) (40 mL) was added little by little with magnetic stirring at room temperature. 4-Phenylbenzyl chloride (1 g) was then added and the mixture was heated at 55 ° C. for 20 hours overnight. The reaction was then cooled to room temperature, the DME was removed under vacuum at 60-70 ° C. and the remaining solvent was separated from the precipitated polymer. The latter was washed with water, then stirred in 1M aqueous AcOH solution (32 mL) and stirred overnight until completely dissolved. Then H the solution₂Dilute with O to a final volume of 200 mL (1% polymer solution) and dipotassium hydrogen phosphate (K)₂HPO₄) (1.74 g) was added with stirring and the pH of the solution was adjusted to pH 7.0 to precipitate the purified polymer. The polymer was collected by filtration through a glass filter and finally dried in vacuo at 50-60 ° C. overnight.</p><p num="0130">[Example 17]<u style="single">CCF clarification at different polymer concentrations</u> In an exemplary experiment, the stimulus-responsive polymers described in Examples 8-16 were evaluated for CCF clarification. The creation of the CCF is described above in Example 7. Polymer solutions from Examples 8-16 were added in amounts of 0.2 g, 0.3 g, 0.4 g and 0.5 g to a 5 mL sample of unclarified cell culture. Samples were mixed at room temperature for 2 minutes. Since the pH dropped to the pH range of 4.5 to 5.5 due to the addition of the polymer, the pH of the mixture was adjusted to pH 7 using a 2M Tris base. Dipotassium hydrogen phosphate (0.043 g) was added to the resulting solution to precipitate the polymer-target molecule, cell and cell debris complex. The precipitate in the form of a dispersed solid suspension was continuously mixed for 5 minutes. The precipitate was then collected by centrifugation (4000 rpm for 1 minute). The supernatant from each sample was then filtered through a 0.2 μm Durapore® filter. The resulting purification is detailed in Table 2.</p><p num="0131">[Example 18]<u style="single">CCF clarification with polymers at different pH</u> In another experiment, polymer solutions from Examples 8-16 (0.4 g each) were added to 4 samples containing the unclarified cell culture medium (5 mL) described in Example 7. .. Samples were mixed at room temperature for 2 minutes. Since the addition of the polymer lowered the pH to the pH range of 4.5-5.5, the pH of the mixture was adjusted to pH 5.5, 6.5, 7.5 and 8.5 using 2M Tris bases, respectively. Dipotassium hydrogen phosphate (0.043 g) was added to the resulting solution to precipitate the polymer-target molecule, cell and cell debris complex. The precipitate in the form of a dispersed solid suspension was continuously mixed for 5 minutes. The precipitate was then collected by centrifugation (4000 rpm for 1 minute). The supernatant from each sample was then filtered through a 0.2 μm Durapore® filter. The purifications that occur are listed in Table 2.</p><p num="0132">[Example 19]<u style="single">CCF clarification with polymers responsive to different levels of multivalent ion stimulation</u> In another experiment, the polymers described in Examples 8-15 were evaluated for clarification of CCF using different amounts of multivalent ion stimulation. Specifically, the polymer solutions (0.4 g each) from Examples 8 to 15 were added to the four samples containing the unclarified cell culture medium (5 mL) described in Example 7. .. Samples were mixed at room temperature for 2 minutes. Since the addition of the polymer lowered the pH to the pH range of 4.5-5.5, the pH of the mixture was adjusted to pH 5.5, 6.5, 7.5 and 8.5 using 2M Tris bases, respectively. To precipitate the polymer-target molecule, cell and cell debris complex, a dipotassium hydrogen phosphate salt in the range 0.031 to 0.043 g (final phosphate concentration of 50 to 70 mM) was added to the resulting solution. The precipitate in the form of a dispersed solid suspension was continuously mixed for 5 minutes. The precipitate was then collected by centrifugation (4000 rpm for 1 minute). The supernatant from each sample was then filtered through a 0.2 μm Durapore® filter. The purifications that occur are listed in Table 2.</p><p num="0133">[Example 20]<u style="single">CCF clarification with conventional flocculants</u> In another experiment, the flocculant chitosan, commonly used in attempts to obtain comparative data, was used for clarification of CCF. Polymer solutions (2 wt%) were prepared according to the procedure described in Riske, F. et al .; Journal of Biotechnology, 128 (2007), 813-823. Polymer solutions were added to the CCF from Example 7 in varying amounts and pH conditions. No irritation was used for this polymer. The purifications that occur are listed in Table 2.</p><p num="0134">[Example 21]<u style="single">Evaluation of purity level after precipitation of CCF with stimulus-responsive polymer</u> In a representative experiment, the polymers described in Examples 8-16 were assayed for IgG recovery using an HPLC assay for affinity protein A analysis. Alternatively, IgG levels in solution were examined using a protein A column for analysis. Poros A / 20 protein A columns (Applied Biosystems) were equilibrated with PBS, eluted with 0.1 M lysine (pH 2) and washed with 6 M guanidine HCl. An IgG standard curve was created using a series of polyclonal IgG (Seracare) with different injection volumes. The sample was injected and the IgG concentration was determined from the standard curve. Samples from Examples 8-16 were sampled from a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Cygnus Technologies Inc., Southport, NC, Cygnus). The host cell protein (HCP) was assayed using # CM015). Samples from Examples 8-16 were also assayed for DNA using the standard picogreen assay and herring semen DNA as standard. Turbidity was examined after centrifugation at 4000 rpm for 1 minute to assess reduction of cells and cell debris.</p><p num="0135"><tables num="2"><img id="000007" he="132" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables><img id="000008" he="222" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000009" he="176" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0136">[Example 22]<u style="single">Synthesis of benzyl-modified polyethyleneimine (BzMPEI)</u> In another experiment, a benzyl-modified polyethyleneimine stimulus-responsive polymer was synthesized as follows. Put PEI (Aldrich, 750kD; 50% wt./wt.) (10g) in a 100ml round bottom flask and add sodium hydroxide (1.2 eq / monomer) (~ 3.34g) to H.₂Add the solution contained in O (25 mL) little by little with magnetic stirring at room temperature. Benzyl chloride (2.30 g, 2.09 mL) is then added, stirred at room temperature for several minutes and then heated at 60 ° C. for 17 hours overnight. The reaction is then cooled to room temperature and the solvent removed. The precipitated polymer is washed with water and then stirred in 1 M aqueous AcOH solution (20 mL) until completely dissolved. Then H the solution₂Dilute to the final volume of 400 mL (1% polymer solution) with O and dipotassium hydrogen phosphate (K)₂HPO₄) (3.48 g) is added with stirring to adjust the pH of the solution to pH 6.8 to precipitate the modified polymer. The polymer is collected by filtration through a glass filter and finally dried in a vacuum oven at 50-60 ° C overnight.