Methods for the production and purification of multivalent immunoglobulin single variable domains

Abstract

To provide a method for producing, purifying, and isolating a multivalent polypeptide comprising at least three or at least four ISVDs, wherein the method reduces or eliminates conformational variants associated with the product.SOLUTION: There is provided a method of isolating or purifying a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs) from a composition comprising the polypeptide and conformational variants thereof, comprising: a) applying conditions to convert the conformational variants into the polypeptide; b) removing the conformational variants; or c) a combination of a) and b).SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. Technical Field The present application relates to the field of immunoglobulin single variable domain (ISVD) production and purification.

[0002] The present application provides methods for producing polypeptides comprising at least three or at least four ISVDs. More specifically, improved methods are provided for producing, purifying, and isolating polypeptides comprising at least three or at least four ISVDs, wherein product-associated conformational variants are reduced or absent. Polypeptides comprising at least three or at least four ISVDs produced / purified according to the present methods have superior product homogeneity due to the reduced or absent product-associated conformational variants. This is beneficial, for example, in the context of therapeutic applications of polypeptides comprising at least three or at least four ISVDs. Thus, the present methods provide for the production of homogeneous polypeptides comprising at least three or at least four ISVDs, where increased homogeneity and / or potency can be obtained. Accordingly, the present application also describes improved compositions for therapeutic applications comprising polypeptides comprising at least three or at least four ISVDs obtainable by the methods of the present invention. [Background technology]

[0003] 2 Background technology For therapeutic applications, immunoglobulins must be of very high product quality. This requires, among other things, structural homogeneity. Furthermore, production costs are strongly influenced by difficulties encountered during the production process. Low yields or lack of homogeneity affect the economics of the production process and, therefore, the overall cost of the therapy. For example, difficulties in separating structural variants of a desired protein from the desired protein may require complex and expensive purification strategies.

[0004] Among other requirements, therapeutic proteins must be fully functional. Protein function depends on, among other factors, the chemical and physical stability of the protein during fermentation, purification, and storage. Chemical instability can be caused by, among other things, deamidation, isomerization, racemization, hydrolysis, oxidation, pyroglutamate formation, carbamylation, beta-elimination, and / or disulfide exchange. Physical instability can be caused by antibody denaturation, aggregation, precipitation, or adsorption. Among them, aggregation, deamidation, and oxidation are known to be the most common causes of antibody degradation (Non-Patent Document 1).

[0005] Limitations in obtaining adequate yields of functional product have been reported for conventional immunoglobulins and their fragments across a wide range of expression systems, including, inter alia, in vitro translation, Escherichia coli, Saccharomyces cerevisiae, Chinese hamster ovary cells, baculovirus systems in insect cells, and Pichia pastoris (Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13).

[0006] In contrast to these observed difficulties, immunoglobulin single variable domains (ISVDs) can be successfully expressed in a variety of host cells, including prokaryotes such as E. coli, lower eukaryotes such as P. pastoris, or higher eukaryotes such as CHO cells. They can be easily expressed in a fully functional form at reasonable rates and levels. As described, for example, in U.S. Patent No. 5,999,949, biopharmaceutical production of ISVDs in higher eukaryotes, such as mammalian cells (e.g., CHO cells), often requires viral clearance / inactivation in downstream purification processes by low pH treatment. In lower eukaryotes, such as yeast, viral inactivation is not an issue. Immunoglobulin single variable domains are characterized by the formation of an antigen-binding site by a single variable domain, which does not require interaction with additional domains (e.g., in the form of VH / VL interactions) for antigen recognition. The production of NANOBODY® ISVDs, as one specific example of immunoglobulin single variable domains, has been extensively described, for example, in U.S. Patent No. 5,999,949.

[0007] Despite these supposed advantages, problems have been reported in producing structurally uniform ISVD products. For example, Patent Document 3 shows that the production of ISVDs can involve product-related variants lacking at least one disulfide bridge. Furthermore, Patent Document 4 describes the existence of structural variants of produced ISVDs containing at least one carbamylated amino acid residue. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO 2010 / 056550 [Patent Document 2] WO 94 / 25591 [Patent Document 3] WO 2010 / 125187 [Patent Document 4] WO2012 / 05600 [Non-patent literature]

[0009] [Non-Patent Document 1] Cleland et al., 1993, Critical Reviews in Therapeutic Drug Carrier Systems 10:307-377 [Non-patent document 2] Ryabova et al., Nature Biotechnology 15:79, 1997 [Non-patent document 3] Humphreys et al., FEES Letters 380:194, 1996 [Non-patent document 4] Shusta et al., Nature Biotech. 16:773, 1998 [Non-patent document 5] Hsu et al., Protein Expr. & Purif. 7:281, 1996 [Non-patent document 6] Mohan et al., Biotechnol. & Bioeng.98:611, 2007 [Non-Patent Document 7] Xu et al., Metabol.Engineer.7:269, 2005 [Non-patent document 8] Merk et al., J. Biochem. 125:328, 1999 [Non-Patent Document 9] Whiteley et al., J. Biol. Chem. 272:22556, 1997 [Non-Patent Document 10] Gasser et al., Biotechnol.Bioeng.94:353, 2006 [Non-Patent Document 11] Demarest and Glaser, Curr. Opin. Drug Discov. Devel. 11(5):675-87, 2008 [Non-Patent Document 12] Honegger, Handb.Exp.Pharmacol.181:47-68, 2008 [Non-Patent Document 13] Wang et al., J.Pharm.Sci. 96(1):1-26, 2007 Summary of the Invention [Problem to be solved by the invention]

[0010] However, the more specific problem of obtaining a structurally homogeneous functional ISVD product containing at least three or at least four ISVDs has not been reported. [Means for solving the problem]

[0011] 3. Summary Product-associated conformational variants have been observed during the production process of multivalent polypeptide products comprising at least three or at least four ISVDs. The product-associated conformational variants have been observed during the production of multivalent polypeptide products comprising at least three or at least four ISVDs in a host, particularly a lower eukaryotic host such as yeast. It has been found that conformational variants of multivalent polypeptide products comprising at least three or at least four ISVDs result from the expression of the polypeptide in a host, particularly a lower eukaryotic host such as yeast. The inventors have been able to identify product-associated conformational variants by specific analytical chromatographic techniques, such as analytical SE-HPLC and / or analytical IEX-HPLC, as provided herein. This technique relates to methods for producing, purifying, and isolating multivalent polypeptides comprising at least three or at least four ISVDs, characterized by a reduction or absence of product-associated conformational variants.

[0012] The present application provides a method for isolating or purifying a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs) from a composition comprising said polypeptide and conformational variants thereof, said method comprising: a) applying conditions that convert conformational variants into said (desired) polypeptide; b) removing conformational variants; or c) A combination of (a) and (b) Includes:

[0013] The polypeptide to be isolated / purified by the method provided herein can be obtained by expression in a host. The polypeptide to be isolated / purified by the method provided herein can be obtained by expression in a host other than a CHO cell. The polypeptide to be isolated / purified by the method provided herein can be obtained by expression in a lower eukaryotic host such as yeast. Conformational variants result from the expression of the polypeptide in a host, particularly a host that is a lower eukaryotic host such as yeast. Without limitation, the yeast can be Pichia (Komagataella), Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis. In one embodiment, the polypeptide to be isolated / purified by the methods provided herein can be obtained by expression in Pichia, particularly Pichia pastoris.

[0014] In one embodiment, the percentage (%) of conformational variants in the composition is reduced to 5% or less. The percentage (%) of variants may be reduced to 4% or less, 3% or less, 2% or less, 1% or less, such as 0.5%, 0.1% or even 0% conformational variants.

[0015] The conformational variants to be converted and / or removed by the methods described herein are characterized by a more compact morphology. The conformational variants to be converted or removed by the methods described herein are also characterized by a reduced hydrodynamic volume. The compact morphology of the conformational variants may be due to a reduced hydrodynamic volume. The conformational variants may also be characterized by an altered surface charge and / or surface hydrophobicity. Thus, the conformational variants may be characterized by a reduced hydrodynamic volume, an altered surface charge, and / or an altered surface hydrophobicity. Without being bound by any hypothesis, the conformational variants to be converted and / or removed by the methods described herein may be characterized by weaker intramolecular interactions between ISVD building blocks present in the polypeptide, which may result in the reduced hydrodynamic volume, altered surface charge, and / or altered surface hydrophobicity of the conformational variant compared to the (desired) polypeptide.

[0016] Due to the differences in the above biophysical parameters, the conformational variants to be converted and / or removed by the methods provided herein are identifiable by chromatographic techniques, such as analytical SE-HPLC and / or analytical IEX-HPLC. Thus, in one embodiment, the conformational variants to be converted and / or removed by the methods provided herein are characterized by an increased retention time in SE-HPLC compared to the polypeptide. In another embodiment, the conformational variants are characterized by an altered retention time in IEX-HPLC compared to the polypeptide. In yet another embodiment, the conformational variants are characterized by an increased retention time in SE-HPLC and an altered retention time in IEX-HPLC compared to the polypeptide.

[0017] In one embodiment, the conformational variant is converted into said polypeptide by applying appropriate conditions, wherein the conditions that convert the conformational variant into said polypeptide are: i) applying a low pH treatment in the isolation and / or purification process steps; ii) applying chaotropic agents in steps of the isolation and / or purification method; iii) applying heat stress during isolation and / or purification process steps; or iv) Any combination of i) to iii) is selected from.

[0018] The low pH treatment to convert the conformational variant to the polypeptide comprises lowering the pH of the composition containing the conformational variant to about pH 3.2 or lower, or about pH 3.0 or lower. In one embodiment, the pH is lowered to between about pH 3.2 and about pH 2.1, between about pH 3.0 and about pH 2.1, between about pH 2.9 and about pH 2.1, between about pH 2.7 and about pH 2.1, or between about pH 2.6 and about pH 2.3. The pH treatment is applied for a sufficient amount of time to convert the conformational variant to the polypeptide. In light of the teachings provided herein, one skilled in the art will recognize that conversion of the conformational variant to the polypeptide will increase over time. However, practically useful levels of conversion of the conformational variant to the polypeptide are already achieved after at least 0.5 hours, e.g., at least about 1 hour, of low pH treatment. Thus, in one embodiment, the low pH treatment is applied for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours. In certain embodiments, pH In another specific embodiment, the pH is lowered to between about pH 3.2 and about pH 2.1, for example, to about pH 3.2, 3.0, 2.9, 2.7, 2.5, 2.3, or 2.1. In another specific embodiment, the pH is lowered to between about pH 3.0 and about pH 2.1, for example, to about pH 3.0, 2.9, 2.7, 2.5, 2.3, or 2.1. In another specific embodiment, the pH is lowered to between about pH 2.9 and about pH 2.1, for example, to about pH 2.9, 2.7, 2.5, 2.3, or 2.1. In another specific embodiment, the pH is lowered to between about pH 2.5 and about pH 2.1, for example, to pH 2.5, pH 2.3, or pH 2.1. In another specific embodiment, the pH is lowered to about pH 3.2 or below for at least 0.5 hours, for example, at least 1 hour. For example, the pH is lowered to between about pH 3.2 and about pH 2.1 for at least about 0.5 hours, e.g., at least about 1.0 hour. In yet another embodiment, the pH is lowered to about pH 3.0 or below for at least 0.5 hours, e.g., at least 1 hour. For example, the pH is lowered to between about pH 3.0 and about pH 2.1 for at least about 0.5 hours, e.g., at least 1.0 hour. In yet another embodiment, the pH is lowered to about pH 2.9 or below for at least 0.5 hours, e.g., at least 1 hour. For example, the pH is lowered to between about pH 2.9 and about pH 2.1 for at least about 0.5 hours, e.g., at least 1.0 hour. In yet another embodiment, the pH is lowered to about pH 2.7 or below for at least 0.5 hours, e.g., at least 1 hour. For example, the pH is lowered to between about pH 2.7 and about pH 2.1 for at least about 0.5 hours, e.g., at least 1.0 hour. In another embodiment, the low pH treatment is terminated by increasing the pH used in the low pH treatment by at least 1 pH unit. In one embodiment, the polypeptide to be isolated / purified can be obtained by expression in Pichia, in particular Pichia pastoris.

[0019] In another specific embodiment, the pH is lowered to about pH 2.5 or below for at least about 1 hour, or at least about 2 hours. In another specific embodiment, the pH is lowered to about pH 2.3 or below for at least about 1 hour. In another embodiment, the low pH treatment is terminated by increasing the pH used in the low pH treatment by at least 1 pH unit. In one embodiment, the polypeptide to be isolated / purified is obtained from expression in Pichia, particularly Pichia pastoris.

[0020] The low pH treatment used to convert the conformational variant into the polypeptide can be applied before or after the chromatography-based purification step. Before the chromatography-based purification step means that the low pH treatment is applied before applying the composition containing the polypeptide to be purified to the stationary phase of the chromatography technique. After the chromatography-based purification step means that the low pH treatment is applied after eluting the polypeptide to be purified from the stationary phase of the chromatography technique. The stationary phase of the chromatography technique is the chromatographic material used, such as a chromatography column containing a resin or a membrane. Thus, the low pH treatment can be applied after eluting the polypeptide from the stationary phase of the chromatography technique used. The low pH treatment can be applied to the eluate obtained by the chromatography-based purification step. In this embodiment, the polypeptide is not bound to or eluted from (i.e., still in contact with) the stationary phase / chromatographic material of the chromatography technique. After elution, the obtained eluate is then adjusted to a low pH treatment for a sufficient amount of time to convert the conformational variant into the polypeptide, as described herein. Thus, in one embodiment, the low pH treatment is applied to the eluate after elution of the polypeptide from the stationary phase of the chromatography-based purification step. In one embodiment, the polypeptide to be isolated / purified can be obtained by expression in Pichia, in particular Pichia pastoris.

[0021] The low pH treatment to convert the conformational variant into the polypeptide may also be applied during the purification step based on chromatography techniques. The term "low-pH treatment" refers to the application of a composition containing a polypeptide to be purified to a stationary phase of a chromatography technique (i.e., contacting the composition containing the polypeptide to be purified with the stationary phase / chromatographic material of the chromatography technique). During the purification process, the composition containing the polypeptide to be purified may be in contact with the stationary phase / chromatographic material (e.g., size-exclusion chromatography) or may be (reversibly) bound to the stationary phase / chromatographic material (e.g., as in affinity chromatography). In one embodiment, the elution buffer has a pH equal to or less than pH 2.5. It is generally known that the actual pH of the eluate is always higher than the initial pH of the low-pH elution buffer. For example, elution with a pH of 3.0 may result in an eluate pH of 3.8. The reason may be that residual liquids present in the stationary phase of the chromatography technique used and having a higher pH (e.g., buffers used for storage, equilibration, or recovery of the stationary phase, or buffers used for binding the polypeptide to the stationary phase) mix with the low-pH buffer used in the low-pH treatment during the purification step based on the chromatography technique. Alternatively, the elution buffer has a pH such that the resulting eluate containing the polypeptide has a pH equal to or less than pH 2.9. In these embodiments, the resulting eluate is optionally adjusted to a pH equal to or less than pH 3.2, e.g., pH 2.7, for at least about 0.5 hours, e.g., at least 1 hour. In one embodiment, the polypeptide to be isolated / purified is obtainable by expression in Pichia, particularly Pichia pastoris.

[0022] Given the teachings provided herein, those skilled in the art will understand that the conversion of conformational variants to the polypeptides described above will increase over time. However, practically useful levels of conversion of conformational variants to the polypeptides described above are already achieved after at least 0.5 hours, e.g., at least about 1 hour, of low pH treatment. In one embodiment, the pH of the eluate is lowered to about pH 3.2 or below for at least 0.5 hours, e.g., at least 1 hour. For example, the pH is lowered between about pH 3.2 and about pH 2.1 for at least about 0.5 hours, e.g., at least about 1.0 hour. In another embodiment, the pH of the eluate is lowered to about pH 3.0 or below for at least 0.5 hours, e.g., at least 1 hour. For example, the pH is lowered between about pH 3.0 and about pH 2.1 for at least about 0.5 hours, e.g., at least about 1.0 hour. In yet another embodiment, the pH of the resulting eluate is lowered to about pH 2.9 or below for at least 0.5 hours, e.g., at least 1 hour. For example, the pH is lowered to between about pH 2.9 and about pH 2.1 for at least about 0.5 hours, e.g., at least 1.0 hour. In yet another embodiment, the pH of the resulting eluate is lowered to about pH 2.7 or below for at least 0.5 hours, e.g., at least 1 hour. For example, the pH is lowered to between about pH 2.7 and about pH 2.1 for at least about 0.5 hours, e.g., at least 1.0 hour. Alternatively, the pH of the resulting eluate containing the polypeptide is lowered to a pH equal to or below pH 2.5. For example, the pH is lowered to pH 2.7 or below for at least 0.5 hours, e.g., at least 1 hour. In one embodiment, the polypeptide to be isolated / purified can be obtained by expression in Pichia, particularly Pichia pastoris.

[0023] In another embodiment, the low pH treatment is terminated by increasing the pH used in the low pH treatment by at least 1 pH unit.

[0024] In another embodiment, the low pH treatment to convert the conformational variant into said polypeptide is applied during a purification step based on Protein A-based affinity chromatography. In one embodiment, the polypeptide to be isolated / purified can be obtained by expression in Pichia, in particular Pichia pastoris. In a particular embodiment, the chromatography technique is Protein A-based affinity chromatography. wherein the elution buffer has a pH of about pH 2.2, and wherein the pH of the resulting eluate is adjusted to a pH of about pH 2.5 for at least about 1.5 hours.

[0025] In one embodiment, the low pH treatment is terminated by increasing the pH to about pH 5.5 or higher. Further, in one embodiment, the low pH treatment is applied after a purification step based on chromatography techniques. Further, in one embodiment, the low pH treatment is applied at room temperature.

[0026] In another embodiment, a chaotropic agent is used to convert the conformational mutant into the polypeptide. In one embodiment, the chaotropic agent is guanidinium chloride (GuHCl). In one embodiment, the GuHCl is at a final concentration of at least about 1 M, e.g., between about 1 M and about 2 M. In one embodiment, the GuHCl is at a final concentration of at least about 2 M. The chaotropic agent treatment is applied for a time sufficient to convert the conformational mutant into the polypeptide. In one embodiment, GuHCl is applied for at least 0.5 hours, or at least 1 hour. The chaotropic agent treatment is terminated by transferring the ISVD polypeptide product to a new buffer system that does not contain a chaotropic agent. In one embodiment, the chaotropic agent treatment is applied after a purification step based on a chromatography technique. In one embodiment, the chaotropic agent is applied at room temperature. In one embodiment, the polypeptide to be isolated / purified can be obtained by expression in Pichia, particularly Pichia pastoris.

[0027] The heat stress applied to convert the conformational variant into the polypeptide comprises incubating the composition containing the conformational variant at about 40°C to about 60°C, about 45°C to about 60°C, or about 50°C to about 60°C. The heat stress is applied for a time sufficient to convert the conformational variant into the polypeptide. In one embodiment, the heat stress is applied for at least about 1 hour. The heat stress is terminated by lowering the temperature to room temperature. In one embodiment, the heat stress is applied after a purification step based on a chromatography technique. In one embodiment, the polypeptide to be isolated / purified can be obtained by expression in Pichia, particularly Pichia pastoris.

[0028] In another embodiment, the conformational mutant is converted into a polypeptide using a combination of the above conditions.

[0029] In another aspect, conformational variants are removed from a composition comprising a multivalent polypeptide comprising at least three or at least four ISVDs by one or more chromatographic techniques. In one aspect, the chromatographic technique is based on hydrodynamic volume, surface charge, or surface hydrophobicity. In one aspect, the chromatographic technique is size-exclusion chromatography (SEC), ion-exchange chromatography (IEX), cation-exchange chromatography (CEX), mixed-mode chromatography (MMC), and / or hydrophobic interaction chromatography (HIC). In one embodiment, the polypeptide to be isolated / purified can be obtained by expression in Pichia, particularly Pichia pastoris.

[0030] In a further aspect, conformational variants are removed by applying a composition comprising a multivalent polypeptide comprising at least three or at least four ISVDs to a chromatography column using a loading rate of at least 20 mg protein per ml of resin, at least 30 mg protein per ml of resin, or at least 45 mg protein per ml of resin. In one embodiment of this aspect, the chromatography column is a Protein A column. In one embodiment, the polypeptide to be isolated / purified is The tides can be obtained by expression in Pichia, particularly Pichia pastoris.

[0031] In another embodiment, one or more of the conditions that convert conformational variants into the above polypeptides are applied alone or in combination with one or more techniques that remove conformational variants.

[0032] Also provided is a method for producing a polypeptide comprising at least three or at least four immunoglobulin single variable domains (ISVDs), the method comprising: a) i) applying a low pH treatment in the isolation and / or purification process steps; ii) applying chaotropic agents in steps of the isolation and / or purification method; iii) applying heat stress during the isolation and / or purification process; or iv) any combination of i) to iii); converting the conformational mutant into the polypeptide by [wherein the conditions are as further described herein]; b) removing conformational variants as further described herein; or c) A combination of a) and b) Includes:

[0033] In particular, the following embodiments are provided: Embodiment 1. A method for isolating or purifying a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs) from a composition comprising said polypeptide and conformational variants thereof, the method comprising: a) applying conditions that convert a conformational variant into said polypeptide; b) removing conformational variants; or c) A combination of (a) and (b) Including, Optionally, the above method, wherein the polypeptide to be isolated or purified herein is obtainable by expression in a host that is not a CHO cell.

[0034] Embodiment 2. The method of embodiment 1, wherein the conformational variant results from expression of the polypeptide in a host that is not a CHO cell, such as a lower eukaryotic host.

[0035] Embodiment 3: The method of embodiment 1, wherein the polypeptide to be isolated or purified is obtainable by expression in a host that is a lower eukaryotic host.

[0036] Embodiment 4: The method of embodiment 2 or embodiment 3, wherein the lower eukaryotic host is a yeast such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces pombe, Cytheromyces, Pachysolen, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, or Endomycopsis.

[0037] Embodiment 5: The method of embodiment 4, wherein the yeast is a Pichia, such as Pichia pastoris.

[0038] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the conformational variant is characterized by a more compact morphology compared to the polypeptide.

[0039] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the conformational variant has a reduced hydrodynamic volume compared to the polypeptide.

[0040] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the conformational variant is characterized by an increased retention time in SE-HPLC compared to the polypeptide.

[0041] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the conformational variant is characterized by an altered retention time in IEX-HPLC compared to the polypeptide.

[0042] Embodiment 10. The method of embodiment 9, wherein the conformational variant is characterized by a decreased retention time in IEX-HPLC compared to the polypeptide.

[0043] Embodiment 11. The method of embodiment 9, wherein the conformational variant is characterized by an increased retention time in IEX-HPLC compared to the polypeptide.

[0044] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the polypeptide comprises or consists of at least three ISVDs.

[0045] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the polypeptide comprises or consists of at least four ISVDs.

[0046] Embodiment 14. The method of any one of embodiments 1 to 11, wherein the polypeptide comprises or consists of three ISVDs, four ISVDs, or five ISVDs.

[0047] Embodiment 15. The conditions for converting a conformational variant into the polypeptide are: i) applying a low pH treatment in a step of the isolation and / or purification method, optionally wherein the low pH treatment comprises lowering the pH of the composition to about pH 3.2 or below, or about pH 3.0 or below; ii) applying a chaotropic agent in a step of the isolation and / or purification method, optionally wherein the chaotropic agent is guanidinium chloride (GuHCl); iii) applying heat stress during the isolation and / or purification process, optionally including incubating the conformational mutant at between 40°C and about 60°C; or iv) Any combination of i) to iii) is selected from 15. The method of any one of embodiments 1 to 14, wherein any of the conditions is applied for an amount of time sufficient to convert the conformational variant into the polypeptide.

[0048] Embodiment 16: The method of embodiment 15, wherein the polypeptide comprises or consists of at least four ISVDs, and the low pH treatment comprises lowering the pH of the composition to about pH 3.0 or below.

[0049] Embodiment 17. The method of embodiment 15 or embodiment 16, wherein the pH is lowered to between about pH 3.2 and about pH 2.1, between about pH 3.0 and about pH 2.1, between about pH 2.9 and about pH 2.1, between about pH 2.7 and about pH 2.1, or between about pH 2.6 and about pH 2.3.

[0050] Embodiment 18. The method of embodiment 17, wherein the pH is lowered to about pH 3.0, about pH 2.9, about pH 2.8, about pH 2.7, about pH 2.6, about pH 2.5, about pH 2.4, about pH 2.3, about pH 2.2, or about pH 2.1.

[0051] Embodiment 19. The method of any one of embodiments 15-18, wherein the low pH treatment is applied for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

[0052] Embodiment 20. The method of any one of embodiments 15-19, wherein the pH is lowered to about pH 2.5 or below.

[0053] Embodiment 21. The method of any one of embodiments 15-19, wherein the pH is lowered to between about pH 3.0 and about pH 2.1 for at least 0.5 hours, at least 1 hour, and optionally at least 2 hours.

[0054] Embodiment 22 The method of embodiment 21, wherein the pH is lowered to between about pH 2.7 and about pH 2.1.

[0055] Embodiment 23. The method of any one of embodiments 15 to 19, wherein the pH is lowered to between about pH 2.7 and about pH 2.1 for at least 1 hour, and optionally at least 2 hours.

[0056] Embodiment 24. The method of embodiment 23, wherein the pH is lowered to between about pH 2.6 and about pH 2.3 for at least 1 hour, and optionally at least 2 hours.

[0057] Embodiment 25 The method of any one of embodiments 15 to 24, wherein the multivalent polypeptide comprises or consists of five ISVDs.

[0058] Embodiment 26 The method of embodiment 25, wherein the pH is lowered to about pH 2.6 or below.

[0059] Embodiment 27 The method of embodiment 25 or 26, wherein the low pH treatment is applied for 1 to 2 hours.

[0060] Embodiment 28. The method of embodiment 27, wherein the polypeptide consists of SEQ ID NO:1.

[0061] Embodiment 29 The method of any one of embodiments 15 to 24, wherein the multivalent polypeptide comprises or consists of four ISVDs.

[0062] Embodiment 30. The method of embodiment 29, wherein the pH is lowered to about pH 2.9 or below, for example to about pH 2.5.

[0063] Embodiment 31 The method of embodiment 29 or 30, wherein the low pH treatment is applied for 1 to 2 hours.

[0064] Embodiment 32. The method of embodiment 31, wherein the polypeptide consists of SEQ ID NO:2.

[0065] Embodiment 33. The method of embodiment 31, wherein the polypeptide consists of SEQ ID NO: 70 or SEQ ID NO: 71.

[0066] Embodiment 34 The method of any one of embodiments 15 to 24, wherein the multivalent polypeptide comprises or consists of three ISVDs.

[0067] Embodiment 35. The method of embodiment 34, wherein the pH is lowered to about pH 3.0 or below, for example to about pH 2.5.

[0068] Embodiment 36 The method of embodiment 34 or 35, wherein the low pH treatment is applied for 2 to 4 hours.

[0069] Embodiment 37. The method of embodiment 36, wherein the polypeptide consists of SEQ ID NO: 69.

[0070] Embodiment 38. The method of any one of embodiments 15 to 37, wherein the low pH treatment is terminated by increasing the pH by at least 1 pH unit, by at least 2 pH units, or by increasing the pH to about pH 5.5 or higher.

[0071] Embodiment 39. The method of any one of embodiments 15 to 38, wherein the low pH treatment is applied before or after a purification step based on chromatographic techniques.

[0072] Embodiment 40 The method of embodiment 39, wherein the low pH treatment is applied before applying the composition to a stationary phase of a chromatographic technique.

[0073] Embodiment 41 The method of embodiment 39, wherein the low pH treatment is applied after eluting the composition from a stationary phase of the chromatographic technique.

[0074] Embodiment 42. The method according to any one of embodiments 15 to 38, wherein the low pH treatment is applied during a purification step based on a chromatography technique, wherein the composition comprising the polypeptide to be purified is in contact with the stationary phase of the chromatography technique.

[0075] Embodiment 43. The method of any one of embodiments 39 to 42, wherein the chromatographic technique is Protein A-based affinity chromatography.

[0076] Embodiment 44. The method of embodiment 43, wherein the chromatographic technique is Protein A-based affinity chromatography, and wherein the elution buffer has a pH equal to or less than pH 2.5.

[0077] Embodiment 45. The method of embodiment 43, wherein the chromatography technique is Protein A-based affinity chromatography, and wherein the elution buffer has a pH such that the resulting eluate containing the polypeptide has a pH equal to or less than pH 2.9.

[0078] Embodiment 46. The method of any one of embodiments 43 to 45, wherein the pH of the eluate containing the polypeptide is adjusted to a pH equal to or less than pH 3.2, for example a pH equal to or less than pH 3.0, or a pH equal to or less than pH 2.7, optionally for at least about 1 hour.

[0079] Embodiment 47. The method of any one of embodiments 43 to 45, wherein the pH of the eluate containing the polypeptide is adjusted to a pH equal to or less than pH 2.5, optionally for at least about 1 hour.

[0080] Embodiment 48. The method of embodiment 42, wherein the chromatography technique is Protein A-based affinity chromatography, wherein the elution buffer has a pH of about pH 2.2, and wherein the pH of the eluate containing the polypeptide is adjusted to a pH of about pH 2.5 for at least about 1.5 hours.

[0081] Embodiment 49. The method of any one of embodiments 42 to 48, wherein the pH of the eluate after the low pH treatment is increased by at least 1 pH unit, increased by at least 2 pH units, or increased to a pH of about pH 5.5 or higher.

[0082] Embodiment 50 The method of any one of embodiments 15 to 49, wherein the low pH treatment is applied at room temperature.

[0083] Embodiment 51. After the low pH treatment: a) adding an appropriate amount of 1 M sodium acetate pH 5.5 to the composition / eluate to obtain a final concentration of about 50 mM sodium acetate; b) adjusting the pH of the composition / elution solution to pH 5.5; and c) adjusting the conductivity of the composition / eluate to about 6 mS / cm or less using water. 51. The method of any one of embodiments 15 to 50, followed by

[0084] Embodiment 52. The method of embodiment 51, wherein the pH in b) is adjusted using NaOH.

[0085] Embodiment 53 The method of embodiment 51 or 52, wherein the polypeptide comprises or consists of five ISVDs.

[0086] Embodiment 54 The method of embodiment 51 or 52, wherein the polypeptide comprises or consists of four ISVDs.

[0087] Embodiment 55. The method of embodiment 54, wherein the polypeptide consists of SEQ ID NO:2.

[0088] Embodiment 56 The method of any one of embodiments 15 to 55, wherein GuHCl is applied at a final concentration of at least about 1 M, or at least about 2 M.

[0089] Embodiment 57 The method of any one of embodiments 15 to 56, wherein GuHCl is applied for at least 0.5 hours, or at least 1 hour.

[0090] Embodiment 58 The method of embodiment 56 or 57, wherein GuHCl is applied at a final concentration of at least about 1 M for at least 0.5 hours.

[0091] Embodiment 59. The method of embodiment 58, wherein GuHCl is applied at a final concentration of at least about 1 M for 0.5 to 1 hour.

[0092] Embodiment 60. The method of embodiment 56 or 57, wherein GuHCl is applied at a final concentration of about 2 M for at least 0.5 hours. Embodiment 61 The method of embodiment 60, wherein GuHCl is applied at a final concentration of at least about 2 M for 0.5 to 1 hour.

[0093] Embodiment 62. The method of any one of embodiments 56 to 61, wherein the polypeptide comprises or consists of at least four ISVDs.

[0094] Embodiment 63. The method of embodiment 61, wherein the polypeptide consists of SEQ ID NO:1.

[0095] Embodiment 64. The method of embodiment 61, wherein the polypeptide consists of SEQ ID NO:2.

[0096] Embodiment 65 The method of any one of embodiments 15 or 56-64, wherein the chaotropic agent treatment is applied at room temperature. Embodiment 66 The method of any one of embodiments 15 or 56-65, wherein the chaotropic agent treatment is applied before or after a purification step based on chromatographic techniques.

[0097] Embodiment 67. The method of embodiment 66, wherein the polypeptide is eluted from a stationary phase of a chromatographic technique, and a chaotropic agent treatment is applied to the resulting eluate.

[0098] Embodiment 68 The method of any one of embodiments 15 to 67, wherein the heat stress is applied for at least about 1 hour, or for about 1 to 4 hours.

[0099] Embodiment 69. The method of embodiment 68, wherein the heat stress is applied at about 40°C to about 60°C, about 45°C to about 60°C, or about 50°C to about 60°C.

[0100] Embodiment 70. The method of embodiment 68, wherein the heat stress is applied at about 40°C to about 55°C, about 45°C to 55°C, or about 48°C to about 52°C.

[0101] Embodiment 71 The method of embodiment 68, wherein the heat stress is applied at about 50°C.

[0102] Embodiment 72 The method of embodiment 71, wherein the heat stress is applied at about 50°C for 1 hour.

[0103] Embodiment 73. The method of embodiment 72, wherein the polypeptide comprises or consists of at least four ISVDs.

[0104] Embodiment 74. The method of embodiment 72, wherein the polypeptide consists of SEQ ID NO:1.

[0105] Embodiment 75. The method of embodiment 72, wherein the polypeptide consists of SEQ ID NO:2.

[0106] Embodiment 76 The method of any one of embodiments 15 or 68 to 75, wherein the heat stress is applied before or after a purification step based on chromatographic techniques.

[0107] Embodiment 77. The method of embodiment 76, wherein the heat stress treatment is applied before applying the composition to a stationary phase of the chromatographic technique or after eluting the composition from the stationary phase of the chromatographic technique.

[0108] Embodiment 78 The method of any one of embodiments 1 to 14, wherein conformational variants are removed by one or more chromatographic techniques.

[0109] Embodiment 79. The method of embodiment 78, wherein the conformational variants are identified by analytical chromatographic techniques, such as SE-HPLC and IEX-HPLC, before being removed by one or more chromatographic techniques.

[0110] Embodiment 80. Chromatographic techniques are based on hydrodynamic volume, surface charge or surface hydrophobicity. 80. The method of embodiment 78 or 79, wherein the method is a chromatographic technique based on the affinity of the target molecule.

[0111] Embodiment 81. The method of embodiment 80, wherein the chromatographic technique is selected from size exclusion chromatography (SEC), ion exchange chromatography (IEX), mixed mode chromatography (MMC), and hydrophobic interaction chromatography (HIC).

[0112] Embodiment 82 The method of embodiment 81, wherein the ion exchange chromatography (IEX) is cation exchange chromatography (CEX).

[0113] Embodiment 83 The method of embodiment 81, wherein the HIC is based on a HIC column resin.

[0114] Embodiment 84 The method of embodiment 83, wherein the HIC resin is selected from Capto Phenyl ImpRes, Capto Butyl ImpRes, Phenyl HP, and Capto Butyl.

[0115] Embodiment 85 The method of embodiment 81, wherein the HIC is based on an HIC membrane.

[0116] Embodiment 86 The method of any one of embodiments 1 to 85, wherein the composition is applied to the chromatography column using a loading rate of at least 20 mg protein per ml of resin, at least 30 mg protein per ml of resin, or at least 45 mg protein per ml of resin, optionally wherein the chromatography column is a Protein A column.

[0117] Embodiment 87. The method of embodiment 86, wherein the composition is applied to a Protein A column using a loading rate of at least 45 mg of protein per ml of resin.

[0118] Embodiment 88. The method of embodiment 87, wherein the polypeptide consists of SEQ ID NO:2.

[0119] Embodiment 89. The method of any one of embodiments 1 to 88, wherein one or more of the conditions that convert conformational variants into said polypeptide are applied alone or in combination with one or more techniques that remove conformational variants.