</p><p num="0137">[Example 23]<u style="single">Synthesis of benzyl-modified polyvinylamine (BzMPVA)</u> In a representative example, a benzyl-modified polyvinylamine was synthesized as follows. Poly (vinylamine) (PVA) hydrochloride (MW = 83,500, Air Products and Chemicals Inc.) (32 g) was weighed into a glass container. H₂Add O (200 mL), add 50% NaOH (26 g) and stir until well mixed. Benzyl chloride (23.5 g) is added and mixed at 70 ° C for 16 hours. As the reaction progresses, a solid white mass separates from the supernatant. The solid is settled and the supernatant is discarded by decanting. Dissolve the solid in 3% acetic acid (350 mL) overnight. H₂Add O (360 mL) and mix for 16 hours until the solution is homogeneous. Deion the entire volume (DI) H₂Dilute to 1% w / v solution by making 3.2 L with O. Sodium phosphate is added to a concentration of 50 mM to initiate the precipitation of the polymer. 1M NaOH is added until pH 6.8 is reached to additionally precipitate the polymer. Filter the dried cake and discard the supernatant. Dry the solid at 70 ° C overnight. Dissolve in 3% acetic acid until a homogeneous solution is produced to make a 5% w / v solution.</p><p num="0138">[Example 24]<u style="single">Comparison of modified and unmodified polymers in clarification in the presence of stimuli</u> Polyallylamine (PAA, Nittobo, 150 kD; 40% wt./wt.) And the polymer from Example 10 (Bz-MPAA) were added to the aqueous solution to give a final polymer concentration of 0.2% wt and 0.4%, respectively. .. Potassium hydrogen phosphate was used as a stimulus and added in different amounts to PAA and the polymer solution of Example 10 to record turbidity and type of agglomerates formed. The results are shown in Table 3 below.</p><p num="0139"><tables num="3"><img id="000010" he="159" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0140">[Example 25]<u style="single">Comparison of modified and unmodified polymers in clarification in the presence of stimuli</u> Unmodified polymer polyallylamine (PAA, Nittobo, 150kD; 40% wt / wt.), Polyethyleneimine (PEI, Aldrich, 750kD; 50%) in aqueous solution to a final polymer concentration of 0.2% by weight and 0.4% by weight, respectively. wt / wt.), Polyvinylamine (poly (vinylamine) (PVA) hydrochloride, MW = 83,500, Air Products and Chemicals Inc.), and modified polymers from Example 10 (Bz-MPAA), from Example 22. A modified polymer (BzMPEI) and a modified polymer from Example 23 (BzMPVA) were added. Potassium hydrogen phosphate (150 mM) was used as a stimulus and added to each of the 0.4% polymer solutions to record turbidity and type of aggregates formed. The results are shown in Table 4 below.</p><p num="0141"><tables num="4"><img id="000011" he="76" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0142">[Example 26]<u style="single">Synthesis of Caproic Acid and tert-Butyl Modified Polyallylamine (HC-t-BuMPAA)</u> 6-Bromohexaneic acid (3.49 g) was dissolved in a solution containing 40 wt% linear polymer poly (allylamine) (150 kDa, NITTOBO) (10 ml) and sodium hydroxide (1M) (30 ml). The mixture was reacted at T = 50 ° C for 18 hours and the product was precipitated as a hydrated gel. The precipitate was dissolved in 100 mg / ml lithium hydroxide solution and mixed with methanol (10 ml) containing tert-butyl glycidyl ether (2.5 ml). The mixture was then reacted at T = 50 ° C for 18 hours. The polymer solution was purified by extensive dialysis (3 days) on deionized (DI) water using a 3.5 kda molecular weight cut-off dialysis tube. The final concentration of the polymer solution was 7.2 wt%. The outline of the synthetic reaction is shown in Fig. 2.</p><p num="0143">[Example 27]<u style="single">Effect of Sodium Chloride on Multivalent Stimulation of Caproic Acid and tert-Butyl Modified Polyallylamine (HC-t-BuMPAA) When Dissolved in Tris Buffer</u> HC-t-BuMPAA (600 μl) from Example 26 was added to 25 mM sodium phosphate (10 ml) containing 0, 0.15 or 0.5 M sodium chloride. The final pH of the solution was 11.6. The solution was titrated with 3M acetic acid and the turbidity of the solution was recorded after each addition. As shown in FIG. 3, a change in pH responsiveness caused by a phase transition was observed by adding sodium chloride in addition to sodium phosphate.</p><p num="0144">[Example 28]<u style="single">CCF clarification with polymer HC-t-BuMPAA at different polymer concentrations</u> In a representative experiment, unclarified CCF was clarified using a polymer according to the present invention as follows. HC-t-BuMPAA (178, 356 or 534 μl) from Example 26 was added to the unclarified cell culture medium (5 ml) from Example 1 containing 25 mM sodium phosphate and 3M acetic acid (178, 356 or 534 μl). The pH was adjusted to 8.7 using 25 μl). After adding the polymer, the final pH of the solution was adjusted to 7.2 with 3M acetic acid to precipitate the polymer-cell complex.</p><p num="0145"> The precipitate in the form of a dispersed solid suspension was continuously mixed for an additional 5 minutes. The precipitate was then collected by centrifugation (4000 rpm for 1 minute) and the supernatant was filtered through a 0.2 μm Durapore® filter. The process recovered 100% of the Mab, regardless of the concentration of polymer used.</p><p num="0146">[Example 29]<u style="single">Synthesis of polyvinylamine (PVA) stimulus-responsive polymers from monomers</u> A stimulus-responsive polymer containing a repeating unit containing a primary amine was synthesized from the monomer as follows. Deionized water (165 g) and N-vinylformamide (NVF) (SIGMA-ALDRICH, 98%) (22.5 g) were placed in a 250 ml round bottom flask. Magnetic stirrer and N in flask₂Equipped with a dipstick. N₂After stirring the solution for 0.5 hours while purging, the solution was heated to 45 ° C for another 0.5 hours while continuously purging. An initiator solution was prepared and dissolved by adding 2,2'-azobis (2-amide propane) dihydrochloride (ABAP) (Aldrich) (0.288 g) to deionized water (10 ml). N initiator solution in a 250 ml round bottom flask₂Added in atmosphere. The solution was heated to 55 ° C for 1 hour with vigorous stirring under nitrogen, then heated at 65 ° C for 2 hours and then at 75 ° C for 1 hour. A viscous homogeneous solution was obtained and cooled to room temperature. Viscosity was measured with a Brookfield Viscosity DV-II + Pro viscometer (setting 100 RPM, 45% torque, spindle # 34). The viscosity of the resulting solution was 278-350 centipoise (cP). Transfer the solution to a 500 ml flask and H₂Dilute with O (330 ml) and add 50% NaOH (40 g) with stirring. The solution was heated at 85 ° C for 8 hours.</p><p num="0147"> Test the susceptibility of small samples to phosphate irritation by adding drop by drop of sodium 2 molar to the hydrolyzed polymer and observing the precipitation of a white solid from the solution when phosphate ions were added. did. 25% HCl was added drop by drop to the hydrolyzed polymer solution until the pH reached about 2. The solution was vigorously stirred overnight to give a homogeneous yellow solution. 4 mol NaOH (100 ml) was added to this solution with isopropyl alcohol (500 ml) with stirring. The polymer was isolated by adding 2 molar sodium phosphate (100 ml), the solid was vacuum filtered and washed with deionized water. The solid polymer was dried overnight at 65 ° C. in a vacuum oven. The partially dried polymer was frozen in liquid nitrogen, ground to a fine powder and further dried in a vacuum oven at 65 ° C. for 24 hours. Finally, 40.7 g of dry powder was recovered and dissolved in 1 mol acetic acid to a final concentration of 5% w / w.</p><p num="0148"> The outline of the polyvinylamine synthesis process is shown in FIG.</p><p num="0149">[Example 30]<u style="single">Synthesis of a series of hydrophobically modified polyvinylamine (PVA) stimulus-responsive polymers</u> Using PVA synthesized from Example 29, three separate hydrophobically modified stimulus-responsive polymers were prepared as follows. The 5% PVA solution (100 ml) from Example 29 was placed in each of three 500 ml glass jars and labeled with jars 1, 2 and 3. To each of the three jars, 4 mol NaOH (100 g) was added with stirring. Then, 1-propanol (50 g) was added to each jar as an auxiliary solvent, the solution was stirred, and then 0.74 g, 1.47 g and 2.94 g of benzyl chloride were added to jars 1, 2 and 3, respectively. Three jars were heated at 60 ° C for 16 hours. The volume of the reaction solution was adjusted to 500 ml with deionized water, the pH was adjusted to 8 with 25% HCl, and sodium 2 molar phosphate (100 g) was added to remove each of the resulting polymers from the individual reaction solutions. Isolated. After the phosphate ion was added, the solid precipitate was collected by vacuum filtration. The solids from each reaction were individually washed with deionized water and dissolved in 1 mol acetic acid (300 ml). Adjust individual solutions to pH 7.4, 2 mol The polymer was precipitated by adding sodium phosphate drop by drop, filtering the resulting solid, washing the solid sequentially with water and isopropyl alcohol, and then drying in a vacuum oven at 65 ° C for 2 days to give the polymer. Further purified. Each sample of the dry polymer was frozen in liquid nitrogen and ground into a fine powder. The recovered dry polymer mass was 1.44, 2.12 and 2.47 g for each of jars 1, 2 and 3, respectively. Each of the individual polymers was dissolved in 1 mol acetic acid to give a 2% solution.</p><p num="0150">[Example 31]<u style="single">Deprotection of amines by hydrolysis of ultra high molecular weight poly (N-vinylacetamide) for the production of polyvinylamine (PVA) stimulus responsive polymers</u> In another exemplary experimental example, an ultrahigh molecular weight stimulus responsive polymer was produced as follows. Ultra high molecular weight polymer usually refers to a polymer having a molecular weight of 1000 KDa or more.</p><p num="0151"> A stimulus-responsive polymer composed of repetitive units containing a primary amine was prepared as follows. In a 2 L glass jar, poly (N-vinylacetamide) linear homopolymer (POLYSCIENCES, INC.) (40 g) having an average molecular weight of 4,060 kDa was dissolved in deionized water (0.8 L) with vigorous stirring for 16 hours. did. Concentrated HCl (140 g) was added to this solution over 1 hour with continuous stirring. The jar was lightly covered and the solution was heated to 99 ° C for 5 days with intermittent rotation to mix the solution. After heating for 5 days, the solution was cooled to room temperature and the total volume was adjusted to 4 liters with deionized water. The solution was adjusted to pH 7 with 8 mol NaOH with vigorous stirring. The hydrolyzed product was precipitated with the addition of 1 drop of sodium 2 molars until no further precipitation was observed. The white precipitate was washed with deionized water and pressed to remove excess water. The recovered polymer was dried in a vacuum oven at 65 ° C. for 2 days. The dried polymer was frozen in liquid nitrogen and pulverized to a fine powder. The collected dry mass weighed 42.5 g. 1 mol of dry powder A 2% solution was prepared by dissolving in acetic acid and 0.08% HCl. The resulting solution was compared to a 2% solution of the starting material (poly (N-vinylacetamide) linear homopolymer) for response to phosphate or citrate stimulation. This was done by adding drop by drop of sodium 2 mol or sodium 0.2 mol citrate to a 50 ml sample of the starting material and the resulting hydrolyzed polymer.</p><p num="0152"> The resulting hydrolyzed polymer precipitated into a white mass by the addition of phosphate or citrate ions, whereas the starting material solution precipitated even with the addition of phosphate or citrate ions one drop at a time. Did not have. This allows the starting material to be non-responsive to stimuli (eg, polyvalent anions such as phosphates or citrates), but a super high molecular weight polymer synthesized as described in this example. Shows that it is responsive to stimuli (eg, polyvalent anions such as phosphates or citrates).</p><p num="0153"> FIG. 5 shows a method for deprotecting a polyamine polymer to form a stimulus-responsive polyvinylamine (PVA).</p><p num="0154">[Example 32]<u style="single">Synthesis of hydrophobically modified polyvinylamine (PVA) stimulus-responsive polymer based on deprotected poly (N-vinylacetamide)</u> Using PVA obtained from the deprotected 4,060 kDa poly (N-vinylacetamide) linear homopolymer from Example 31, an ultrahigh molecular weight hydrophobically modified stimulus responsive polymer was prepared as follows.</p><p num="0155"> A 2% solution (100 g) of deprotected 4,060 kDa poly (N-vinylacetamide) was placed in a glass jar. 4 mol NaOH (100 g) was added to this glass jar to adjust the pH to about 13. 1-Propanol (20 g) was then added to the jar as an auxiliary solvent. Finally, benzyl chloride (0.58 g) was added and the jar was covered. The reaction was heated at 60 ° C. for 3 hours with vigorous stirring. After 3 hours, the reaction mixture was cooled to room temperature and the product was precipitated with acetone before collection. The resulting solid was washed successively with deionized water and isopropyl alcohol, and dried in a vacuum oven at 65 ° C. for 2 days. The dried solid was ground to a fine powder and the final dried mass of the collected polymer was 1.44 g. A 2% solution was prepared by dissolving the dry powder in 1 mol acetic acid and 0.08% HCl. 2 mol Sodium phosphate or 0.2 mol in 5 mL sample of 0.5% polymer solution The susceptibility of the solution produced by adding sodium citrate salt drop by drop to polyvalent ion stimulation was tested. A white precipitate was observed when phosphate or citrate ions were added. This indicates that the polymer was responsive to multivalent anion stimulation.