[0120] Embodiment 90. A method for isolating or purifying a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs), the method comprising: i) applying a low pH treatment to a composition comprising the polypeptide in a step of an isolation or purification method, optionally wherein the low pH treatment comprises lowering the pH of the composition to about pH 3.2 or below, or pH 3.0 or below; ii) applying a chaotropic agent to a composition comprising the polypeptide in a step of the isolation or purification method, optionally wherein the chaotropic agent is GuHCl; iii) subjecting a composition comprising the polypeptide to heat stress during an isolation or purification process, optionally including incubating the conformational mutant at 40°C to about 60°C; iv) applying the composition comprising the polypeptide to a chromatography column using a loading rate of at least 20 mg / ml, at least 30 mg / ml, or at least 45 mg / ml; optionally, the chromatography column is a Protein A column; or v) Any combination of i) to iv) and Optionally, the above method, wherein the polypeptide to be isolated or purified herein is obtainable by expression in a host that is not a CHO cell.

[0121] Embodiment 91: The method of embodiment 90, wherein the conformational variant results from expression of the polypeptide in a host that is not a CHO cell, such as a lower eukaryotic host.

[0122] Embodiment 92. The method of embodiment 90, wherein the polypeptide to be isolated or purified is obtainable by expression in a host that is a lower eukaryotic host.

[0123] Embodiment 93. The method of embodiment 91 or embodiment 92, wherein the lower eukaryotic host is a yeast such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Cytheromyces, Pachysolen, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, or Endomycopsis.

[0124] Embodiment 94. The method of embodiment 93, wherein the yeast is Pichia, e.g., Pichia pastoris.

[0125] Embodiment 95. The method of any one of embodiments 90 to 94, wherein the pH is lowered to between about pH 3.2 and about pH 2.1, between about pH 3.0 and about pH 2.1, between about pH 2.9 and about pH 2.1, between about pH 2.7 and about pH 2.1, or between about pH 2.6 and about pH 2.3.

[0126] Embodiment 96. The method of embodiment 95, wherein the pH is lowered to about pH 3.0, about pH 2.9, about pH 2.8, about pH 2.7, about pH 2.6, about pH 2.5, about pH 2.4, about pH 2.3, about pH 2.2, or about pH 2.1.

[0127] Embodiment 97. The method of any one of embodiments 90-96, wherein the low pH treatment is applied for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

[0128] Embodiment 98 The method of any one of embodiments 90-97, wherein the pH is lowered to about pH 2.5 or below.

[0129] Embodiment 99. The method of any one of embodiments 90-97, wherein the pH is lowered to between about pH 3.0 and about pH 2.1 for at least 0.5 hours, at least 1 hour, or at least 2 hours.

[0130] Embodiment 100. The method of embodiment 99, wherein the pH is lowered to between about pH 2.7 and about pH 2.1.

[0131] Embodiment 101. The method of any one of embodiments 90-97, wherein the pH is lowered to between about pH 2.7 and about pH 2.1 for at least 1 hour, and optionally at least 2 hours.

[0132] Embodiment 102. The method of embodiment 101, wherein the pH is lowered to between about pH 2.6 and about pH 2.3 for at least 1 hour, and optionally at least 2 hours.

[0133] Embodiment 103. The multivalent polypeptide of any one of embodiments 90 to 102, wherein the multivalent polypeptide comprises or consists of three ISVDs, four ISVDs, or five ISVDs. method.

[0134] Embodiment 104. The method of any one of embodiments 90 to 103, wherein the polypeptide comprises or consists of at least four ISVDs.

[0135] Embodiment 105. The method of any one of embodiments 90 to 104, wherein the polypeptide comprises or consists of five ISVDs.

[0136] Embodiment 106 The method of embodiment 105, wherein the pH is lowered to about pH 2.6 or below.

[0137] Embodiment 107. The method of any one of embodiments 103 to 106, wherein the low pH treatment is applied for 1 to 2 hours.

[0138] Embodiment 108. The method of embodiment 107, wherein the polypeptide consists of SEQ ID NO:1.

[0139] Embodiment 109. The method of any one of embodiments 90 to 104, wherein the multivalent polypeptide comprises or consists of four ISVDs.

[0140] Embodiment 110. The method of embodiment 109, wherein the pH is lowered to about pH 2.9 or below, for example to about pH 2.5.

[0141] Embodiment 111 The method of embodiment 109 or 110, wherein the low pH treatment is applied for 1 to 2 hours.

[0142] Embodiment 112. The method of embodiment 111, wherein the polypeptide consists of SEQ ID NO:2.

[0143] Embodiment 113. The method of embodiment 111, wherein the polypeptide consists of SEQ ID NO: 70 or SEQ ID NO: 71.

[0144] Embodiment 114 The method of any one of embodiments 90 to 103, wherein the multivalent polypeptide comprises or consists of three ISVDs.

[0145] Embodiment 115. The method of embodiment 114, wherein the pH is lowered to about pH 3.0 or below, for example to about pH 2.5.

[0146] Embodiment 116 The method of embodiment 114 or 115, wherein the low pH treatment is applied for 2 to 4 hours.

[0147] Embodiment 117. The method of embodiment 116, wherein the polypeptide consists of SEQ ID NO: 69.

[0148] Embodiment 118. The method of any one of embodiments 90 to 117, wherein the low pH treatment is terminated by increasing the pH by at least 1 pH unit, by at least 2 pH units, or by increasing the pH to about pH 5.5 or higher.

[0149] Embodiment 119. The method of any one of embodiments 90 to 118, wherein the low pH treatment is applied before or after a purification step based on chromatographic techniques.

[0150] Embodiment 120. The method of embodiment 119, wherein the low pH treatment is applied before applying the composition to a stationary phase of a chromatographic technique.

[0151] Embodiment 121 The method of embodiment 119, wherein the low pH treatment is applied after elution of the composition from the stationary phase of the chromatographic technique.

[0152] Embodiment 122. The method according to any one of embodiments 90 to 118, wherein the low pH treatment is applied during a purification step based on a chromatography technique, wherein the composition comprising the polypeptide to be purified is in contact with the stationary phase of the chromatography technique.

[0153] Embodiment 123. The method of any one of embodiments 119 to 122, wherein the chromatographic technique is Protein A-based affinity chromatography.

[0154] Embodiment 124. The method of embodiment 123, wherein the chromatographic technique is Protein A-based affinity chromatography, and wherein the elution buffer has a pH equal to or less than pH 2.5.

[0155] Embodiment 125. The method of embodiment 123, wherein the chromatography technique is Protein A-based affinity chromatography, and wherein the elution buffer has a pH such that the resulting eluate containing the polypeptide has a pH equal to or less than pH 2.9.

[0156] Embodiment 126. The method of any one of embodiments 123 to 125, wherein the pH of the eluate containing the polypeptide is adjusted to a pH equal to or less than 3.0, optionally for at least 1 hour, for example to a pH equal to or less than pH 2.7, optionally for at least 0.5 hours or about 1 hour.

[0157] Embodiment 127. The method of any one of embodiments 123 to 125, wherein the pH of the eluate containing the polypeptide is adjusted to a pH equal to or less than pH 2.5, optionally for at least about 0.5 or 1 hour.

[0158] Embodiment 128. The method of embodiment 122, wherein the chromatography technique is Protein A-based affinity chromatography, wherein the elution buffer has a pH of about pH 2.2, and wherein the pH of the eluate containing the polypeptide is adjusted to a pH of about pH 2.5 for at least about 1.5 hours.

[0159] Embodiment 129. The method of any one of embodiments 119 to 128, wherein the pH of the eluate after the low pH treatment is increased by at least 1 pH unit, or by at least 2 pH units, or to a pH of about pH 5.5 or higher.

[0160] Embodiment 130. The method of any one of embodiments 90 to 129, wherein the low pH treatment is applied at room temperature.

[0161] Embodiment 131. After the low pH treatment: a) adding an appropriate amount of 1 M sodium acetate pH 5.5 to the composition / eluate to obtain a final concentration of about 50 mM sodium acetate; b) adjusting the pH of the composition / elution solution to pH 5.5; and c) adjusting the conductivity of the composition / eluate to about 6 mS / cm or less using water. 131. The method of any one of embodiments 90 to 130, followed by the step of:

[0162] Embodiment 132. The method of embodiment 131, wherein the pH in b) is adjusted using NaOH.

[0163] Embodiment 133. The method of embodiment 131 or 132, wherein the polypeptide comprises or consists of five ISVDs.

[0164] Embodiment 134. The method of embodiment 131 or 132, wherein the polypeptide comprises or consists of four ISVDs.

[0165] Embodiment 135. The method of embodiment 134, wherein the polypeptide consists of SEQ ID NO:2.

[0166] Embodiment 136 The method of any one of embodiments 90 to 135, wherein GuHCl is applied at a final concentration of at least about 1 M, or at least about 2 M.

[0167] Embodiment 137 The method of embodiment 90 or 136, wherein GuHCl is applied for at least 0.5 hours, or at least 1 hour.

[0168] Embodiment 138 The method of embodiment 136 or 137, wherein GuHCl is applied at a final concentration of at least about 1 M for at least 0.5 hours.

[0169] Embodiment 139. The method of embodiment 138, wherein GuHCl is applied at a final concentration of at least about 1 M for 0.5 to 1 hour.

[0170] Embodiment 140 The method of embodiment 136 or 137, wherein GuHCl is applied at a final concentration of about 2 M for at least 0.5 hours.

[0171] Embodiment 141. The method of embodiment 140, wherein GuHCl is applied at a final concentration of at least about 2 M for 0.5 to 1 hour.

[0172] Embodiment 142. The method of any one of embodiments 90 or 136 to 141, wherein the polypeptide comprises or consists of at least four ISVDs.

[0173] Embodiment 143. The method of embodiment 142, wherein the polypeptide consists of SEQ ID NO:1.

[0174] Embodiment 144. The method of embodiment 142, wherein the polypeptide consists of SEQ ID NO:2.

[0175] Embodiment 145 The method of any one of embodiments 90 or 136-144, wherein the chaotropic agent treatment is applied at room temperature.

[0176] Embodiment 146 The method of any one of embodiments 90 or 136 to 145, wherein the chaotropic agent treatment is applied before or after a purification step based on chromatographic techniques.

[0177] Embodiment 147 The method of embodiment 146, wherein the polypeptide is eluted from a stationary phase of a chromatographic technique, and a chaotropic agent treatment is applied to the resulting eluate. How to post.

[0178] Embodiment 148 The method of any one of embodiments 90 to 147, wherein the heat stress is applied for at least about 1 hour, or for about 1 to 4 hours.

[0179] Embodiment 149. The method of embodiment 148, wherein the heat stress is applied at about 40°C to about 60°C, about 45°C to about 60°C, or about 50°C to about 60°C.

[0180] Embodiment 150. The method of embodiment 148, wherein the heat stress is applied at about 40°C to about 55°C, about 45°C to 55°C, or about 48°C to about 52°C.

[0181] Embodiment 151 The method of embodiment 148, wherein the heat stress is applied at about 50°C.

[0182] Embodiment 152 The method of embodiment 151, wherein the heat stress is applied at about 50°C for 1 hour.

[0183] Embodiment 153. The method of any one of embodiments 148 to 152, wherein the polypeptide comprises or consists of at least four ISVDs.

[0184] Embodiment 154. The method of embodiment 152, wherein the polypeptide consists of SEQ ID NO:1.

[0185] Embodiment 155. The method of embodiment 152, wherein the polypeptide consists of SEQ ID NO:2.

[0186] Embodiment 156 The method of any one of embodiments 90 or 148 to 155, wherein the heat stress is applied before or after a purification step based on chromatographic techniques.

[0187] Embodiment 157. The method of embodiment 156, wherein the heat stress treatment is applied before applying the composition to the stationary phase of the chromatographic technique or after eluting the composition from the stationary phase of the chromatographic technique.

[0188] Embodiment 158. A method for producing a polypeptide comprising at least three or at least four immunoglobulin single variable domains (ISVDs), comprising purifying and / or isolating a polypeptide according to any one of embodiments 1 to 154.

[0189] Embodiment 159. The following steps: a) optionally culturing a host or host cells under conditions such that the host or host cells grow; b) maintaining the host or host cell under conditions such that the host or host cell expresses and / or produces the polypeptide; and c) isolating and / or purifying the secreted polypeptide from the culture medium, comprising one or more of the isolation or purification methods according to any one of embodiments 1 to 154. 159. The method of embodiment 158, comprising at least:

[0190] Embodiment 160. The method of embodiment 158 ​​or 159, wherein the host is a lower eukaryotic host.

[0191] Embodiment 161. The method of embodiment 160, wherein the lower eukaryotic host is a yeast such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Cytheromyces, Pachysolen, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, or Endomycopsis.

[0192] Embodiment 162. The method of embodiment 161, wherein the yeast is Pichia, such as Pichia pastoris.

[0193] Embodiment 163. A method for isolating or purifying a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs) from a composition comprising said polypeptide and conformational variants thereof, the method comprising: (1) Identifying conformational variants by analytical chromatographic techniques such as SE-HPLC and IEX-HPLC; (2) adjusting the chromatographic conditions to allow for the specific removal of conformational variants; and (3) removing conformational variants from a composition comprising the polypeptide and its conformational variants by one or more chromatographic techniques; wherein optionally the polypeptide to be isolated or purified is obtainable by expression in a host other than a CHO cell.

[0194] Embodiment 164. A method for optimizing one or more chromatography techniques to allow the isolation or purification of a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs) from a composition comprising said polypeptide and conformational variants thereof, comprising: (1) Identifying conformational variants by analytical chromatographic techniques such as SE-HPLC and IEX-HPLC; (2) Optimizing chromatographic conditions to enable specific removal of conformational variants Including, Optionally, the above method, wherein the polypeptide to be isolated or purified herein is obtainable by expression in a host that is not a CHO cell.

[0195] Embodiment 165. The method of embodiment 163 or 164, wherein the conformational variant results from expression of the polypeptide in a host that is not a CHO cell, such as a lower eukaryotic host.

[0196] Embodiment 166: The method according to embodiment 163 or 164, wherein the polypeptide to be isolated or purified is obtainable by expression in a host which is a lower eukaryotic host.

[0197] Embodiment 167: The method of embodiment 165 or embodiment 166, wherein the lower eukaryotic host is a yeast such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Cytheromyces, Pachysolen, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, or Endomycopsis.

[0198] Embodiment 168: The method of embodiment 167, wherein the yeast is Pichia, such as Pichia pastoris.

[0199] Embodiment 169. The method of any one of embodiments 163 to 168, wherein the conformational variant is characterized by a more compact morphology compared to the polypeptide.

[0200] Embodiment 170 The method of any one of embodiments 163 to 169, wherein the conformational variant has a reduced hydrodynamic volume compared to the polypeptide.

[0201] Embodiment 171. The method of any one of embodiments 163 to 170, wherein the conformational variant is characterized by an increased retention time in SE-HPLC compared to the polypeptide.

[0202] Embodiment 172. The method of any one of embodiments 163 to 171, wherein the conformational variant is characterized by an altered retention time in IEX-HPLC compared to the polypeptide.

[0203] Embodiment 173 The method of embodiment 172, wherein the conformational variant is characterized by a decreased retention time in IEX-HPLC compared to the polypeptide.

[0204] Embodiment 174 The method of embodiment 172, wherein the conformational variant is characterized by an increased retention time in IEX-HPLC compared to the polypeptide.

[0205] Embodiment 175. The method of any one of embodiments 163 to 174, wherein the polypeptide comprises or consists of at least three ISVDs.

[0206] Embodiment 176. The method of any one of embodiments 163 to 175, wherein the polypeptide comprises or consists of at least four ISVDs.

[0207] Embodiment 177. The method of any one of embodiments 163 to 176, wherein the polypeptide comprises or consists of three ISVDs, four ISVDs, or five ISVDs.

[0208] Embodiment 178. The method of any one of embodiments 163 to 177, wherein the chromatographic technique is a chromatographic technique based on hydrodynamic volume, surface charge, or surface hydrophobicity.

[0209] Embodiment 179. The method of embodiment 178, wherein the chromatographic technique is selected from size exclusion chromatography (SEC), ion exchange chromatography (IEX), mixed mode chromatography (MMC), and hydrophobic interaction chromatography (HIC).

[0210] Embodiment 180 The method of embodiment 179, wherein the ion exchange chromatography (IEX) is cation exchange chromatography (CEX).

[0211] Embodiment 181 The method of embodiment 179, wherein the HIC is based on a HIC column resin.

[0212] Embodiment 182 The method of embodiment 181, wherein the HIC resin is selected from Capto Phenyl ImpRes, Capto Butyl ImpRes, Phenyl HP, and Capto Butyl.

[0213] Embodiment 183. The method of embodiment 179, wherein the HIC is based on an HIC membrane. .