</p><p num="0156"> FIG. 6 shows an outline of the synthesis method described in this example.</p><p num="0157">[Example 33]<u style="single">Synthesis of stimulus-responsive vinyl amine / vinyl butyl ether copolymer (VA-co-VBE)</u> In another experiment, a stimulus-responsive copolymer was produced in which one of the monomeric units contained a hydrophobic group.</p><p num="0158"> A stimulus-responsive polymer composed of repeating units containing primary amines and butyl ether was synthesized from the monomers as follows. Octane (90g), Span-85 (SIGMA) (2.5g), N-vinylformamide (NVF) (ALDRICH, 98%) (16g), N-butyl vinyl ether (SIGMA) (5g) and deionized water (30g) Was placed in a 250 ml round bottom flask. Magnetic stirrer and N in flask₂Equipped with a dipstick. Stir the solution and the temperature will rise to 55 ° C so N₂Was purged for 1 hour.</p><p num="0159"> An initiator solution was prepared and dissolved by adding 2,2'-azobis (2-amidinopropane) dihydrochloride (ABAP) (ALDRICH) (0.10 g) in deionized water (10 ml). The initiator solution was filled in a 250 ml round bottom flask containing the reaction solution in a nitrogen-purged atmosphere. The solution was heated to 55 ° C for 1 hour, then at 60 ° C for 1 hour, then at 70 ° C for 1 hour, and then at 80 ° C for 1 hour, with vigorous stirring and continuous nitrogen purging. Heated for hours. This resulted in a two-phase solution with a viscous gel layer on the bottom. The upper layer was decanted and discarded. Deionized water (200 ml) was added to the lower layer, and 50% NaOH (20 g) was added with vigorous stirring. The solution was heated at 80 ° C for 6 hours. After 6 hours, the solution was removed from heat and cooled to room temperature. The volume was increased to 2 L with deionized water. 2 mol Isolation of the product by adding sodium phosphate drop by drop resulted in a large white precipitate. The precipitate was collected by decanting the supernatant and the precipitate was washed with deionized water. The isolated polymer was dissolved in deionized water (500 ml), acetic acid (10 g) and concentrated HCl (2 g). The susceptibility of the resulting solution to polyvalent ion stimulation was tested by adding one drop of sodium 2 molar or sodium 0.2 molar citrate to a 5 mL sample of 0.5% polymer solution. A white precipitate was observed when phosphate or citrate ions were added. This indicates that the polymer was responsive to multivalent anion stimulation.</p><p num="0160"> FIG. 7 outlines the reactions described in this example. This example shows that copolymers containing amines or charged functional groups copolymerized with hydrophobic monomers are responsive to polyvalent ion stimuli.</p><p num="0161">[Example 34]<u style="single">Synthesis of ultra-high molecular weight polyvinylamine (PVA) stimulus-responsive polymers from NVF monomers by reverse emulsion polymerization</u> A stimulus-responsive polymer composed of repeating units containing a primary amine was synthesized from the monomer as follows. Octane (90 g), Span-85 (SIGMA) (2.5 g), N-vinylformamide (NVF) (ALDRICH, 98%) (16 g) and deionized water (30 g) were placed in a 250 ml round bottom flask. Magnetic stirrer and N in flask₂Equipped with a dipstick. The solution was agitated and the temperature rose to 55 ° C, so N₂Was purged for 1 hour. An initiator solution was prepared by adding 2,2'-azobis (2-amidinopropane) dihydrochloride (ABAP) (ALDRICH) (0.20 g) in deionized water (20 ml) and dissolving it. The initiator solution was filled in a 250 ml round bottom flask containing the reaction solution in a nitrogen-purged atmosphere. The solution was heated to 60 ° C. for 2 hours and then to 75 ° C. for 1 hour with vigorous stirring and continuous purging of nitrogen. A two-phase solution with a viscous gel layer on the bottom was produced. The upper layer was decanted and discarded. Deionized water (500 ml) was added to the lower layer, and 50% NaOH (48 g) was added with vigorous stirring. The solution was heated to 80 ° C for 16 hours. After 16 hours, the solution was removed from heat and cooled to room temperature. The volume was increased to 1 L with deionized water. 2 mol Isolation of the product by adding sodium phosphate drop by drop resulted in a large white precipitate. The precipitate was collected by decanting the supernatant, the precipitate was washed with deionized water, immersed in isopropyl alcohol for 2 hours and finally washed again with deionized water. The resulting solid mass was dried in a vacuum oven at 65 ° C. for 3 days. The dried polymer was frozen in liquid nitrogen, pulverized to a fine powder, and dried for another day. The resulting dry powder mass was 21.5 g. A 2% solution was prepared by dissolving the dry powder in 1 mol acetic acid and 0.08% HCl. The susceptibility of the resulting solution to polyvalent ion stimulation was tested by adding one drop of sodium 2 mol or sodium 0.2 mol citrate to a 50 mL sample of 1% polymer solution. A white precipitate was observed when phosphate or citrate ions were added.</p><p num="0162">[Example 35]<u style="single">Synthesis of Ultra High Molecular Weight Hydrophobic Modified Polyvinyl Amine (PVA) Stimulus Responsive Polymers</u> Using a solution of Lupamin 9095 (linear polyvinylamine, average MW = 340 kDa, 20% solid, pH 7-9) obtained from BASF, a hydrophobically modified stimulus-responsive polymer was prepared as follows. Lupamin 9095 (about 60 g of polyvinylamine) (300 g) was added to a 2 L glass jar. The NaOH pellets (40 g) and deionized water (500 ml) were then dissolved and added to the jar. After this, 1,2-dimethoxyethane (SIGMA) (500 ml) was added as an auxiliary solvent, and the solution was vigorously stirred until it became homogeneous. Benzyl chloride (ACROS ORGANICS, 99%) (17.66 g) was then added to the reaction jar with stirring. The solution was heated to 60 ° C. for 16 hours with magnetic agitation. The solution was then cooled to room temperature and transferred to a 5 L beaker. Then, deionized water (1500 ml) was added with stirring. The pH was adjusted to 5 with glacial acetic acid. The product was precipitated by the slow addition of 2 molar sodium phosphate (250 ml), the solid was collected and washed with deionized water.</p><p num="0163"> The polymer was further purified by the following method. The solid was dissolved in 1 mol acetic acid (2 L) with stirring. The total volume was adjusted to 10 L with deionized water, and the pH was adjusted to 7 by adding 50% NaOH drop by drop. 2 Mole sodium phosphate (600 g) was added to precipitate the product. The solid was isolated by vacuum filtration and washed with deionized water. The resulting solid mass was dried in a vacuum oven at 65 ° C. for 3 days. The dried polymer was frozen in liquid nitrogen, ground to a fine powder, and dried for another day. The resulting dry powder mass weighed 46 g. 1 mol CD of small sample₃COOD / D₂Dissolved in O acid,¹The H-NMR spectrum was obtained. This spectrum is shown in FIG.¹The H-NMR peak was integrated and the amount of benzyl modification was found to be 18%.</p><p num="0164">[Example 36]<u style="single">Synthesis of 200g scale of stimulus-responsive polymer containing hydrophobically modified polyallylamine as a main component</u> The exemplary experiments described herein have demonstrated that the polymers described herein can be produced on a large scale.</p><p num="0165"> Using a solution of polyallylamine (PAA, NITTOBO, 150kD; 40% wt / wt.), A hydrophobically modified stimulus-responsive polymer was prepared as follows. Polyallylamine (PAA, NITTOBO, 150 kD; 40% wt./wt.) (500 g) (about 200 g of polyallylamine) was added to a 4 L glass jar. The NaOH pellets (80 g) and deionized water (1000 ml) were then dissolved and added to the jar. After this, 1,2-dimethoxyethane (SIGMA) (1000 ml) was added as an auxiliary solvent, and the solution was vigorously stirred until it became homogeneous. Benzyl chloride (ACROS ORGANICS, 99%) (114 g) was then added to the reaction jar. The solution was heated at 60 ° C. for 16 hours with magnetic agitation. The solution was then cooled to room temperature and transferred to a 10 L beaker.</p><p num="0166"> Deionized water (1000 ml) was then added with stirring to precipitate a viscous solid mass from the solution. The product was further precipitated by the slow addition of 2 molar sodium phosphate (200 ml), the solid was collected and washed with deionized water. The polymer was further purified by the following method. The solid was dissolved in 1 mol acetic acid (3 L) with stirring. The total volume was adjusted to 10 L with deionized water, and the pH was adjusted to 7 by adding 50% NaOH drop by drop. The product was precipitated by adding sodium 2 morphophosphate (800 g), the solid was collected and washed with deionized water. The polymer was further purified by the following method. The solid was dissolved in 1 mol acetic acid (3 L) with stirring. The total volume was adjusted to 10 L with deionized water, and the pH was adjusted to 7 by adding 50% NaOH drop by drop. The product was precipitated by adding sodium 2 morphophosphate (800 g), the solid was collected and washed with deionized water. The resulting solid mass was dried in a vacuum oven at 65 ° C. for 3 days. The dried polymer was frozen in liquid nitrogen, ground to a fine powder, and dried for another day. The resulting dry powder mass was 250 g. 1 mol of small sample CD₃COOD / D₂It dissolves in O acid and is shown in FIG.¹The H-NMR spectrum was obtained.¹The H-NMR peak was integrated and the amount of benzyl modification was found to be 33%.</p><p num="0167">[Example 37]<u style="single">Measurement of aggregation performance and supernatant quality with increasing addition of stimulus-responsive polymer-to-cationic polymer electrolyte in CHO cell culture</u> In the exemplary experiments described herein, stimulus-responsive polymers according to the invention were compared to known polymers for some desired properties, namely chitosan.</p><p num="0168"> CHO cell cultures were prepared using the method described in Example 1. A solution of a 2% w / w solution of medium molecular weight chitosan (MMW chitosan) (Sigma-Aldrich) was prepared in 1 mol acetic acid. A 2% w / w solution of a stimulus-responsive (33% -BnPAA) polymer containing hydrophobically modified polyallylamine as the main component was prepared according to Example 36. CHO cell culture (10 ml) was dispensed into 15 ml conical tubes. For each LMW chitosan and 33% -BnPAA, 0.0, 0.1, 0.2, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.14, 0.18, 0.22 in each conical tube containing CHO cell culture. And 0.4% w / v added amount of polymer was added. Only for conical tubes containing 33% -BnPAA, the pH was adjusted to 7.2 and stimulation with 150 mM sodium phosphate was applied. All conical tubes were centrifuged at 3000 RPM for 2 minutes, the supernatant was decanted and the turbidity was measured.</p><p num="0169"> The results of representative experiments are summarized in Table 5 and FIG. 10 below, and non-stimulation-responsive polymers (eg, chitosan) need to be added in optimized amounts for efficient aggregation, but stimulation according to the present invention. It is clearly stated that the responsive polymer does not seem to require addition amount optimization. In other words, in the case of non-irritating responsive polymers such as chitosan, the optimum amount of polymer is added once for efficient aggregation, and the turbidity increases when the optimum amount is exceeded, which is not desirable. On the other hand, in the case of stimulus-responsive polymers as described herein, the stimulus-responsive polymer remains an effective flocculant / precipitant regardless of increased additions.</p><p num="0170"><tables num="5"><img id="000012" he="208" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0171">[Example 38]<u style="single">Measurement of aggregation performance and supernatant quality with increased addition of stimulus-responsive polymer in the presence of polyvalent anion stimulation in CHO cell cultures without counterstimulation</u> CHO cell cultures were prepared using the method described in Example 1. A 2% w / w solution of a stimulus-responsive (33% -BnPAA) polymer containing hydrophobically modified polyallylamine as the main component was prepared according to Example 36. For 33% -BnPAA polymer, add 0.0, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.14, 0.18, 0.22 and 0.4% w / v to each conical tube containing the CHO cell culture. Individual polymers were added in three ways (one phosphate, one citrate, one non-irritating). The pH was adjusted to 7.2 with or without stimulation with 150 mM sodium phosphate or 150 mM sodium citrate. All conical tubes were centrifuged at 3000 RPM for 2 minutes, the supernatant was decanted, and the centrifugation turbidity was measured. The results of the experiments described in this example are shown in Table 6 and FIG.</p><p num="0172"> The results of representative experiments are summarized in Table 6 and FIG. 11, and the stimulus-responsive polymer (eg, described in Example 36) is non-stimulus responsive by optimizing the amount of polymer added without irritation. It is clearly shown that it can act as a flocculant (similar to the chitosan data in Example 37). However, when a polyvalent ion stimulus such as phosphate or citrate is applied, the stimulus-responsive polymer needs to be optimized because the centrifugation turbidity is not affected even if the polymer addition amount is increased. do not.