[0214] 4. DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0215] [Figure 1] Figure 1: SE-HPLC chromatograms of eluates after capture using Protein A or non-Protein A capture resin (including zoom, bottom panel). [Figure 2] Figure 2: SE-HPLC chromatograms (including zoom, bottom panel) of the eluates after Protein A capture using elution buffers A, B, C, and D as described in Table 2. [Figure 3-1] Figure 3: SE-HPLC chromatograms of eluates after Protein A capture with or without pH neutralization using elution buffer A in (1) and buffer B in (2) (including zoom, bottom panel). [Figure 3-2] Continued from Figure 3-1. [Figure 4] Figure 4: Chromatographic profile of compound A on the cation exchange resin used for polishing development. [Figure 5] FIG. 5: SE-HPLC chromatograms (including zoom, bottom panel) of the load, side and top fractions obtained in the preparative CEX described in Example 1 and FIG. [Figure 6] Figure 6: IEX-HPLC chromatograms (including zoom, bottom panel) of conformational variant-enriched side fractions and conformational variant-depleted top fractions obtained in the preparative CEX described in Example 1 and Figure 4. [Figure 7-1]Figure 7: SE-HPLC chromatograms (including zoom, bottom panel) of conformational variant-enriched material (1) and depleted material (2) after low pH treatment (pH 2.5). [Figure 7-2] Continued from Figure 7-1. [Figure 8] Figure 8: IEX-HPLC chromatograms (including zoom, bottom panel) of conformational variant enriched material after low pH treatment (pH 2.5). [Figure 9] Figure 9: SE-HPLC chromatograms (including zoom, bottom panel) of conformational variant enriched material treated with 2 M or 3 M GuHCl chaotropic agent for 0.5 h at room temperature. [Figure 10] Figure 10: IEX-HPLC chromatograms (including zoom, bottom panel) of conformational variant enriched material treated with 2 M or 3 M GuHCl chaotropic agent treatment for 0.5 h at room temperature. [Figure 11] Figure 11: SE-HPLC chromatogram of conformational variant enriched material treated at 50 °C for 1 h (zoom included, bottom panel). [Figure 12] Figure 12: IEX-HPLC chromatogram of conformational variant enriched material treated at 50 °C for 1 h (zoom included, bottom panel). [Figure 13] Figure 13: SE-HPLC chromatograms of capture eluates using various elution conditions as described in Example 4 (including zoom, bottom panel). [Figure 14] Figure 14: IEX-HPLC chromatograms of capture eluates using various elution conditions as described in Example 4 (including zoom, bottom panel). [Figure 15-1] Figure 15: SE-HPLC chromatograms of captured eluates after low pH incubation and pH adjustment immediately after low pH (T0) in (1) and (2); and after low pH incubation and pH adjustment after 1 hour incubation at low pH (T1h) in (3) and (4) (zoom, including bottom panel). [Figure 15-2] Continued from Figure 15-1. [Figure 15-3] Continued from Figure 15-2. [Figure 15-4] Continued from Figure 15-3. [Figure 16A] Figure 16A: SE-HPLC chromatograms of the sample after application of two different sets of pH-adjusted stock solutions (including zoom, bottom panel). [Figure 16B] FIG. 16B: Effect of pH on product quality of Compound A analyzed by IEX-HPLC as described in Example 4 (first experiment). [Figure 16C] FIG. 16C: Effect of pH on product quality of Compound A analyzed by IEX-HPLC as described in Example 4 (second experiment). [Figure 17-1] Figure 17: SE-HPLC chromatograms of captured eluate and captured filtrate from 10 L scale (1) and 100 L scale (2) (zoom, including bottom panel). [Figure 17-2] Continued from Figure 17-1. [Figure 18] Figure 18: IEX-HPLC chromatograms of captured eluate and captured filtrate from the 10 L scale (zoom included, bottom panel). [Figure 19] Figure 19: IEX-HPLC chromatograms of captured eluate and captured filtrate from the 100 L scale. [Figure 20] Figure 20: Chromatographic MMC profile used for the removal of conformational variants of compound A. Grey box: fractions F8 and F11 selected for analysis. [Figure 21-1] FIG. 21: SE-HPLC chromatograms (including zoom, lower panel) of load and fraction F8 in (1) and load and fraction F11 in (2) obtained in MMC as described in Example 6. [Figure 21-2] Continuation of Figure 21-1. [Figure 22-1]Figure 22: IEX-HPLC chromatograms (including zoom, lower panel) of load and fraction F8 in (1) and load and fraction F11 in (2) obtained in MMC as described in Example 6. [Figure 22-2] Continuation of Figure 22-1. [Figure 23] Figure 23: Chromatographic HIC profile on TSK Phenyl Gel 5 PW(30) resin used for the removal of conformational variants of compound A. Grey box: fractions F26 and F41 selected for analysis. [Figure 24-1] Figure 24: SE-HPLC chromatograms (including zoom, lower panel) of the load and fraction F26 in (1) and the load and fraction F41 in (2) obtained in HIC using TSK Phenyl Gel 5 PW(30) resin. [Figure 24-2] Continuation of Figure 24-1. [Figure 25] Figure 25: SE-HPLC chromatograms (including zoom, bottom panel) of the top fractions and loads obtained in HIC using Capto Butyl Impres resin used with an ammonium sulfate gradient. [Figure 26] Figure 26: Chromatographic HIC profile on Capto Butyl ImpRes resin used for removal of conformational variants of compound A. Gray box: fractions F15, F20, and F29 selected for analysis. [Figure 27] Figure 27: SE-HPLC chromatograms of the load and fractions F15, F20 and F29 obtained in HIC using Capto Butyl ImpRes resin (zoom, including bottom panel). [Figure 28] Figure 28: SE-HPLC chromatogram of the captured eluate after membrane-based HIC on a Sartobind Phenyl membrane (filter plate) (zoom included, bottom panel). [Figure 29]FIG. 29: Chromatographic HIC profile on Sartobind Phenyl membrane used for removal of conformational variants of Compound A. [Figure 30] Figure 30: SE-HPLC chromatograms of load, fraction pool 2, and strip fractions obtained in HIC on Sartobind Phenyl membrane (zoom, including bottom panel). [Figure 31] Figure 31: IEX-HPLC chromatogram of compound B (zoom, including bottom panel). [Figure 32] Figure 32: Chromatographic CEX profile of compound B during the polishing process step. Grey box: fractions selected for analysis. [Figure 33] Figure 33: IEX-HPLC chromatograms (including zoom, bottom panel) of fraction 2C4 and pooled fractions 2C7-2C11 obtained in CEX as described in Example 7. [Figure 34] Figure 34: SE-HPLC chromatograms (including zoom, bottom panel) of fraction 2C4 and pooled fractions 2C7-2C11 obtained in the CEX described in Example 7. [Figure 35] Figure 35: IEX-HPLC chromatogram (including zoom, bottom panel) of the capture eluate of Compound B after 1 hour of low pH treatment at pH 2.3 followed by adjustment to pH 5.5 with 1 M sodium acetate. Capture eluate immediately adjusted to pH 5.5 with 1 M sodium acetate was used as a control. [Figure 36] Figure 36: SE-HPLC chromatogram (including zoom, bottom panel) of the capture eluate of compound B after 1 hour of low pH treatment at pH 2.3 followed by adjustment to pH 5.5 with 1 M sodium acetate. Capture eluate immediately adjusted to pH 5.5 with 1 M sodium acetate was used as a control. [Figure 37] Figure 37: IEX-HPLC chromatogram of captured eluate of compound B after 4 hours of low pH 2.5 treatment (zoom included, bottom panel). [Figure 38] Figure 38: SE-HPLC chromatogram of captured eluate of compound B after 4 hours of low pH 2.5 treatment (zoom included, bottom panel). [Figure 39] Figure 39: IEX-HPLC chromatogram of the captured eluate of compound B after GuHCl chaotropic agent treatment for 0.5 hours at room temperature (zoom, including bottom panel). [Figure 40] Figure 40: IEX-HPLC chromatogram of the captured eluate of compound B after 1 hour heat treatment at 50°C (zoom, including bottom panel). [Figure 41] Figure 41: SE-HPLC chromatogram of the captured eluate of compound B after heat treatment at 50°C for 1 hour (zoom, including bottom panel). [Figure 42A] Figure 42A: SE-HPLC chromatograms of captured eluates of Compound B after treatment at pH 2.3 and then adjusting to pH 5.5 either immediately or after 1 hour (zoom, including bottom panel). [Figure 42B] Figure 42B: SE-HPLC chromatograms of captured eluates of Compound B after treatment at pH 2.5 and then adjusting to pH 5.5 immediately or 1 hour later (zoom, including bottom panel). [Figure 43A] Figure 43: Effect of low pH treatment on product quality analyzed as a function of time by IEX-HPLC. (A) Initial experiments with 2 and 4 hours of low pH treatment at pH 2.3 and pH 2.5; (B) Additional experiments with 2 and 4 hours of low pH treatment at pH 2.7, pH 2.9, pH 3.1, pH 3.3, pH 3.5, and pH 2.7. [Figure 43B] Figure 43: Effect of low pH treatment on product quality analyzed as a function of time by IEX-HPLC. (A) Initial experiments with 2 and 4 hours of low pH treatment at pH 2.3 and pH 2.5; (B) Additional experiments with 2 and 4 hours of low pH treatment at pH 2.7, pH 2.9, pH 3.1, pH 3.3, pH 3.5, and pH 2.7. [Figure 44]Figure 44: SE-HPLC chromatograms of captured eluates of Compound B after treatment at pH 2.4 and pH 2.6 for 2 hours and then adjusting to pH 5.5 (zoom, including bottom panel). [Figure 45] Figure 45: SE-HPLC chromatogram of the captured eluate of compound B after treatment at pH 2.6 for 2 hours and then adjusting to pH 5.5 (zoom, including bottom panel). [Figure 46] Figure 46. Chromatographic CEX profile used for removal of conformational variants of compound B. Grey box: fractions selected for analysis. [Figure 47] Figure 47: Chromatographic HIC profile on Capto Butyl ImpRes resin used for removal of conformational variants of compound B. Grey box: fractions selected for analysis. [Figure 48] Figure 48: SDS-PAGE analysis of selected fractions of the HIC run on Capto Butyl ImpRes as shown in Figure 47. [Figure 49] Figure 49: Predictive profiler of the DOE model showing the effect of loading rate on product quality assessed by IEX-HPLC analysis. [Figure 50] Figure 50: SE-HPLC chromatograms of a representative cycle 1 capture eluate and a representative cycle 1 capture filtrate from the 10 L scale-up (zoom included, bottom panel). [Figure 51] Figure 51: SE-HPLC chromatograms of a representative cycle 1 capture eluate and a representative cycle 1 capture filtrate from the 100 L scale-up (zoom included, bottom panel). [Figure 52] Figure 52: Schematic of the hypothetical model. [Figure 53A]Figure 53: (A) SE-HPLC chromatograms of captured eluates of compound C produced in Pichia pastoris after 0, 2, and 4 hours of low pH 3.0 treatment (zoom, includes bottom panel). (B) SE-HPLC chromatograms of captured eluates of compound C after 0, 2, and 4 hours of low pH 2.5 treatment (zoom, includes bottom panel). [Figure 53B] Figure 53: (A) SE-HPLC chromatograms of captured eluates of compound C produced in Pichia pastoris after 0, 2, and 4 hours of low pH 3.0 treatment (zoom, includes bottom panel). (B) SE-HPLC chromatograms of captured eluates of compound C after 0, 2, and 4 hours of low pH 2.5 treatment (zoom, includes bottom panel). [Figure 54] FIG. 54: Effect of pH on product quality of Compound C analyzed by SE-HPLC as described in Example 14. [Figure 55] Figure 55: SE-HPLC chromatograms of captured eluates of Compound C produced in CHO cells after low pH treatment at pH 2.6 and pH 3.0 compared to treatment at pH 5.5 after 2 hours of incubation (zoom, including bottom panel). [Figure 56] FIG. 56: Effect of pH on product quality of Compound D analyzed by SE-HPLC as described in Example 16. [Figure 57] FIG. 57: Effect of pH on product quality of Compound E analyzed by SE-HPLC as described in Example 17. DETAILED DESCRIPTION OF THE INVENTION

[0216] 5. Detailed Description The present disclosure describes the surprising finding of conformational variants of polypeptides comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs). The conformational variants of the polypeptides were observed during production of the polypeptides in a host. In particular, conformational variants were observed during production of polypeptides comprising or consisting of at least three or at least four ISVDs in a host, such as a lower eukaryotic host as described herein. It has been found that conformational variants of multivalent polypeptide products comprising at least three or at least four ISVDs result from expression of the polypeptides in a host, particularly a lower eukaryotic host such as yeast. While the molecular weight of the polypeptide and its conformational variants is the same, the conformational variants exhibit altered charge / surface characteristics, resulting in different physicochemical behavior, e.g., different retention times in analytical size-exclusion chromatography and / or analytical ion-exchange chromatography. Thus, conformational variants of a polypeptide comprising or consisting of at least three or at least four ISVDs can be identified as a shoulder post-peak or a separate post-peak (SE-HPLC post-peak 1) of the main peak containing the polypeptide in analytical size exclusion chromatography, and / or as a peak containing the polypeptide in analytical ion exchange chromatography. This could be observed as a post-peak shoulder of the main peak or as a separate post-peak (IEX-HPLC post-peak 1). Such different physicochemical behavior was not due to scrambled disulfide bridges.

[0217] Based on these observations, we hypothesized that polypeptides comprising or consisting of at least three or at least four ISVDs allow certain structural flexibilities that result in intramolecular interactions, such that the polypeptides can exist as conformational variants with conformational arrangements of ISVD building blocks that result in a more compact morphology compared to the arrangement of the ISVD building blocks of the polypeptide (see Figure 52). Although ISVDs themselves are very stable molecules, we surprisingly observed that as the valency of a polypeptide increases to at least three or at least four ISVDs (i.e., the number of ISVD building blocks increases to three, four, or more), the polypeptide can become more prone to intramolecular interactions. Without being bound by any hypothesis, we concluded that polypeptides comprising or consisting of at least three or at least four ISVDs allow intramolecular interactions between at least two ISVDs within the polypeptide, allowing for the formation of conformational variants of the polypeptide with a compact morphology. A compact morphology is characterized by a reduced hydrodynamic volume compared to the polypeptide. Furthermore, it has been found that the compact morphology can be characterized by altered surface charge and / or altered surface hydrophobicity / hydrophobic exposure. Thus, polypeptides comprising or consisting of at least three or at least four ISVDs and conformational variants thereof can be distinguished based on analytical chromatographic techniques. In particular, polypeptides comprising or consisting of at least three or at least four ISVDs and conformational variants thereof can be distinguished based on shifts in hydrodynamic volume and / or surface charge by analytical chromatographic techniques such as size-exclusion high-performance liquid chromatography (SE-HPLC) and / or ion-exchange high-performance liquid chromatography (IEX-HPLC).

[0218] It has further been shown that conformational variants can be converted into (desired) polypeptides using the treatment conditions disclosed in this application. Furthermore, based on the observed biochemical / biophysical differences between the polypeptide and its conformational variants, it has been found that conformational variants can be removed from compositions comprising the polypeptide and its conformational variants using known preparative chromatographic techniques based on hydrodynamic volume, surface charge, and / or surface hydrophobicity, as described herein.

[0219] 5.1 Definition Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, as would be apparent to one skilled in the art. See, for example, Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. 1-3, Cold Spring Harbor Laboratory Press), Ausubel et al., 1987 (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York), Lewin, 1985 (Genes II, John Wiley & Sons, New York, NY), Old et al., 1981 (Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd ed., University of California Press, Berkeley, CA), Roitt et al., 2001 (Immunology, 6th Ed., Mosby / Elsevier, Edinburgh), Roitt et al., 2001 (Roitt's Essential Immunology, 10th ed., Blackwell Publishing, NY). Reference is made to standard manuals such as Janeway et al. 2005 (Immunobiology, 6th ed., Garland Science Publishing / Churchill Livingstone, New York), as well as the general background cited herein.

[0220] It will be clear to those skilled in the art that, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se. Again, reference is made, for example, to the standard manuals and general background art mentioned herein and the further references cited therein; further, for example, to the following reviews: Presta 2006 (Adv. Drug Deliv. Rev. 58:640), Levin and Weiss 2006 (Mol. Biosyst. 2:49), Irving et al. 2001 (J. Immunol. Methods 248:31), Schmitz et al. 2000 (Placenta 21 Suppl. A:S106), Gonzales et al. 2005 (Tumor Biol. 26:31), which describe techniques for protein engineering such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0221] The term "about" as used in the context of parameters or parameter ranges provided herein shall have the following meaning: Unless otherwise indicated, when the term "about" is applied to a particular value or range, that value or range shall be interpreted as being as accurate as the method used to measure it. Unless a tolerance is specified in the application, the last decimal place of a numerical value indicates its precision. Unless another tolerance is indicated, the maximum permissible error is ascertained by applying a rounding conversion to the last decimal place; for example, for a pH value of approximately pH 2.7, the tolerance is 2.65 to 2.74. However, for the following parameters, specific tolerances shall apply: temperatures specified in °C without decimal places shall have a tolerance of ±1°C (e.g., a temperature value of approximately 50°C means 50°C ±1°C); times specified in hours shall have a tolerance of 0.1 hours regardless of the decimal place (e.g., a time value of approximately 1.0 hour means 1.0 hour ±0.1 hour; a time value of approximately 0.5 hour means 0.5 hour ±0.1 hour).

[0222] In the present application, any parameter indicated with the term "about" is also intended to be disclosed without the term "about." In other words, embodiments that refer to a parameter value using the term "about" also describe embodiments that relate to the numerical value of the parameter itself. For example, an embodiment that specifies a pH of "about pH 2.7" also discloses an embodiment that specifies the pH of "pH 2.7" itself; an embodiment that specifies a pH range between "about pH 2.7 and about pH 2.1" also describes an embodiment that specifies a pH range such as "between pH 2.7 and pH 2.1."

[0223] 5.2 Immunoglobulin Single Variable Domains The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which the antigen-binding site is present on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal molecules) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (V) is present on and formed by a single immunoglobulin domain. H ) and the light chain variable domain (V L ) act together to form the antigen-binding site. In this case, V H and V L Both complementarity determining regions (CDRs) of the nucleotides contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0224] In view of the above definitions, in these cases, binding to an individual epitope of an antigen does not usually occur by one (single) immunoglobulin domain, but by a pair (associated) immunoglobulin domains, e.g., a light chain and a heavy chain variable domain, i.e., the V H -V L The antigen-binding domain of a traditional four-chain antibody (e.g., an IgG, IgM, IgA, IgD, or IgE molecule; known in the art), or the antigen-binding domain of a Fab fragment, F(ab')2 fragment, Fv fragment, such as a disulfide-linked Fv or scFv fragment, or a diabody (all known in the art) derived from such a four-chain antibody, are not normally considered to be an immunoglobulin single variable domain because they occur in pairs and bind together to their respective epitopes of the antigen.

[0225] In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain consists of a single VH , a single V HH or a single V L It is formed by domains.

[0226] Thus, a single variable domain can be any light chain variable domain sequence (e.g., V), as long as it is capable of forming a single antigen-binding unit; L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V H -sequence or V HH sequence) or a suitable fragment thereof (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0227] Immunoglobulin single variable domains (ISVDs) include, for example, camelized V H or humanized V HH Including V H , V HH In one embodiment, this may be a heavy chain ISVD such as V HH and camelization V H or humanized V HH The heavy chain ISVD can be derived from a traditional four-chain antibody or a heavy chain antibody.