</p><p num="0173"><tables num="6"><img id="000013" he="213" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0174">[Example 39]<u style="single">Measurement of aggregation performance and supernatant quality with increasing addition of unmodified stimulus-responsive polymer in CHO cell culture</u> In another experiment, the ability to aggregate and supernatant quality in unmodified stimulus-responsive polymers in CHO cell cultures was measured as described herein.</p><p num="0175"> CHO cell cultures were prepared using the method described in Example 1. A 2% w / w solution of an unmodified polyallylamine-based stimulus-responsive polymer was prepared by dissolving polyallylamine (PAA, NITTOBO, 150kD; 40% wt./wt.) In 1 mol acetic acid. .. Distribute CHO cell culture (10 ml) into 15 ml conical tubes. Polymers added in 0.0, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.14, 0.18, 0.22 and 0.4% w / w were added to each conical tube containing the CHO cell culture. The pH was adjusted to 7.2 and stimulation with 150 mM sodium phosphate was applied. All conical tubes were centrifuged at 3000 RPM for 2 minutes, the supernatant was decanted and the turbidity was measured. The results of the experiments described in this example are shown in Table 7.</p><p num="0176"><tables num="7"><img id="000014" he="84" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0177">[Example 40]<u style="single">Measurement of stimulus-responsive polymer aggregation performance, sedimentation time and supernatant quality in CHO cell cultures</u> CHO cell cultures were prepared using the method described in Example 1. A 10% w / w solution of a stimulus-responsive (33% -BnPAA) polymer containing hydrophobically modified polyallylamine as a main component was prepared in the same manner as in Example 36. The CHO cell culture (50 ml) was placed in a 100 ml glass graduated cylinder in two ways. To one of the cylinders, a stimulus-responsive polymer was added in an amount of 0.5%, 2 mortris bases were added drop by drop to adjust the pH to 7, and a solution of sodium 2 morphophosphate was added. The sodium phosphate concentration was adjusted to 50 mM and the solution was stirred for 2 minutes. Complexes of stimulus-responsive polymers and cells, cell debris, impurities, and residual polymers were precipitated, solids aggregated, and sodium phosphate and pH adjustments were performed to increase particle size. After adding sodium phosphate and adjusting the pH, large aggregated particles were observed in the feed material treated with a stimulus-responsive polymer. The other graduated cylinder was agitated for 2 minutes and nothing was added. Both cylinders were left for 1 hour. After 1 hour, the supernatant was aspirated and the turbidity was recorded.</p><p num="0178"> Solids in cylinders with smart polymers settle faster than solids in untreated cylinders, which have a clear settling front and give an unclear settling front (sharp solid to liquid phase transition). Was observed.</p><p num="0179"> The results of one of the above experiments are summarized in Table 8. In the case of untreated cylinders, the settling fronts were largely scattered and unclear, so measurements on the settling fronts were rough estimates.</p><p num="0180"><tables num="8"><img id="000015" he="92" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0181">[Example 41]<u style="single">Comparison of clarification performance using polyamines with different molecular weights</u> A series of polymers with different molecular weights were obtained, modified and used to aggregate, precipitate and purify cell cultures. Polymers with primary amine repeating units with molecular weights of 15 kD, 85 kD, 150 kD, 350 kD, 600-950 kD and 2000-4000 kD were obtained and / or modified by the following methods.</p><p num="0182"> The 15kD polyallylamine polymer was obtained from NITTOBO and benzylated using the same method as in Example 36 (the benzyl group was covalently bonded and purified). The 85kD benzylated polyvinylamine polymer was prepared according to Example 23. 150 kD benzylated polyallylamine was prepared according to Example 36. The 350 kD benzylated polyvinylamine polymer was prepared according to Example 35. The 950 kD polyvinylamine polymer backbone was prepared by hydrolyzing Polymin VZ (BASF) with 2 equivalents of base at 80 ° C for 8 hours. Unmodified 2000-4000 kD PVA was prepared according to Example 31. The benzylated 2000-4000 kD polymer was prepared according to Example 32. The polymer is about 12x10⁶Cell / mL CHO DG44 cell cultures were used for aggregation, precipitation and purification and collected with &lt;50% cell viability. Aggregation was performed with polymer additions of 0.2% and 0.4% w / w. Solution stimulation of 50 mM sodium phosphate and pH adjustment to 7 with a 2 maltris base were applied.</p><p num="0183"> Observations on flock size were recorded, with + as small flock and +++++ as very large aggregates. Vials labeled +++++ were completely zoned rather than a suspension of agglomerates. It is also noted that large aggregates / particles with sharp solid-liquid interfaces settle faster. The results are shown in Table 9. This table shows the results of aggregation using polyamines of different molecular weights.</p><p num="0184"><tables num="9"><img id="000016" he="67" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0185">[Example 42]<u style="single">Comparison of clarification performance using polyamines with different hydrophobic modifications</u> CHO cell cultures were prepared using the method described in Example 1. Samples of the polymers described in Examples 31, 32, 33 and 35 were added to the cell cultures described in Example 38. However, the amount of polymer added was 0.1 wt% to 0.6 wt% as shown in Table 10. The initial cell culture turbidity was ~ 900 NTU and the turbidity of the centrifuge without polymer treatment was 212 NTU. The pH was adjusted to 7.2 and stimulation with 50 mM sodium phosphate was applied. All conical tubes were centrifuged at 3000 RPM for 2 minutes, the supernatant was decanted, and the centrifugation turbidity was measured. The results of the experiments described in this example are shown in Table 10. This table illustrates the performance of various hydrophobic modifications at different polymer additions.</p><p num="0186"> Table 10 demonstrates that varying the type of hydrophobic group and / or the molecular weight of the polymer can alter the response to stimuli and the resulting centrifugal turbidity.</p><p num="0187"><tables num="10"><img id="000017" he="103" wi="159" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0188">[Example 43]<u style="single">Effect of low turbidity on downstream filtration obtained with stimulus-responsive polymers</u> The following steps were performed to assess the effect of the stimulus-responsive polymer on subsequent centrifugation and depth filtration steps. Approximately 15 × 10 DG44 Chinese hamster ovary (CHO) cell lines expressing PTG1 antibody in a 10 L bioreactor (NEW BRUNSWICK SCIENTIFIC)⁶It was grown to a cell / mL density and collected at &lt;50% viability. A 10% w / w solution of a stimulus-responsive (33% -BnPAA) polymer containing hydrophobically modified polyallylamine as a main component is prepared in the same manner as in Example 36.