[0228] For example, an immunoglobulin single variable domain can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) or a NANOBODY® ISVD (as defined herein, including but not limited to V HH ); other single variable domains, or any suitable fragment of any one of them.

[0229] In particular, the immunoglobulin single variable domain is a NANOBODY® ISVD (e.g., a humanized V HH or Camelization V H Contains V HH) or a suitable fragment thereof [Note: NANOBODY® is a registered trademark of Ablynx NV].

[0230] V HH , V HH Antibody fragments, and V HH Also known as antibodies, "V HH The term "V domain" was originally described as the antigen-binding immunoglobulin variable domain of a "heavy chain antibody" (i.e., an antibody lacking a light chain; Hamers-Casterman et al. Nature 363:446-448, 1993). HH The "V domain" refers to these variable domains as compared to the heavy chain variable domains (herein referred to as "V" domains) present in conventional four-chain antibodies. H domain”), and the light chain variable domain (referred to herein as “V L It was chosen to distinguish it from the "domain" (called a "V"). HH For further explanation, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0231] Typically, the production of immunoglobulins involves immunizing laboratory animals, fusing immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening natural or synthetic libraries, such as phage display libraries.

[0232] The generation of immunoglobulin sequences such as VHHs has been extensively described in various publications, including WO 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001). In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. The repertoire of VHHs obtained from this immunization is further screened for VHHs that bind to the target antigen.

[0233] In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from natural sources or during recombinant production.

[0234] Immunization and / or screening for immunoglobulin sequences can be carried out using peptide fragments of such antigens.

[0235] Immunoglobulin sequences of various origins, including mouse, rat, rabbit, donkey, human, and camel immunoglobulin sequences, can be produced, purified, and / or isolated by the methods described herein. Also, fully human, humanized, or chimeric sequences can be produced, purified, and / or isolated by the methods described herein. For example, camel immunoglobulin sequences and humanized camel immunoglobulin sequences, or camelized domain antibodies, such as the camelized dAbs described by Ward et al. (see, e.g., WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994, and Prot. Eng., 9:531-537, 1996), can be produced, purified, and / or isolated by the methods described herein. Furthermore, ISVDs can be fused to comprise or consist of at least three or at least four ISVDs to form multivalent and / or multispecific constructs (one or more VSVDs can be fused to form multivalent and / or multispecific constructs). HH(For multivalent, multispecific polypeptides containing domains and their production, see Conrath et al., J. Biol. Chem., Vol. 276, 10, 7346-7350, 2001, and also, e.g., WO 96 / 34103 and WO 99 / 23221.) The ISVD sequence may include a tag or other functional moiety, such as a toxin, label, radiochemical, etc.

[0236] "Humanized V HH " is a naturally occurring V HH The amino acid sequence of the V domain corresponds to that of a conventional four-chain antibody of human origin, but is "humanized," i.e., H one or more of the amino acid residues present at the corresponding positions in the naturally occurring V domain HH The amino acid sequence of the humanized V is preferably a V-like protein, and more preferably a V-like protein. The V-like protein may be a V-like protein, for example, a V-like protein, or a V-like protein. The V-like protein may be a V-like protein, for example, a V-like protein, or a V-like protein. The V-like protein may be a V-like protein, for example, a V-like protein, or a V-like protein. The V-like protein may be a V-like protein, for example, a V-like protein, or a V-like protein. HH It should be noted that the VHH domain-containing polypeptides can be obtained by any suitable method known per se and are therefore not strictly limited to polypeptides obtained using naturally occurring VHH domain-containing polypeptides as starting material.

[0237] "Camelization V H " is a naturally occurring V H The amino acid sequence of the V domain corresponds to that of the naturally occurring V from a conventional four-chain antibody, but is "camelized", i.e. H One or more amino acid residues in the amino acid sequence of the domain are replaced by the V HH Do This includes the amino acid sequence of V by substituting one or more amino acid residues present at the corresponding positions in the V domain. This can be done in a manner known per se, which will be clear to the skilled artisan, for example, based on the further description herein and the prior art (e.g., Davies and Riechmann (1994 and 1996), supra). Such "camelizing" substitutions can be made by substituting one or more amino acid residues present at the corresponding positions in the V domain. H -V L Forming a boundary and / or V H -V L Insertions are made at amino acid residues at boundaries and / or at so-called Camelidae hallmark residues as defined herein (see, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996), supra). In one embodiment, camelized V H V, which is used as a starting material or starting point for generating or designing H The sequence is V from mammals H sequences, e.g., human V H Array, e.g. V H 3 sequence. However, such camelized V H can be obtained by any suitable method known per se, and therefore does not require the naturally occurring V as starting material. H It should be noted that the invention is not strictly limited to polypeptides obtained using polypeptides containing the domain.

[0238] It should be noted that one or more ISVD sequences can be linked to each other and / or to other amino acid sequences (e.g., via disulfide bridges) to generate peptide constructs (e.g., Fab' fragments, F(ab')2 fragments, scFv constructs, "bispecific antibodies" and other multispecific constructs) that are also useful in the methods of the invention. See, e.g., the review by Holliger and Hudson, Nat Biotechnol. 2005 Sep;23(9):1126-36). Generally, if the polypeptide is intended to be administered to a subject (e.g., for prophylactic, therapeutic and / or diagnostic purposes), it will contain immunoglobulin sequences that do not naturally occur in said subject.

[0239] The structure of an immunoglobulin single variable domain sequence can be considered to be composed of four framework regions ("FRs"), which are referred to in the art and herein as "framework region 1" ("FR1"); "framework region 2" ("FR2"); "framework region 3" ("FR3"); and "framework region 4" ("FR4"); these framework regions are interrupted by three complementarity-determining regions ("CDRs"), which are referred to in the art and herein as "complementarity-determining region 1" ("CDR1"); "complementarity-determining region 2" ("CDR2"); and "complementarity-determining region 3" ("CDR3");

[0240] As further described in paragraph q) of pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), the amino acid residues of immunoglobulin single variable domains are similar to those of camel-derived V in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195; see e.g., Figure 2 of this publication). HH As applied to the domain, V shown by Kabat et al. HThey may be numbered according to the common numbering system for domains ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). -V H Domain and V HH It should be noted that, as is well known in the art for domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This generally means that the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. H Domain and V HH The total number of amino acid residues in a domain is usually 110. It will be in the range of 1 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0241] CDR sequences can be determined according to AbM numbering as described in Kontermann and Duebel (eds. 2010, Antibody Engineering, vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 comprises amino acid residues at positions 1-25, CDR1 comprises amino acid residues at positions 26-35, FR2 comprises amino acid residues at positions 36-49, CDR2 comprises amino acid residues at positions 50-58, FR3 comprises amino acid residues at positions 59-94, CDR3 comprises amino acid residues at positions 95-102, and FR4 comprises amino acid residues at positions 103-113.

[0242] The CDR regions can also be determined according to various methods. In determining the CDRs according to Kabat, FR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 1 to 30, CDR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 31 to 35, FR2 of an immunoglobulin single variable domain comprises the amino acid residues at positions 36 to 49, CDR2 of an immunoglobulin single variable domain comprises the amino acid residues at positions 50 to 65, FR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 66 to 94, CDR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 95 to 102, and FR4 of an immunoglobulin single variable domain comprises the amino acid residues at positions 103 to 113.

[0243] In such immunoglobulin sequences, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be apparent to those skilled in the art, for example, based on the standard manuals and further disclosure and prior art referred to herein.

[0244] The framework sequences are immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization) (appropriate combinations of). For example, the framework sequences may be those of a light chain variable domain (e.g., V L -sequence) and / or heavy chain variable domain (e.g., V H -sequence or V HH In one particular embodiment, the framework sequences are derived from V HH -framework sequences derived from conventional V sequences (wherein said framework sequences are optionally partially or fully humanized) or have been camelized (as defined herein) H It can be either an array.

[0245] In particular, the framework sequences present in the ISVD sequences used in the methods described herein may contain one or more of the Hallmark residues (as defined herein), such that the ISVD sequence is a humanized V HH or Camelization V H Contains V HH and NANOBODY® ISVDs such as: Non-limiting examples of (suitable combinations of) such framework sequences will become apparent from the further disclosure herein.

[0246] Again, it is also possible to use any suitable fragment (or combination of fragments) of the foregoing, such as a fragment containing one or more CDR sequences (e.g., in the same order as those CDRs and framework sequences would be present in the full-size immunoglobulin sequence from which the fragment is derived), suitably flanked and / or linked via one or more framework sequences, as generally described herein for immunoglobulin sequences.

[0247] It should be noted, however, that the ISVDs comprised in the multivalent ISVD polypeptides used in the methods of the invention are not limited with respect to the origin of the ISVD sequence (or of the nucleotide sequence used to express it), nor with respect to the manner in which the ISVD sequence or nucleotide sequence is generated (produced) or obtained (obtained). Thus, the ISVD sequence may be a naturally occurring sequence (from an appropriate species) or a synthetic or semi-synthetic sequence. In specific, but non-limiting, embodiments, the ISVD sequence is a naturally occurring sequence (from any appropriate species) or a synthetic or semi-synthetic sequence, including, but not limited to, a "humanized" (as defined herein) immunoglobulin sequence (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, and particularly a partially or fully humanized VHV sequence). HH sequences), "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized V Hsequences), as well as ISVDs obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for manipulating immunoglobulin sequences well known to those skilled in the art; or any suitable combination of any of the foregoing.

[0248] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may be, for example, a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence produced by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, for example mismatch PCR), a nucleotide sequence produced by PCR using overlapping primers, or a nucleotide sequence produced using techniques for DNA synthesis known per se.

[0249] As noted above, the ISVD may be a NANOBODY® ISVD or a suitable fragment thereof. For a general description of NANOBODY® ISVDs, reference is made to the prior art cited herein, as well as to the further description below. However, in this respect, this description and the prior art are not intended to be limiting. H 3 Class" NANOBODY® ISVD (i.e., V such as DP-47, DP-51, or DP-29) H It should be noted that the present disclosure primarily describes ISVDs (ISVDs with a high degree of sequence homology to three classes of human germline sequences). However, in its broadest sense, the ISVD polypeptides used in the methods described herein can generally be any type of NANOBODY® ISVD, and can be any type of ISVD, such as those described in WO2007 / 118670.H NANOBODY® ISVD (i.e., V such as DP-78) belongs to the "Class 4" H It should be noted that four classes of ISVDs with a high degree of sequence homology to human germline sequences can also be used.

[0250] Generally, NANOBODY® ISVDs (especially (partially) humanized V HH Sequence and camelized V H V containing arrays HH A NANOBODY® ISVD may be characterized by the presence of one or more "hallmark residues" (as described herein) in one or more of the framework sequences (again, as further described herein). Thus, in general, a NANOBODY® ISVD may have the (generic) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and where FR1 to FR4 refer to framework regions 1 to 4, respectively, and where CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and where one or and more as further defined herein.

[0251] In particular, nanobodies have the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1-FR4 refer to framework regions 1-4, respectively, and wherein CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and wherein the framework sequences are as further defined herein.

[0252] More particularly, NANOBODY® ISVD has the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein: One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the Hallmark Residues mentioned in Table A below.

[0253] [Table 1]

[0254] 5.3 Multivalent ISVD Polypeptides and Conformational Variants Thereof Methods are provided for the purification or isolation of multivalent ISVD polypeptides comprising or consisting of at least three or at least four ISVDs. The multivalent ISVD polypeptides to be isolated / purified by the method can be obtained by expression in a host. In particular, the multivalent ISVD polypeptides can be obtained by expression in a host that is not a CHO cell. The multivalent ISVD polypeptides can be obtained by expression in a lower eukaryotic host, such as Pichia pastoris, as described herein. Methods are provided for the production, purification, and isolation of multivalent ISVD polypeptides comprising or consisting of at least three or at least four ISVDs. The multivalent ISVD polypeptides to be isolated / purified / produced by the method can be produced in a host, such as a lower eukaryotic host, as described herein. In one embodiment, the multivalent ISVD polypeptides to be isolated / purified / produced by the method can be produced in a yeast host, such as Pichia, e.g., Pichia pastoris, as described herein.

[0255] In general, the term "multivalent" refers to the presence of multiple ISVDs (binding units) in a polypeptide. In one embodiment, the polypeptide is at least "trivalent," i.e., comprises or consists of at least three ISVDs. In another embodiment, the polypeptide is at least "tetravalent," i.e., comprises or consists of at least four ISVDs. Accordingly, the polypeptides produced, purified, and / or isolated in the methods described herein can be "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," "octavalent," "nonevalent," etc., i.e., the polypeptide comprises or consists of three, four, five, six, seven, eight, nine, etc. ISVDs, respectively. In one embodiment, the multivalent ISVD polypeptide is trivalent. In another embodiment, the multivalent ISVD polypeptide is tetravalent. In yet another embodiment, the multivalent ISVD polypeptide is pentavalent.

[0256] Multivalent ISVD constructs comprising or consisting of at least three or at least four ISVDs can be multispecific. The term "multispecific" refers to binding to multiple different target molecules. Thus, multivalent ISVD constructs can be "bispecific," "trispecific," "tetraspecific," etc., i.e., capable of binding to two, three, four, etc. different target molecules, respectively.

[0257] For example, a polypeptide can be bispecific-trivalent, such as a polypeptide comprising or consisting of three ISVDs, where two ISVDs bind to human TNFα and one ISVD binds to human serum albumin (e.g., Compound C, SEQ ID NO: 69). In another example, a polypeptide can be trispecific-tetravalent, such as a polypeptide comprising or consisting of four ISVDs, where one ISVD binds to human TNFα, two ISVDs bind to human IL23p19, and one ISVD binds to human serum albumin (e.g., Compound B, SEQ ID NO: 2); or, such as a polypeptide comprising or consisting of four ISVDs, where one ISVD binds to human TNFα, two ISVDs bind to human IL6, and one ISVD binds to human serum albumin (e.g., Compound D, SEQ ID NO: 70; or Compound E, SEQ ID NO: 71). In yet another example, the polypeptide can be trispecific-pentavalent, for example, a polypeptide comprising or consisting of five ISVDs, where two ISVDs bind to human TNFα, two ISVDs bind to human OX40L, and one ISVD binds to human serum albumin (e.g., Compound A; SEQ ID NO: 1).

[0258] The polypeptide consisting of at least three or at least four ISVDs to be produced / purified / isolated by the methods described herein can be linked by one or more suitable linkers, such as peptide linkers. The use of linkers to connect two or more (poly)peptides is well known in the art. Exemplary peptide linkers are shown in Table B. One frequently used class of peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues, and usually have the structure GGGGS (SEQ ID NO: 4). The amino acid sequence includes one or more repeats of a peptide motif such as the amino acid sequence (e.g., a repeat of the formula (Gly-Gly-Gly-Gly-Ser) n(where n can be 1, 2, 3, 4, 5, 6, 7 or more). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 7), the 15GS linker (n=3) and the 35GS linker (n=7). See, e.g., Chen et al., Adv. Drug Deliv. Rev. 2013 Oct. 15; 65(10):1357-1369; and Kle See, e.g., in Wang et al., Protein Eng. Des. Sel. (2014) 27(10):325-330. In one embodiment, the polypeptide uses a 9GS linker to link components of the polypeptide to each other. In one embodiment, at least three or at least four ISVDs are connected to each other in a linear (i.e., unbranched) sequence, optionally via one or more peptide linkers.

[0259] The polypeptides consisting of at least three or at least four ISVDs to be produced / purified / isolated by the methods of the invention may also contain other groups, residues, moieties, or binding units. These other groups, residues, moieties, or binding units may confer an increased half-life to the polypeptide compared to a corresponding polypeptide that does not contain said one or more other groups, residues, moieties, or binding units. For example, the binding units may be ISVDs that bind to serum proteins, such as human serum albumin (see, e.g., WO2012 / 175400, WO2015 / 173325, WO2017 / 080850, WO2017 / 085172, WO2018 / 104444, WO2018 / 134234, WO2018 / 134235). Furthermore, the polypeptide consisting of at least three or at least four ISVDs to be produced / purified / isolated by the method of the present invention may also contain other suitable groups, residues, moieties or binding units (e.g. tags such as His tags) required for any purification process.

[0260] A polypeptide comprising or consisting of at least three or at least four ISVDs to be produced / purified / isolated by the methods of the invention may also form part of a protein or polypeptide which, for example, comprises one or more further amino acid sequences which are not ISVDs but which provide other functions (all optionally linked via one or more suitable linkers). For example and without limitation, at least three or at least four ISVDs may be used as binding units in such proteins or polypeptides, which may optionally contain one or more further amino acid sequences which are not ISVDs but which may serve as binding units (i.e. for one or more other targets) and / or as functional units.

[0261] [Table 2]

[0262] The multivalent ISVD polypeptide comprising or consisting of at least three or at least four ISVDs to be produced, purified, and / or isolated is the desired product of the production / purification / isolation methods described herein. In this regard, the term "(multivalent ISVD) polypeptide comprising or consisting of at least three or at least four ISVDs" is used interchangeably within the present application with "said polypeptide," "desired polypeptide (product)," "ISVD polypeptide," "desired ISVD polypeptide," "(multivalent) ISVD polypeptide (product)," or "(multivalent) ISVD construct." The desired polypeptide product is also referred to as "product," "intact product," or "intact (ISVD) form." The intact form appears as the main peak in analytical chromatographic techniques such as SE-HPLC and IEX-HPLC.

[0263] "Conformational variants" of multivalent ISVD polypeptides comprising or consisting of at least three or at least four ISVDs are undesirable and should be converted to the desired ISVD polypeptides by the methods described herein and / or interconverted. The conformational variants should be removed from compositions containing the intact product and the conformational variants. The conformational variants are characterized by a more compact form compared to the intact product. Thus, the term "conformational variant" is used interchangeably within this application with "variant," "compact variant," "compact conformational variant," or "compact form."