</p><p num="0189"> One fraction of the DG44 CHO cell culture is treated with 0.2% stimulus-responsive polymer 33% -BnPAA, and another fraction of the DG44 CHO cell culture is not treated. To cell culture treated with a stimulus-responsive polymer, the sodium phosphate concentration was increased to 50 mM by adding 1 drop of sodium 2 morpholate, and the pH was increased to 7.2 by adding 1 drop of 2 maltris base. Adjust. Sodium phosphate and pH regulation are performed to precipitate a complex of stimulus-responsive polymers and cells, cell debris, impurities, residual polymers, to aggregate solids and increase particle size. After sodium phosphate addition and pH regulation, large floc-like particles are observed in the feed material treated with the stimulus-responsive polymer. Both fractions were centrifuged at 3000 RPM for 5 minutes, the supernatant was decanted and the turbidity was measured. The centrifuge turbidity for the feed material treated with 33% -BnPAA polymer was measured to be 10 NTU and the centrifuge turbidity for the untreated feedstock was measured to be 60 NTU.</p><p num="0190"> The depth filter treatment amount for each feed material was measured by the following method. 23 cm for each material supplied²A X0HC Millistak +® Pod Disposable Depth Filter (MILLIPORE) with a surface area of is used. The depth filter was equipped with a peristaltic pump and an in-line pressure sensor. Deionized water was flowed through the filter according to the instructions, the feedstock was pumped at 100 LMH and the filtrate was pooled. The pooled turbidity for the feed material treated with 33% -BnPAA polymer was 6 NTU and the pooled turbidity for the filtrate of the untreated feed material was 9 NTU. Filtered amount for feedstock treated with 33% -BnPAA polymer is 1304 L / m at 10 psi²At this point, the experiment was stopped due to supply limits. Filtering amount for untreated feed material is 206 L / m at 20 psi²At this point, the experiment was stopped due to the pressure limit.</p><p num="0191">[Example 44]<u style="single">Purification with a stimulus-responsive polymer of the model protein stream followed by capture with an affinity resin</u> To better assess the effect of the stimulus-responsive polymer on subsequent purification steps, model feed materials are created and the following steps are performed. A CHO cell culture is prepared in Example 1 using the same method. The initial feed material HCP level was ~ 210,000 ppm. A 10% w / w solution of a stimulus-responsive (33% -BnPAA) polymer containing hydrophobically modified polyallylamine as a main component is prepared in the same manner as in Example 36. One fraction of the CHO cell culture was treated with 0.1% stimulus-responsive polymer 33% -BnPAA and another fraction of the CHO cell culture was treated with 0.4% stimulus-responsive polymer 33% -BnPAA. The third fraction of the CHO cell culture is not processed. To the cell culture treated with the stimulus-responsive polymer, 2 mol of sodium phosphate was added drop by drop to bring the sodium phosphate concentration to 50 mM, and 2 mol. Adjust the pH to 7.2 by adding tris bases drop by drop. Sodium phosphate and pH regulation are performed to precipitate a complex of stimulus-responsive polymers and cells, cell debris, impurities, residual polymers, to aggregate solids and increase particle size. After sodium phosphate addition and pH regulation, large floc-like particles are observed in the feed material treated with the stimulus-responsive polymer. Each fraction of cell culture is centrifuged at 3000 RPM for 5 minutes using a laboratory-scale bucket centrifuge. The turbidity of each centrifuge is recorded and reported in Table 11. The centrifuge is filtered through a 0.2 μm Durapore® filter.</p><p num="0192"> The filtrate pool was subjected to Protin A Affinity Chromatography (ProSep Ultra Plus®), Binding-Eluent Cationic Exchange Chromatography (ProRes S®), and Membrane Adsorbent Anion Exchange Chromatography in Passage Mode (ChromaSorb Purified by purification based on 3-step chromatography composed of registered trademark)). Purification was performed on a chromatography workstation according to the method in Table 12. Step-by-step pools by ELISA for host cell protein (CHOP), ELISA for exuded protein A (L ProA), PicoGreen® assay for residual DNA, turbidity, aggregation protein (AGG) % Was analyzed by size exclusion ELISA and protein concentration was analyzed by UV absorption.</p><p num="0193"><tables num="11"><img id="000018" he="214" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0194"><tables num="12"><img id="000019" he="243" wi="144" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0195">[Example 45]<u style="single">Purification with a stimulus-responsive polymer of the model protein stream followed by capture with a cation exchange resin</u> To better assess the effect of the stimulus-responsive polymer on subsequent purification steps, the following procedure was performed. Approximately 15 x 10 DG44 Chinese Hamster Ovary (CHO) cell lines expressing PTG1 antibody in a 10 L bioreactor (New Brunswick Scientific)⁶It was grown to a cell / mL density and collected at &lt;50% viability at HCP levels of ~ 142000 ppm.</p><p num="0196"> A 10% w / w solution of a stimulus-responsive (33% -BnPAA) polymer containing hydrophobically modified polyallylamine as a main component was prepared in the same manner as in Example 36. One fraction of the CHO cell culture was treated with 0.1% stimulus-responsive polymer 33% -BnPAA and another fraction of the CHO cell culture was treated with 0.4% stimulus-responsive polymer 33% -BnPAA. The third fraction of the CHO cell culture was not treated. To cell culture treated with a stimulus-responsive polymer, the sodium phosphate concentration was increased to 50 mM by adding 1 drop of sodium 2 morpholate, and the pH was increased to 7.2 by adding 1 drop of 2 maltris base. Adjusted. Sodium phosphate and pH regulation were performed to precipitate a complex of stimulus-responsive polymers and cells, cell debris, impurities, and residual polymers to aggregate the solids and increase their particle size. After sodium phosphate addition and pH adjustment, large aggregated particles were observed in the feed material treated with the stimulus-responsive polymer. Centrifuge each fraction of cell culture at 3000 RPM for 5 minutes in a laboratory-scale bucket centrifuge. The turbidity of each centrifuge is recorded and reported in Table 13. Centrifugal solution 0.2 / μm Filtered through a Durapore® filter.