[0264] Compact variants are characterized by a reduced hydrodynamic volume compared to the desired polypeptide product. Generally, hydrodynamic volume is the apparent volume occupied by an expanded or swollen molecular coil together with absorbed solvent. In other words, hydrodynamic volume is how much space a particular polymer molecule occupies when it is in solution (the effective hydration volume of a polymer in solution). The hydrodynamic volume of a polymer can be estimated from its behavior in solution, for example, from its retention time in size-exclusion chromatography (SEC), and is therefore a size-based mechanical property of a polymer. By measuring the hydrodynamic volume of a protein / polypeptide, SEC can analyze the protein tertiary structure (or even the quaternary structure if appropriate native conditions that preserve macromolecular interactions are used) and can distinguish between folded and unfolded versions, or even folded and unfolded domains of the same protein / polypeptide (but not molecular weight). For example, the apparent hydrodynamic radius of a typical protein domain can be 14 Å and 36 Å for the folded and unfolded forms, respectively. SEC allows the separation of these two forms, as the folded form elutes much slower due to its smaller size.

[0265] Compact variants are characterized by altered surface charge and / or altered hydrophobic exposure (surface hydrophobicity) compared to the desired polypeptide product.

[0266] Without being bound by hypothesis, the compact conformation of the variant results from intramolecular interactions between at least two of the at least three or at least four ISVD building blocks of the polypeptide (compared to the desired polypeptide product). Thus, a conformational variant can be characterized by at least two ISVDs that interact with each other to result in a reduced hydrodynamic volume compared to the desired polypeptide product. Thus, a compact variant can further be characterized by at least two ISVDs that interact with each other to result in an altered surface charge and / or altered surface hydrophobicity compared to the desired polypeptide product.

[0267] Therefore, conformational variants can be distinguished from the desired polypeptide product by a shift in hydrodynamic volume. Furthermore, conformational variants can be distinguished from the desired polypeptide product by a shift in surface charge and / or surface hydrophobicity. Conformational variants and the desired polypeptide product do not differ in their molecular weights. Therefore, conformational variants and the desired polypeptide product cannot be distinguished by their molecular weights. Furthermore, conformational variants and the desired polypeptide product do not differ in their disulfide bridges. Therefore, conformational variants and the desired polypeptide product cannot be distinguished by scrambled disulfide bridges.

[0268] Due to the changes described above, conformational variants and the desired polypeptide product can be distinguished by the altered retention time of the conformational variant compared to the desired polypeptide product observed in analytical and / or preparative chromatographic techniques. For example, conformational variants can be distinguished from the desired polypeptide product by one or more analytical chromatographic techniques, such as SE-HPLC and / or IEX-HPLC. In particular, conformational variants can be distinguished from the desired polypeptide product by a shift in hydrodynamic volume, as described above. The change in surface charge of the conformational variant compared to the intact product is indicated by an increased retention time in analytical SE-HPLC. Furthermore, conformational variants can be distinguished from the desired polypeptide product by a shift in surface charge, which is indicated by an altered retention time in analytical IEX-HPLC. The increased retention time of the conformational variant compared to the intact product can be identified in analytical SE-HPLC as a post-peak shoulder or as a separated post-peak in the SE-HPLC chromatogram. The change in surface charge of the conformational variant compared to the intact product can be identified in analytical IEX-HPLC as a pre-peak shoulder or a separated pre-peak, respectively, in analytical IEX-HPLC, or as a post-peak shoulder or a separated post-peak in the IEX-HPLC chromatogram. As will be apparent to those skilled in the art, whether the retention time of the conformational variant compared to the intact product decreases or increases depends on both the quality and quantity of the surface charge of the conformational variant compared to the intact product, as well as on the conditions used in IEX-HPLC (e.g., resin, buffer, pH, salt concentration / ionic strength, etc.). Thus, in one embodiment, the conformational variant is characterized by an increased retention time in IEX-HPLC. In another embodiment, the conformational variant is characterized by a decreased retention time in IEX-HPLC. Thus, the conformational variant is characterized by an increased retention time in SE-HPLC compared to the intact product. The conformational variant may also be characterized by an altered (decreased or increased) retention time in IEX-HPLC compared to the intact product.

[0269] Due to the above changes, conformational variants can also be distinguished from the intact product by one or more preparative chromatographic techniques, such as size exclusion chromatography (SEC), ion exchange chromatography (IEX), e.g., cation exchange chromatography (CEX), mixed-mode chromatography (MMC), and / or hydrophobic interaction chromatography (HIC). In particular, conformational variants can be distinguished from the (desired) polypeptide by their presence in different fractions obtained from the above preparative chromatographic techniques (due to the altered retention time of the conformational variant compared to the desired polypeptide product observed in the above preparative chromatographic techniques). For example, a conformational variant can be characterized by its presence in side fractions in preparative IEX (e.g., CEX), preparative MMC (e.g., based on a hydroxyapatite resin), and / or HIC (e.g., based on an HIC column resin or HIC membrane) compared to the desired polypeptide product eluting as the top fraction. As will be apparent to those skilled in the art, whether a conformational variant elutes as a pre- or post-side fraction, i.e., whether it elutes at a decreased or increased retention time, respectively, will depend on both the quality and quantity of the difference in surface charge and / or surface hydrophobicity of the conformational variant compared to the desired polypeptide product, and also on the conditions (e.g., resin, buffer, pH, salt concentration / ionic strength, etc.) used in each preparative chromatographic technique employed.

[0270] Thus, after identification of conformational variants by specific analytical chromatographic techniques provided herein, such as SE-HPLC and / or IEX-HPLC, one skilled in the art can adjust / optimize preparative chromatographic techniques to remove conformational variants.

[0271] In a further embodiment, conformational variants can be distinguished from the desired polypeptide product by altered potency, where the conformational variant has decreased potency (as defined herein) compared to the desired polypeptide product.

[0272] Furthermore, conformational variants can be distinguished from desired polypeptide products by their ability to be converted to the desired polypeptide product in processing methods such as those described herein. More particularly, conformational variants are those that: i) applying a low pH treatment in one or more steps of the isolation and / or purification process; ii) applying a chaotropic agent in one or more steps of the isolation and / or purification method; iii) applying heat stress during one or more steps of the isolation and / or purification process; or iv) Any combination of i) to iii) The present invention is characterized by its ability to be converted to the desired polypeptide product upon analytical chromatographic analysis using one or more analytical chromatographic techniques, such as SE-HPLC and / or IEX-HPLC, where the conversion is demonstrated by one or more analytical chromatographic techniques, such as SE-HPLC and / or IEX-HPLC. In particular, the conversion is demonstrated by a reduction or even disappearance of a post-peak shoulder or isolated post-peak in an analytical SE-HPLC chromatogram. Additionally, or alternatively, the conversion is demonstrated by a reduction or even disappearance of a pre-peak shoulder or isolated pre-peak, or a post-peak shoulder or isolated post-peak in an analytical IEX-HPLC chromatogram.

[0273] Additionally, or alternatively, conversion is demonstrated by partial or complete restoration of potency relative to the potency of the desired polypeptide product.

[0274] 5.4 Manufacturing / purification / isolation methods Methods for isolating or purifying the above-described multivalent ISVD polypeptide products are provided, wherein the multivalent ISVD polypeptide to be isolated or purified can be obtained by expression in a host. In one embodiment, the host is not a CHO cell. In one embodiment, the host is a lower eukaryotic host as described herein (Section 5.3 "Multivalent ISVD Polypeptides and Conformational Variants Thereof"). As used herein, the terms "purify," "purify," or "purifying" refer to a composition comprising a desired multivalent ISVD polypeptide product and conformational variants being free of contaminants, including conformational variants. As used herein, the terms "isolate," "isolated," or "isolating" refer to the separation or separation of a desired multivalent polypeptide product from a composition comprising both the desired multivalent ISVD polypeptide product and its conformational variants in addition to contaminants.

[0275] Additionally provided are methods for producing a multivalent ISVD polypeptide product in a host. In one embodiment, the host is not a CHO cell. In one embodiment, the host is a lower eukaryotic host as described herein. The method comprises transforming / transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, followed by one or more isolation and / or purification steps. In particular, the method for producing a multivalent ISVD polypeptide product comprises: a) expressing a nucleic acid sequence encoding said polypeptide in a suitable host cell or host organism or in another suitable expression system; followed by: b) isolating and / or purifying the desired polypeptide may include:

[0276] In a significant fraction of multivalent ISVD polypeptides produced by hosts, such as lower eukaryotic host cells, the presence of product-associated conformational variants has been observed. The presence of these conformational variants can affect the quality and selectivity of the final multivalent ISVD polypeptide product. However, high quality and uniformity of the product is essential, for example, for therapeutic uses of these multivalent ISVD polypeptide products.

[0277] The present application describes methods for the production / purification / isolation of compositions comprising multivalent ISVD polypeptide products with improved quality (i.e., with reduced levels or no conformational variants). Quality is improved by applying specific conditions: (1) conformational variants are converted to the desired polypeptide product; and / or (2) conformational variants are removed during the isolation or purification process of the multivalent ISVD polypeptide. Thus, methods are provided herein for converting product-associated conformational variants to a desired ISVD-containing polypeptide product. Methods are also provided for removing product-associated conformational variants from compositions comprising a (desired) polypeptide product and its conformational variants. Methods are provided for converting product-associated conformational variants to a (desired) ISVD polypeptide product and for removing product-associated conformational variants from compositions comprising a (desired) ISVD polypeptide product and its conformational variants.

[0278] 5.4.1 Production of Polypeptides Comprising or Consisting of at Least Three or at Least Four ISVDs The present inventors have identified conformational variants of a polypeptide comprising or consisting of at least three or at least four ISVDs upon production of that polypeptide in a host, which conformational variants have been observed upon production in a host, particularly a host that is a lower eukaryotic host as set forth herein.

[0279] Those skilled in the art are well aware of general methods for producing immunoglobulin single variable domains in host cells.

[0280] In a typical embodiment, the method for producing a polypeptide comprising at least three or at least four immunoglobulin single variable domains (ISVDs) comprises one or more purification / isolation steps that result in the conversion of conformational variants to the desired ISVD polypeptide product and / or the removal of conformational variants from a composition comprising the desired ISVD polypeptide product and its conformational variants, as further detailed below in Sections 5.4.3 "Conversion of Conformation Variants to Desired Polypeptide Products" and 5.4.4 "Removal of Conformation Variants."

[0281] More particularly, the method for producing a polypeptide comprising at least three or at least four ISVDs comprises the steps of: a) optionally culturing the host or host cells under conditions such that the host or host cells grow; b) maintaining the host or host cell under conditions such that the host or host cell expresses and / or produces the polypeptide; and c) isolating and / or purifying the secreted polypeptide from the culture medium, wherein said isolating and / or purifying comprises one or more purification / isolation steps that result in conversion of the conformational variant to the desired ISVD polypeptide product and / or removal of the conformational variant from a composition comprising the desired ISVD polypeptide product and its conformational variant; At least includes.

[0282] The ISVD polypeptide to be isolated / purified by the methods described herein can be produced in a host. The host can be a host that is not a CHO cell. In particular, the host can be a lower eukaryotic host, such as a yeast organism. The polypeptide to be isolated / purified can be produced in a host that is not a CHO cell. In particular, the host can be a lower eukaryotic host, such as a yeast organism. Suitable yeast organisms for the production of polypeptides include Pichia (Komagataella), Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citromyces, Pachysolen, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, and Endomycopsis. In certain embodiments, the polypeptide to be purified / isolated is produced in Pichia, particularly Pichia pastoris.

[0283] Production of ISVDs in lower eukaryotic hosts, such as Pichia pastoris, is described in Frenken et al. (2000) (J. Biotechnol. 78:11-21), WO 94 / 25591, WO 2010 / 125187, WO 2012 / 056000, WO 2012 / 152823, and WO 2017 / 137579. The contents of these applications are expressly incorporated by reference with respect to general culture techniques and methods, including appropriate media and conditions. Those skilled in the art are also able to construct appropriate genetic constructs for expression of the domains in host cells based on well-known general knowledge.

[0284] The terms "host organism" and "host cell" are referred to herein jointly as "host." Any host (organism) or host cell may be used in the production methods described herein, provided that they are suitable for producing an ISVD-containing polypeptide. In particular, hosts (e.g., lower eukaryotic hosts) are described in which portions of the polypeptide are produced in the form of product-related conformational variants.

[0285] Examples of suitable hosts include prokaryotes such as Coryneform bacteria or Enterobacteriaceae. Insect cells, particularly insect cells suitable for baculovirus-mediated recombinant expression, such as cells from Trioplusiani or Spodoptera frugiperda, including but not limited to BTI-TN-5B1-4 High Five TMInsect cells (Invitrogen), SF9, or Sf21 cells; mammalian cells, such as CHO cells; and lower eukaryotic hosts, including yeast, such as Pichia (Komagataella), Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Cytheromyces, Pachysolenes, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, and Endomycopsis. In one embodiment, yeast, such as Pichia pastoris, is used as a host.

[0286] The host used in the production method is capable of producing an ISVD-containing polypeptide. Typically, it is genetically modified to contain one or more nucleic acid sequences encoding one or more ISVD-containing polypeptides. Non-limiting examples of genetic modification include, for example, transformation with a plasmid or vector, or transduction with a viral vector. Some hosts can be genetically modified by fusion techniques. Genetic modification includes the introduction of a separate nucleic acid molecule into the host, e.g., a plasmid or vector, as well as direct modification of the host's genetic material, e.g., by integration into the host's chromosome, e.g., by homologous recombination. A combination of both often occurs; for example, the host is transformed with a plasmid that is (at least partially) integrated into the host's chromosome upon homologous recombination. Those skilled in the art will know suitable genetic modification methods to enable the host to produce an ISVD-containing polypeptide.

[0287] Specific conditions and gene constructs for the expression of nucleic acids and production of polypeptides have been described in the art, for example in WO 94 / 25591, Gasser et al. Biotechnol. Bioeng. 94:535, 2006; Gasser et al. Appl. Environ. Microbiol. 73:6499, 2007; or Damasceno et al. Microbiol. Biotechnol. 74:381, 2007 General culture methods, plasmids, promoters and leader sequences.

[0288] 5.4.2 Purification of Polypeptides Comprising or Consisting of at Least Three or at Least Four ISVDs Those skilled in the art will recognize that ISVD polypeptides (e.g., V H and V HH ) are well known in the art.

[0289] For example, the purification of ISVD is described in WO 2010 / 125187 and WO 2012 / 056000.

[0290] After production / expression of the polypeptide, the host can be removed from the medium by conventional means. For example, the host can be removed by centrifugation or filtration. The solution obtained by removing the host from the medium is also called a culture supernatant or a clarified culture supernatant.

[0291] The multivalent ISVD product can be purified from the culture supernatant by standard methods. Standard methods include, but are not limited to, chromatographic methods, including size-exclusion chromatography (SEC), ion-exchange chromatography (IEX), affinity chromatography (AC), hydrophobic interaction chromatography (HIC), and mixed-mode chromatography (MMC). These methods can be performed alone or in combination with other purification methods, such as precipitation. Those skilled in the art can devise appropriate combinations of purification methods for ISVDs and ISVD-containing polypeptides based on well-known general knowledge. For specific examples, reference is made to the techniques cited herein.

[0292] As described in detail below (Sections 5.4.3 "Conversion of Conformational Variants to Desired Polypeptide Product" and 5.4.4 "Removal of Conformational Variants"), it is contemplated that any or a combination of conditions that convert or remove conformational variants may be applied before, at or during any step, or after any step of these purification methods.

[0293] Any or all of the chromatography steps can be performed by any mechanical means. Chromatography can be performed, for example, in a column. The column can be flowed from top to bottom or bottom to top, with or without pressure. The direction of fluid flow in the column can be reversed during the chromatography process. Chromatography can also be performed in a batch process, where the solid medium is separated from the liquid used to load, wash, and elute the sample by any suitable means, including gravity, centrifugation, or filtration.

[0294] Chromatography can also be performed by contacting the sample with a filter that absorbs or retains some molecules in the sample more strongly than others. In the following description, various embodiments are described largely in the context of chromatography performed in a column. However, it is understood that the use of a column is only one of several chromatography modes that can be used, and that the description using a column does not limit application to column chromatography, as one of skill in the art can readily apply the teachings to other modes, such as batch processes or modes using filters.

[0295] Suitable supports may be any currently available or later developed material having the necessary characteristics to carry out the claimed methods, and may be based on any synthetic, organic, or natural polymer. For example, commonly used support materials include cellulose, polystyrene, agarose, sepharose, polyacrylamide, poly(vinyl alcohol), and the like. Suitable solid supports include organic materials such as methacrylates, dextran, and starch, and inorganic materials such as charcoal, silica (glass beads or sand), and ceramic materials. Suitable solid supports are, for example, those listed in Zaborsky "Immobilized Enzymes" CRC Press, 1973, pp. 28-46, Table IV.

[0296] General method conditions, solutions and / or buffers, as well as their concentration ranges for use in various chromatographic processes, can be determined by those skilled in the art of chromatography based on standard chromatographic manuals (e.g., Guenter Jagschies, Eva Lindskog (eds.) Biopharmaceutical Processing, Development, Design, and Implementation of Manufacturing Processes, 1st ed. 2017, Elsevier).

[0297] The first step in an ISVD polypeptide purification process is often referred to as the "capture step." The purpose of the capture step is to have a first reduction of process-related impurities (e.g., but not limited to, host cell proteins (HCPs), color, and DNA) and to capture the ISVD polypeptide product while maintaining high recovery. In one embodiment, the capture step refers to the initial purification with Protein A chromatography in bind and elute mode.

[0298] The second step of the purification process, often referred to as a "polishing step," aims to improve purity. For example, as the second purification step of the ISVD polypeptide purification process, an ion exchange chromatography step in bind-and-elute mode can be used to remove / reduce product-related variants (e.g., but not limited to, high molecular weight (HMW) species, low molecular weight (LMW) species, and other charged variants), as well as some process-related impurities still present after the capture step (e.g., but not limited to, HCP, residual Protein A, DNA).

[0299] In one exemplary embodiment, multivalent ISVD polypeptides can be purified from culture supernatants by a combination of affinity chromatography with Protein A, ion exchange chromatography, and size exclusion chromatography. Any reference to a "purification step" includes, but is not limited to, these specific methods.

[0300] Protein A-based chromatography In one embodiment, an ISVD polypeptide-containing preparation can be purified by protein A chromatography. Staphylococcal protein A (SpA) is a 42 kDa protein composed of five nearly homologous domains designated E, D, A, B, and C, from the N-terminus (Sjodhal Eur. J. Biochem. 78:471-490 (1977); Uhlen et al. J. Biol. Chem. 259:1695-1702 (1984)). These domains contain approximately 58 residues and share approximately 65% ​​to 90% amino acid sequence identity with each other. Binding studies between Protein A and antibodies have shown that all domains of SpA (E, D, A, B, and C) bind to IgG via their Fc regions, but domains D and E show significant Fab binding (Ljungberg et al. Mol. Immunol. 30(14):1279-1285 (1993); Roben et al. J. Immunol. 154:6437-6445 (1995); Starovasnik et al. Protein Sei. 8:1423-1431 (1999)). The Z domain, a functional analog and energy-minimized version of the B domain (Nilsson et al. Protein Eng. 1:107-113 (1987)), has been shown to have negligible binding to antibody variable domain regions (Cedergren et al. Protein Eng. 6(4):441-448 (1993); Ljung Berg et al. (1993) supra; Starovasnik et al. (1999) supra).

[0301] Until recently, commercially available Protein A stationary phases employed SpA (isolated from Staphylococcus aureus or recombinantly expressed) as their immobilized ligand. The use of non-proteinaceous ligands precluded the use of alkaline conditions for column regeneration and sanitization, as is typically done with other modes of chromatography (Ghose et al., Biotechnology and Bioengineering Yol. 92 (6):665-73 (2005)). New resins (MabSELECT) have been developed to withstand stronger alkaline conditions. TM A new ligand, SuRe, was developed (Ghose et al. (2005) supra). Using protein engineering techniques, multiple asparagine residues were replaced in the Z domain of protein A, and the new ligand was generated as a tetramer of four identically modified Z domains (Ghose et al. (2005) supra).

[0302] Thus, the purification method can be performed using a commercially available Protein A column according to the manufacturer's specifications. TM Column or MabSELECT TM SuRe columns (GE Healthcare Products) can be used. TM is a commercially available resin that contains recombinant SpA as its immobilized ligand. Other commercially available sources of Protein A columns can be usefully employed, including, but not limited to, PROSEP-ATM (Millipore, UK), which consists of Protein A covalently coupled to controlled pore glass. Other useful Protein A formulations include Protein A Sepharose FAST FLOW TM (Amersham Biosciences, Piscataway, NJ), Amsphere TM A3 (JSR Life Sciences), and TOYOPEARL TM 650M Protein A (TosoHaas Co., Philadelphia, PA).

[0303] Protein purification by Protein A-based chromatography can be performed on a column containing immobilized Protein A ligand (typically a column packed with agarose beads or a modified support of methacrylate copolymers to which an adsorbent consisting of Protein A or its functional derivatives has been immobilized). The column is typically equilibrated with a buffer, and a sample containing a mixture of proteins (target protein and contaminating proteins) is loaded onto the column. As the compound passes through the column, the target protein binds to the adsorbent (Protein A or its derivative) within the column, while some unbound impurities and contaminants flow through. The bound protein is then eluted from the column. In this process, the target protein is bound to the column while the impurities flow through. The target protein is then recovered from the eluate.

[0304] In a general embodiment, a method for purifying / isolating a polypeptide comprising at least three or at least four immunoglobulin single variable domains (ISVDs) is provided, wherein the method comprises one or more purification / isolation steps that result in the conversion of conformational variants to the desired ISVD polypeptide product and / or the removal of conformational variants from a composition comprising the desired ISVD polypeptide product and its conformational variants, as further detailed in Section 5.4.3 "Conversion of Conformation Variants to a Desired Polypeptide Product" and Section 5.4.4 "Removal of Conformation Variants."

[0305] 5.4.3 Conversion of Conformational Variants to Desired Polypeptide Products In one embodiment, a composition comprising a polypeptide product and a conformational variant thereof is prepared by applying conditions that convert the conformational variant into the desired polypeptide product. It is more refined.

[0306] In this aspect, the conditions for converting the conformational mutants to the desired polypeptide product can be selected from a) applying a low pH treatment, b) applying a chaotropic agent, c) applying heat stress, and d) applying a combination of any of the treatments a) to c). For example, in one aspect, the conformational mutants are converted to the desired polypeptide product by applying a low pH treatment and a chaotropic agent. In another embodiment, the conformational mutants are converted to the desired polypeptide product by applying a low pH treatment and a heat treatment. In a further embodiment, the conformational mutants are converted to the desired polypeptide product by applying heat stress and a chaotropic agent. In yet another embodiment, the conformational mutants are converted to the desired polypeptide product by applying a low pH treatment, a chaotropic agent, and heat stress.

[0307] Conditions that convert conformational variants to the desired polypeptide product can be applied (without limitation) to the culture supernatant containing the multivalent ISVD polypeptide (before the capture step), during the capture step, before the capture step but before the polishing step, during the polishing step, or after the polishing step. Conditions that convert conformational variants to the desired polypeptide product can be applied to a partially or highly purified preparation of the multivalent ISVD polypeptide. Conditions that convert conformational variants to the desired polypeptide product can also be applied on a column, to a clarified supernatant, or to a partially or highly purified preparation of the ISVD-containing polypeptide. Conditions that convert conformational variants to the desired polypeptide product can also be applied during another step, such as before or after a filtration step, or any other step in the purification.

[0308] Below, conditions that convert the conformational variant into the desired polypeptide product are discussed in more detail. The application of these conditions is also referred to as "treating" the multivalent ISVD polypeptide.

[0309] Low pH treatment The conformational mutant can be converted to the desired polypeptide by low pH treatment.

[0310] The low pH treatment can be applied at any time during the method for purifying / isolating a multivalent ISVD polypeptide. In one embodiment, the low pH treatment is applied before a purification step based on a chromatography technique. In another embodiment, the low pH treatment is applied during a purification step based on a chromatography technique, such as Protein A-based affinity chromatography (AC). For example, the low pH treatment can be applied during the ISVD polypeptide capture step of Protein A-based affinity chromatography. In another embodiment, the low pH treatment is applied after a purification step based on a chromatography technique. For example, the low pH treatment can be applied after the ISVD polypeptide capture step of Protein A-based affinity chromatography (and before the ISVD polypeptide polishing step). Alternatively, the low pH treatment can be applied after the ISVD polypeptide polishing step.

[0311] Low pH treatment involves lowering the pH of a composition containing a desired polypeptide product and its conformational variants to about pH 3.2 or below for a sufficient amount of time to convert the conformational variants to intact ISVD polypeptide products.

[0312] The low pH treatment reduces the pH of the composition containing the desired polypeptide product and its conformational variants to about pH 3.0 or below, so that the conformational variants remain intact in the ISV form. The method comprises lowering the enzyme for a sufficient amount of time to convert the enzyme to a D polypeptide product.

[0313] Thus, low pH treatment includes lowering the pH of a composition comprising an intact polypeptide product and its conformational variants (e.g., a capture eluate following a (Protein A) capture step) to about pH 3.2 or less, about pH 3.1 or less, about pH 3.0 or less, about pH 2.9 or less, about pH 2.8 or less, about pH 2.7 or less, about pH 2.6 or less, about pH 2.5 or less, about pH 2.4 or less, about pH 2.3 or less, about pH 2.2 or less, or even about pH 2.1 or less. Specifically, the pH of the composition may be lowered to about pH 2.9, about pH 2.8, about pH 2.7, about pH 2.6, about pH 2.5, about pH 2.4, about pH 2.3, about pH 2.2, or about pH 2.1. In one embodiment, the pH is reduced to between about pH 3.2 and about pH 2.1, between about pH 3.0 and about pH 2.1, between about pH 2.9 and about pH 2.1, or between about pH 2.7 and about pH 2.1. In another embodiment, the pH is reduced to between about pH 2.6 and about pH 2.3. In another embodiment, the pH is reduced to between about pH 2.5 and about pH 2.1.

[0314] In the low pH treatment, the pH can be lowered by any conventional means. For example, the pH of a composition containing a desired polypeptide product and its conformational variants can be lowered using HCl (e.g., at a stock concentration of 0.1 M to 3 M, e.g., 0.1 M, 1 M, 3 M, or 2.7 M) or glycine (e.g., at a stock concentration of 0.1 M). Those skilled in the art can easily select other suitable means.

[0315] In one embodiment, the low pH treatment is applied during a purification step based on a chromatographic technique, e.g., Protein A-based affinity chromatography. The elution buffer used for Protein A-based affinity chromatography may have a pH equal to or less than about pH 2.5. Alternatively, the elution buffer used for Protein A-based affinity chromatography may have a pH such that the resulting eluate containing the polypeptide has a pH equal to or less than about pH 3.2, e.g., less than about pH 2.9. After elution of the polypeptide from the Protein A column using an elution buffer as described above, the pH of the resulting eluate containing the polypeptide may (optionally) be further lowered to a pH equal to or less than about pH 2.5. In another embodiment, the pH of the resulting eluate may be adjusted to a pH equal to or less than about pH 3.2 for at least about 0.5 hours, e.g., 1 or 2 hours. In another embodiment, the pH of the resulting eluate may be adjusted to a pH equal to or less than about pH 2.9 for at least about 0.5 hours, e.g., 1 or 2 hours. In yet another embodiment, the pH of the resulting eluate may be adjusted to a pH of about pH 2.7 or less for at least about 1 hour. In another embodiment, the chromatographic technique is Protein A-based affinity chromatography, wherein the elution buffer has a pH of about pH 2.2, and wherein the pH of the resulting eluate is adjusted to a pH of about pH 2.5 for at least about 1.5 hours.

[0316] The present technology also provides a method for identifying conformational variants of polypeptides comprising or consisting of at least three or at least four ISVDs by analytical chromatographic methods such as SE-HPLC and IEX-HPLC. The present technology further provides a concept for converting conformational variants into intact products by low pH treatment. Therefore, based on the concept provided herein, one skilled in the art can adjust the low pH treatment described herein in terms of the optimal acidic pH and incubation time for any polypeptide comprising or consisting of at least three or at least four ISVDs.

[0317] The low pH treatment can be terminated by increasing the pH of the composition containing the polypeptide. The low pH treatment can be terminated by increasing the pH of the low pH-treated composition by at least one pH unit. For example, if the low pH treatment is performed at about pH 2.7, the treatment can be terminated by increasing the pH to at least about pH 3.7. The low pH treatment can be terminated by increasing the pH of the low pH-treated composition by at least two pH units. For example, if the low pH treatment is performed at about pH 2.7, the treatment can be terminated by increasing the pH to at least about pH 4.7. Thus, the low pH treatment can be terminated by increasing the pH to about pH 3.5 or higher, about pH 4.0 or higher, about pH 4.5 or higher, about pH 5.0 or higher, about pH 5.5 or higher, about pH 6.0 or higher, about pH 6.5 or higher, about pH 7.0 or higher, about pH 7.5 or higher, about pH 8.0 or higher, etc. However, increasing the pH too high (e.g., to about pH 9 or above) may result in (serious) degradation of the polypeptide product. Therefore, the low pH treatment is terminated by increasing the pH to between about pH 4 and about pH 8, or between about pH 5 and about pH 7.5. As will be clear to those skilled in the art, the pH increase can be adapted to the pH required for possible subsequent purification, formulation, or storage steps. In this application, termination of the low pH treatment is used interchangeably with "pH neutralization."

[0318] To terminate the low pH treatment, the pH can be increased by any conventional means. For example, without limitation, the pH of the composition can be increased using NaOH (e.g., at a stock concentration of 0.1 M or 1 M) or sodium acetate (e.g., at a stock concentration of 1 M). Those skilled in the art can easily select other suitable means.

[0319] Based on the methods described herein, one skilled in the art can determine the time necessary to convert a conformational variant into a desired polypeptide product. For example, low pH treatment is applied for a sufficient amount of time until the conformational variant is essentially no longer detectable by the chromatographic techniques described herein. For example, low pH treatment is applied for a sufficient amount of time until neither a post-peak shoulder nor a separated post-peak (indicating a conformational variant) is essentially observable in a chromatogram of the composition after low pH treatment using analytical SE-HPLC. Additionally, or alternatively, low pH treatment is applied for a sufficient amount of time until neither a pre / post-peak shoulder nor a separated pre / post (indicating a conformational variant) is essentially observable in a chromatogram of the composition after low pH treatment using analytical IEX-HPLC. In this regard, the low pH treatment can be applied for at least about 0.5 hours, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 2.5 hours, at least about 3 hours, at least about 3.5 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, or at least about 24 hours. For example, the low pH treatment can be applied for about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, or about 24 hours. In certain embodiments, the low pH treatment can be applied for at least about 1 hour, or at least about 2 hours, or at least about 4 hours.

[0320] In one embodiment, the pH is lowered to between about pH 3.2 and about pH 2.1 for at least 0.5 hours, between about pH 2.9 and about pH 2.1 for at least 0.5 hours, between about pH 2.7 and about pH 2.1 for at least 0.5 hours, for example, to about pH 2.9, about pH 2.7, about pH 2.5, or about pH 2.3 for 0.5 hours. In another embodiment, the pH is lowered to between about pH 3.2 and about pH 2.1 for at least 1 hour, between about pH 2.9 and about pH 2.1 for at least 1 hour, between about pH 2.7 and about pH 2.1 for at least 1 hour, for example, to about pH 2.9, about pH 2.7, about pH 2.5, or about pH 2.3 for 1 hour. In yet another embodiment, the pH is lowered to between about pH 3.2 and about pH 2.1 for at least 2 hours, between about pH 2.9 and about pH 2.1 for at least 2 hours, or to about pH 2.9. The pH is lowered to between about pH 3.2 and about pH 2.1 for at least 2 hours, between about pH 2.7 and about pH 2.1 for at least 2 hours, e.g., to about pH 2.9, about pH 2.7, about pH 2.5, or about pH 2.3 for 2 hours. In yet another embodiment, the pH is lowered to between about pH 3.2 and about pH 2.1 for at least 4 hours, between about pH 2.9 and about pH 2.1 for at least 4 hours, between about pH 2.7 and about pH 2.1 for at least 4 hours, e.g., to about pH 2.9, about pH 2.7, about pH 2.5, or about pH 2.3 for 4 hours. In another embodiment, the pH is lowered to between about pH 2.6 and about pH 2.3 for at least 1 hour, or at least 2 hours, e.g., to about pH 2.6 for 1 or 2 hours. In another embodiment, the pH is lowered to between about pH 2.5 and about pH 2.1 for at least 1 hour, or at least 2 hours, e.g., to about pH 2.4 or pH 2.5 for 2 hours.

[0321] Low pH treatment can be applied over a wide range of temperatures, provided that the temperature does not irreversibly denature or degrade the ISVD polypeptide. Non-limiting examples include temperatures between about 4°C and about 30°C. Thus, low pH treatment can be applied at temperatures of about 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, or 4°C. Those skilled in the art can readily select a suitable temperature for low pH treatment. In one embodiment, low pH treatment is applied at a temperature between about 15°C and about 30°C. In another embodiment, low pH treatment is applied at a temperature between about 4°C and about 12°C. In another embodiment, the low pH treatment is applied at room temperature (RT), ie, between about 20°C and 25°C.

[0322] Chaotropic agent treatment Conformational mutants can also be converted to the desired polypeptide product by applying a chaotropic agent.

[0323] Chaotropic agents generally disrupt inter- and intramolecular interactions mediated by non-covalent forces such as hydrogen bonding, van der Waals forces, and hydrophobic interactions, thereby increasing the entropy of the system. With respect to biomolecules, chaotropic agents can disrupt and denature the structure of macromolecules such as proteins and nucleic acids (e.g., DNA and RNA). Chaotropic agents are well known to those skilled in the art and include (but are not limited to) n-butanol, ethanol, guanidinium chloride (GuHCl), lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. In one embodiment, conformational mutants are converted to the desired polypeptide product by applying a chaotropic agent that is GuHCl or urea. In a specific embodiment, conformational mutants are converted to the desired polypeptide product by applying a chaotropic agent that is GuHCl.

[0324] The chaotropic agent can be applied at any time during the method for purifying / isolating a multivalent ISVD polypeptide. In one embodiment, the chaotropic agent is applied before a purification step based on a chromatography technique (e.g., before an ISVD polypeptide capture step or before an ISVD polypeptide polishing step). In another embodiment, the chaotropic agent is applied after a purification step based on a chromatography technique (e.g., after an ISVD polypeptide capture step or after an ISVD polypeptide polishing step). In another embodiment, the chaotropic agent is applied immediately after a purification step based on a chromatography technique, where the chromatography technique is Protein A-based affinity chromatography (e.g., used in an ISVD polypeptide capture step). Thus, in one embodiment, the chaotropic agent is applied immediately after a Protein A-based ISVD polypeptide capture step and before a polishing step. In another embodiment, the chaotropic agent is applied immediately after an ISVD polypeptide polishing step. .

[0325] Those skilled in the art are well aware that chaotropic agents must be applied at a concentration that allows for the conversion of conformational variants to the desired polypeptide product without causing their irreversible denaturation or degradation. Based on the methods described herein, those skilled in the art can determine which concentration of chaotropic agent is appropriate for converting conformational variants to the desired polypeptide product. An appropriate concentration has been applied when the conformational variants are no longer detectable by the chromatographic techniques described herein. For example, an appropriate concentration has been applied when, using analytical SE-HPLC, essentially no post-peak shoulder or separated post-peaks (indicating conformational variants) are observed in the chromatogram of the composition after chaotropic agent treatment. Additionally or alternatively, an appropriate concentration has been applied when, using analytical IEX-HPLC, essentially no pre / post-peak shoulder or separated pre / post-peaks (indicating conformational variants) are observed in the chromatogram of the composition after chaotropic agent treatment. Irreversible denaturation or degradation of the ISVD polypeptide product by the chaotropic agent can be ruled out if the respective SE-HPLC or IEX-HPLC chromatograms do not show the formation of high molecular weight species (HMW species) (pre-peak in SE-HPLC) and / or a decrease in the total area (loss of product) or main peak in IEX-HPLC and / or SE-HPLC.