</p><p num="0197"> Purification of the filtrate pool based on two-step chromatography consisting of binding-elution cation exchange chromatography (ProRes S®) and membrane adsorbent anion exchange chromatography in transit mode (ChromaSorb®). Purified by Purification was performed on a chromatography workstation according to the method in Table 12. Step-by-step pools by ELISA for host cell protein (CHOP), ELISA for exuded protein A (L ProA), PicoGreen® assay for residual DNA, turbidity, aggregation protein (AGG) % Was analyzed by size exclusion ELISA and protein concentration was analyzed by UV absorption.</p><p num="0198"><tables num="13"><img id="000020" he="152" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0199">[Example 46]<u style="single">Polyethylene membrane surface modified with 2-hydroxyethyl methacrylate (PAHEMA) phosphate</u> In another experiment, the membrane was modified to incorporate multivalent ion stimuli. This membrane can be used to remove residual stimulus responsive polymers A 16% aqueous mixture of PAHEMA was prepared using PAHEMA (Aldrich # 695890,75% PAHEMA and 25% BisHEMPA) (16 g), Irgacure 2959 (0.2 g) and water (93.8 g). Polyethylene membranes (0.65um, UPDP MILLIPORE) were pre-moistened with methanol, replaced with water and treated with PAHEMA preparation. The sample was exposed to UV light, washed with methanol and water and dried. The weight added to the film by this surface modification was 4.4%. The infrared spectrum of the membrane showed strong methacrylate carbonyl absorption. Staining the film with methylene blue (a positively charged dye) produced a deep blue color with a cyan optical density of 1.43.</p><p num="0200">[Example 47]<u style="single">Hydrophilic polyethylene membrane surface modified with 2-hydroxyethyl methacrylate (PAHEMA) phosphate</u> A 16% aqueous mixture of PAHEMA was prepared using PAHEMA (Aldrich # 695890,75% PAHEMA and 25% BisHEMPA) (16 g), Irgacure 2959 (0.2 g) and water (93.8 g). The hydrophilic membrane (0.65um, MPLC MILLIPORE) was brought into direct contact with the PAHEMA solution. When exposed to UV light and washed as described above, 7.6% was added to the membrane. The infrared spectrum of the membrane showed strong methacrylate carbonyl absorption. Staining the film with methylene blue (a positively charged dye) produced a deep blue color with a cyan optical density of 1.45.</p><p num="0201">[Example 48]<u style="single">Hydrophilic polymethacrylate resin modified with hydroxyethyl methacrylate (PAHEMA) phosphate</u> In another experiment, the resin was modified to include irritation. This modified resin can then be used to remove the residual polymer.</p><p num="0202"> A solution having a composition of allyl glycidyl ether (AGE) (60 ml), 4M NaOH (110 g) and sodium sulfate (12 g) is prepared. Toyo Pearl 65C medium (60 ml) is added to this solution and the mixture is placed in a rotary hybridizer at 50 ° C. for 16 hours. The medium is separated and washed according to standard procedures. A PAHEMA grafted solution of PAHEMA (1.0 g), ammonium persulfate (0.06 g) and water (9.0 g) is prepared. Add AGE-modified Toyo Pearl medium (5 ml) to this solution. Place the mixture in the hybridizer at 80 ° C for 16 hours. Separated and washed according to standard procedures to give a PAHEMA modified resin. Treatment of this product with a 0.01% aqueous solution of the positively charged dye methylene blue results in a dark blue stain.</p><p num="0203">[Example 49]<u style="single">Binding of stimulus-responsive polymer to hydrophilic polyethylene membrane surface modified with 2-hydroxyethyl methacrylate (PAHEMA) phosphate</u> Using a PAHEMA-modified MPLC membrane (Example 47), polyallylamine (PAA) polymers were captured from three solutions containing 100, 10 and 1 ppm PAA, respectively, using the following experimental procedure. A 20 mm diameter membrane disc was processed in a 15 cc cell holder. The PAA solution was processed at 1.5 cc / sec. The membrane was washed inside and outside the cell holder with PBS buffer (100 mL). The processed membrane was stained with Ponceau S.</p><p num="0204"> Ponceau S is a negatively charged dye that strongly adsorbs to positively charged surfaces. When PAA was adsorbed on the PAHEMA modified membrane, the surface changed from negative charge to positive charge. This change is easily observed by staining the processing solution with Ponceau S. A good measure of staining with Ponceau S is the magenta optical density measured with a Macbeth densitometer. Table 14 shows the magenta optical density values for various processed films both upstream and downstream (ie, filled with PAA).</p><p num="0205"> All of the PAA is trapped upstream of the membrane, as seen at 1 ppm. For a 15 mL processing solution, this is 4.8 μg PAA / cm²-Corresponds to the film surface.<tables num="14"><img id="000021" he="41" wi="158" file="JP6055893B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0206"> Therefore, based on the results of this example, the modified membranes and resins described herein can be used to reduce the level of residual polymer or to completely remove the residual polymer. We can conclude.</p><p num="0207"> The specification is best understood in the light of the teachings of the literature cited herein herein by reference. The embodiments in the specification give an explanation of the embodiments of the present invention and should not be construed as limiting the scope of the present invention. Those skilled in the art will readily recognize that many other embodiments are included in the present invention. All publications and patent documents are incorporated by full-text reference. As long as the material incorporated by reference denies or contradicts this specification, this specification supersedes the above material. References to the literature herein are not tolerated as prior art to the present invention.</p><p num="0208"> Unless otherwise specified, all numbers representing the amounts of ingredients, cell cultures, treatment conditions, etc. used in the specification, including claims, are all modified by the term "about". Should be understood. Therefore, unless otherwise indicated, the numerical parameters are approximate and may vary depending on the desired properties sought to be obtained by the present invention. Unless otherwise indicated, it should be understood that the term "at least" before a set of elements refers to any element in the set. One of ordinary skill in the art is aware of many equivalents to the specific embodiments of the invention described herein and can only be confirmed using routine experiments. These equivalents are intended to be covered by the claims below.</p><p num="0209"> As will be appreciated by those skilled in the art, many modifications and modifications of the present invention may be made without departing from their spirit and scope. The specific embodiments described herein are presented by way of example only and are by no means limited. The specification and examples are considered as examples only, and the true scope and gist of the present invention is intended to be set forth in the following claims.</p>