[0326] In one embodiment, the chaotropic agent is GuHCl at a final concentration of between about 0.5 molar (M) and about 3 M, between about 0.5 M and about 2.5 M, between about 1 M and about 2.5 M, between about 1 M and about 2 M, e.g., about 1 M, about 2 M, about 2.5 M, or about 3 M. In another embodiment, the chaotropic agent is GuHCl at a final concentration of at least about 1 M, or at least about 2 M.

[0327] Based on the methods described herein, one skilled in the art can determine the time required to convert a conformational variant into a desired polypeptide product. For example, chaotropic agent treatment is applied for a sufficient amount of time until the conformational variant is no longer essentially detectable by the chromatographic techniques described herein. For example, chaotropic agent treatment is applied for a sufficient amount of time until essentially no post-peak shoulder or separated post-peak (indicating a conformational variant) is observed in a chromatogram of the composition after chaotropic agent treatment using analytical SE-HPLC. Additionally, or alternatively, chaotropic agent treatment is applied for a sufficient amount of time until essentially no pre- / post-peak shoulder or separated pre- / post-peak (indicating a conformational variant) is observed in a chromatogram of the composition after chaotropic agent treatment using analytical IEX-HPLC. Those skilled in the art are well aware that a chaotropic agent must be applied for a time that allows for the conversion of a conformational variant into a desired polypeptide product without causing irreversible denaturation or degradation of the conformational variant. Irreversible denaturation or degradation of the ISVD polypeptide product by the chaotropic agent can be ruled out if the respective SE-HPLC or IEX-HPLC chromatograms show no formation of high molecular weight species (HMW species) (pre-peak in SE-HPLC) and / or no decrease in total area (loss of product) or main peak in IEX-HPLC and / or SE-HPLC. In this regard, chaotropic agent treatment can be applied for at least about 0.5 hours, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 2.5 hours, at least about 3 hours, at least about 3.5 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, or at least about 12 hours. For example, the chaotropic agent can be applied for about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 6 hours, about 8 hours, or about 12 hours. In one embodiment, the chaotropic agent is applied for at least about 0.5 hours, or It may be applied for at least about 1 hour.

[0328] In this aspect, GuHCl is applied for at least about 0.5 hours, or for at least about 1 hour. In one embodiment, the chaotropic agent is GuHCl at a final concentration of between about 1 M and about 2 M for about 0.5 hours. In another embodiment, the chaotropic agent is GuHCl at a final concentration of between about 1 M and about 2 M for about 1 hour.

[0329] The present technology provides a method for identifying conformational variants of polypeptides comprising or consisting of at least three or at least four ISVDs by analytical chromatographic methods such as SE-HPLC and IEX-HPLC. The present technology further provides the concept of converting conformational variants into intact products by treatment with a chaotropic agent. Therefore, based on the concept provided herein, one skilled in the art can adjust the chaotropic agent treatment described herein in terms of chaotropic agent concentration as well as incubation time for any polypeptide comprising or consisting of at least three or at least four ISVDs.

[0330] The chaotropic agent treatment can be terminated by transferring the ISVD polypeptide product into a new buffer system (free of chaotropic agents). This transfer can be accomplished by conventional means, such as dialysis, diafiltration, or chromatographic methods (e.g., size exclusion or buffer exchange chromatography). For example, the ISVD polypeptide product can be transferred into PBS by dialysis. The ISVD polypeptide product can also be transferred into physiological saline. One of skill in the art can readily select other appropriate buffer systems. The choice of buffer can depend on the buffer conditions required for potential subsequent purification, formulation, or storage steps.

[0331] Chaotropic agent treatment can be applied over a wide range of temperatures, provided that the temperature does not irreversibly denature or degrade the ISVD polypeptide. Examples include, but are not limited to, temperatures between about 4°C and about 30°C. Thus, chaotropic agent treatment can be applied at about 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, or 4°C. One skilled in the art can readily select an appropriate temperature for chaotropic agent treatment. In one embodiment, chaotropic agent treatment is applied at a temperature between about 15°C and about 30°C. In another embodiment, chaotropic agent treatment is applied at a temperature between about 4°C and about 12°C. In another embodiment, the chaotropic agent treatment is applied at room temperature, ie, between about 20°C and 25°C.

[0332] Heat Treatment The conformational mutants can also be converted to the desired polypeptide product by applying heat stress. The terms "heat treatment" and "heat stress" are used interchangeably herein.

[0333] Heat stress can be applied at any time during the method for purifying / isolating multivalent ISVD polypeptides. In one embodiment, heat stress is applied before a purification step based on a chromatography technique. In another embodiment, heat stress is applied after a purification step based on a chromatography technique. For example, heat stress can be applied after the ISVD polypeptide capture step of Protein A-based affinity chromatography (and before the ISVD polypeptide polishing step). Alternatively, heat stress can be applied after any ISVD polypeptide polishing step.

[0334] The heat stress is applied at an appropriate temperature between 40°C and 60°C that allows the conversion of the conformational mutant into the desired polypeptide product, but does not result in its irreversible denaturation or degradation. Based on the methods described herein, one skilled in the art can determine which temperature is appropriate for converting a conformational variant into the desired polypeptide product. An appropriate temperature has been applied when the conformational variant is no longer essentially detectable by the chromatographic techniques described herein. For example, an appropriate temperature has been applied when, using analytical SE-HPLC, essentially no post-peak shoulder or isolated post-peak (indicating a conformational variant) is observed in the chromatogram of the composition after heat stress. Additionally, or alternatively, an appropriate temperature has been applied when, using analytical IEX-HPLC, essentially no pre / post-peak shoulder or isolated pre / post-peak (indicating a conformational variant) is observed in the chromatogram of the composition after heat stress. Irreversible denaturation or degradation of the ISVD polypeptide product due to heat stress can be ruled out if the respective SE-HPLC or IEX-HPLC chromatograms do not show high molecular weight species (HMW species) (pre-peak in SE-HPLC) and / or a decrease in the total area (loss of product) or main peak in IEX-HPLC and SE-HPLC. Thus, the heat stress applied to convert the conformational variant to the desired polypeptide product includes incubating the composition at about 40° C. to about 60° C., about 45° C. to about 60° C., or about 50° C. to about 60° C. Heat stress can also include incubating the composition at about 40° C. to about 55° C., about 45° C. to 55° C., or about 48° C. to about 52° C., e.g., about 50° C.

[0335] Based on the methods described herein, one skilled in the art can determine the time required to convert a conformational variant into a desired polypeptide product. Heat stress is applied for a sufficient amount of time until the conformation is no longer essentially detectable by the chromatographic techniques described herein. For example, heat stress is applied for a sufficient amount of time until essentially no post-peak shoulder or isolated post-peak (indicating a conformational variant) is observed in a chromatogram of the composition after heat stress using analytical SE-HPLC. Additionally, or alternatively, heat stress is applied for a sufficient amount of time until essentially no pre / post-peak shoulder or isolated pre / post-peak (indicating a conformational variant) is observed in a chromatogram of the composition after heat stress using analytical IEX-HPLC. One skilled in the art will appreciate that heat stress must be applied for a time that allows for conversion of the conformational variant into the desired polypeptide product, but does not result in its irreversible denaturation or degradation. Irreversible denaturation or degradation of the ISVD polypeptide product due to heat stress can be ruled out if the respective SE-HPLC or IEX-HPLC chromatograms show no formation of high molecular weight species (HMW species) (pre-peak in SE-HPLC) or no decrease in the total area (loss of product) or main peak in IEX-HPLC and SE-HPLC. In this regard, heat stress should not be applied for longer than 4 hours. Accordingly, heat stress can be applied for at least about 0.5 hours, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 2.5 hours, at least about 3 hours, at least about 3.5 hours, or about 4 hours, but not longer than 4 hours. In particular, heat stress can be applied for about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, or about 4 hours. In one embodiment, heat stress is applied for at least about 0.5 hours or at least about 1 hour, e.g., at 50°C for about 1 hour. In another embodiment, the heat stress is applied for about 4 hours, for example, at 50° C. for about 4 hours.

[0336] The present technology provides a method for identifying conformational variants of polypeptides comprising or consisting of at least three or at least four ISVDs by analytical chromatographic methods such as SE-HPLC and IEX-HPLC. The present technology further provides the concept of converting conformational variants into intact products by heat treatment. Therefore, based on the concept provided herein, one skilled in the art can identify conformational variants of at least three or at least four ISVDs in terms of both the optimal heat stress temperature as well as the incubation time. The heat treatment can be adjusted for any polypeptide that comprises or consists of four ISVDs.

[0337] Heat stress can be terminated by adjusting the temperature of the composition comprising the ISVD polypeptide product to a temperature below about 30°C, i.e., any temperature between about 4°C and about 30°C. Thus, heat treatment is terminated by adjusting the temperature of the composition to about 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, or 4°C. In one embodiment, heat treatment is terminated by adjusting the temperature of the composition to between about 15°C and about 30°C. In another embodiment, heat treatment is terminated by adjusting the temperature of the composition to between about 4°C and about 12°C. In another embodiment, heat treatment is terminated by adjusting the temperature of the composition to room temperature, i.e., between about 20°C and about 25°C. The temperature adjustment (for the end of the heat treatment) may be adapted to the temperature required for possible subsequent purification, formulation or storage steps.

[0338] General aspects regarding conditions for converting conformational variants into desired polypeptide products The above-described treatment conditions for converting conformational variants into desired polypeptide products can be applied using a wide range of buffers suitable for protein purification / formulation, particularly any known buffer suitable for antibody purification / formulation. Examples include, but are not limited to, PBS, phosphate buffer, acetate, histidine buffer, Tris-HCl, and glycine buffer. The ISVD polypeptide can also be present in physiological saline. Those skilled in the art can easily select other suitable buffer systems.

[0339] Any of the conditions described above that convert conformational variants into the desired polypeptide product, or any combination thereof, can be combined with any method of removing conformational variants as further described below.

[0340] Based on the concepts of low pH treatment, treatment with chaotropic agents, and heat treatment provided herein, one skilled in the art can adjust the treatment conditions described herein in terms of optimal pH, chaotropic agent concentration, and / or heat stress temperature, as well as incubation time, for any polypeptide comprising or consisting of at least three or at least four ISVDs.

[0341] 5.4.4 Elimination or reduction of conformational variants By removed or reduced, we mean that the product-associated conformational variants are physically separated from a composition containing both the desired ISVD polypeptide product and the product-associated conformational variants. The exact meaning will be clear from the context. In the prior art, those skilled in the art were unaware of the existence of conformational variants of polypeptides comprising or consisting of at least three or at least four ISVDs when produced in a lower eukaryotic host as provided herein. Only based on this knowledge provided by the present application, those skilled in the art can adjust / optimize the assay conditions used to remove or reduce the conformational variants present in a composition containing the desired ISVD polypeptide product and the product-associated conformational variants. Thus, the identification of conformational variants of a polypeptide comprising or consisting of at least three or at least four ISVDs by the specific methods provided herein (see the section "Analytical Methods" below) is a prerequisite for those skilled in the art to specifically adjust / optimize prior art purification methods to specifically remove the conformational variants.

[0342] The desired polypeptide product is isolated / purified by applying conditions that remove conformational variants from a composition comprising the desired polypeptide and its conformational variants. In this embodiment, the conformational variants are removed by one or more preparative chromatographic techniques. The chromatographic techniques can be preparative chromatographic techniques based on hydrodynamic volume, surface charge, and / or hydrophobic exposure / surface hydrophobicity. In one embodiment, the preparative chromatographic technique is selected from size exclusion chromatography (SEC), ion exchange chromatography (IEX), e.g., cation exchange chromatography (CEX), mixed-mode chromatography (MMC), and hydrophobic interaction chromatography (HIC).

[0343] According to one embodiment, conformational variants are removed by preparative chromatographic separation based on hydrodynamic volume. Therefore, conformational variants are removed using preparative size-exclusion chromatography (SEC). In SEC, a chromatographic column is packed with fine porous beads composed of (but not limited to) dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P). The pore size of these beads is used to estimate the size of macromolecules. Non-limiting examples of SEC resins include Sephadex-based products (GE Healthcare, Merck), BioGel-based products (Bio-Rad), Sepharose-based products (GE Healthcare), and Superdex-based products (GE Healthcare).

[0344] In another embodiment, conformational variants are removed by preparative chromatographic separation based on surface charge. Thus, conformational variants are removed using preparative ion exchange chromatography (IEX) (e.g., cation exchange chromatography (CEX)). Non-limiting examples of IEX resins include Poros 50HS (ThermoFischer), Poros 50HQ (ThermoFischer), SOURCE 30S (GE Healthcare), SOURCE 15S (GE Healthcare), SP Sepharose (GE Healthcare), Capto S (GE Healthcare), Capto SP Impres (GE Healthcare), Capto S ImpAct (GE Healthcare), Q Sepharose (GE Healthcare), Capto Q (GE Healthcare), DEAE Sepharose (GE Healthcare), Poros XS (Thermo Scientific TM), AG® 50W (Bio-Rad), AG® MP-50 (Bio-Rad), Nuvia HR-S (Bio-Rad), UNOsphere TM S (Bio-Rad), and UNOsphere Rapid S (Bio-Rad).

[0345] In another embodiment, conformational variants are removed by preparative chromatographic separation based on surface hydrophobicity / hydrophobic exposure. Thus, conformational variants are removed using preparative hydrophobic interaction chromatography (HIC). In one embodiment, HIC is based on HIC column resins. Examples of HIC resins include, but are not limited to, Capto Phenyl ImpRes (GE Healthcare), Capto Butyl ImpRes (GE Healthcare), Phenyl HP (GE Healthcare), Capto Butyl (GE Healthcare), Capto Octyl (GE Healthcare), Toyopearl PPG-600 (Tosoh Biosciences), Toyopearl phenyl-600 (Tosoh Biosciences), Toyopearl phenyl-650 (Tosoh Biosciences), Toyopearl butyl-600 (Tosoh Biosciences), and Toyopearl butyl-65. 0 (Tosoh Biosciences), TSKgel Phenyl 5-PW (Tosoh Biosciences). In another embodiment, the HIC is based on an HIC membrane. Without limitation, the HIC membrane can be Adsorber Q (GE Healthcare), Adsorber S (GE Healthcare), Adsorber Phen (GE Healthcare), Mustang Q System (Pall), NatriFlo HD-Q Membrane Chromatography (Natrix Separations), Sartobind STIC (Sartorius), Sartobind Q (Sartorius), or Sartobind Phenyl (Sartorius).

[0346] In yet another embodiment, conformational variants are removed by preparative chromatographic separation based on hydrodynamic volume, surface charge, and / or surface hydrophobicity / hydrophobic exposure. Thus, conformational variants are removed using mixed-mode chromatography (MMC). MMC refers to a chromatographic method that utilizes more than one form of interaction between the stationary phase and the analytes to achieve their separation. Thus, MMC resins are based on media functionalized with ligands that are inherently capable of several different types of interactions: ion exchange, affinity, size exclusion, and hydrophobicity. Various hydroxyapatite chromatography resins are commercially available, and any available form of material can be used. Detailed descriptions of conditions suitable for hydroxyapatite chromatography are provided in WO 2005 / 044856 and WO 2012 / 024400, the contents of which are incorporated herein by reference in their entireties.

[0347] In one embodiment, the hydroxyapatite is in crystalline form. Hydroxyapatite can aggregate to form particles and be sintered at high temperatures to form a stable porous ceramic mass. The particle size of the hydroxyapatite can vary widely, but typical particle sizes can range from 1 μm to 1000 μm in diameter, and can be 10 μm to 100 μm. In one embodiment, the particle size is 20 μm. In another embodiment, the particle size is 40 μm. In yet another embodiment, the particle size is 80 μm.

[0348] Numerous chromatographic supports can be used in the manufacture of ceramic hydroxyapatite columns, the most widely used being type I and type II hydroxyapatite. Type I has a high protein binding capacity and better capacity for acidic proteins. However, type II has a lower protein binding capacity but better resolution of nucleic acids and certain proteins. Type II material also has a very low affinity for albumin and is particularly suitable for the purification of many immunoglobulin species and classes. The choice of a particular hydroxyapatite type can be determined by one skilled in the art.

[0349] Without limitation, hydroxyapatite resins include CHT ceramic hydroxyapatite, type I (20, 40, or 80 μm) (BioRad), CHT ceramic hydroxyapatite type II (20, 40, or 80 μm) (BioRad), MPC TM Ceramic hydroxyfluoroapatite type I (40 μm), Ca ++ Pure-HA(Tosoh BioScience).

[0350] Additionally or alternatively, conformational variants may be removed using any sequential combination of preparative SEC, IEX, HIC, or MMC described above.

[0351] In view of the present disclosure, one skilled in the art will be able to identify and then find suitable chromatographic conditions for removing (or at least reducing) conformational variants of multivalent ISVD polypeptides. Having identified the conformational variants described herein, one skilled in the art will be able to determine the parameters and conditions (gradient, etc.) of the selected chromatographic method. One could then adapt the chromatography conditions (e.g., buffer, concentration) and then take the appropriate fractions of the peak. For example, but not limited to, the chromatographic conditions used in the examples herein can be used to remove (or at least reduce) conformational variants of a multivalent ISVD polypeptide comprising at least three or at least four ISVDs. The chromatographic conditions used in the examples can at least serve as a reference point for the development of suitable chromatographic conditions for removing (or at least reducing) conformational variants of a particular multivalent ISVD polypeptide comprising at least three or at least four ISVDs.

[0352] In particular, based on the teachings of the present application, the removal or reduction of conformational variants from a composition comprising both a multivalent ISVD polypeptide and its conformational variants can be achieved by: i) applying preparative chromatographic techniques; ii) analyzing the fractions obtained from step (i) for the presence of multivalent ISVD polypeptides; iii) selecting the fractions of step (ii) that contain only multivalent ISVD polypeptides but not conformational variants. Includes:

[0353] Steps i) and ii) can be performed by means known to those skilled in the art of antibody purification, particularly in the field of ISVD purification. The methods can be specifically adapted / optimized for both the identification of conformational variants and the removal / reduction of conformational variants as provided herein. Suitable exemplary analytical and preparative chromatography techniques are described herein. These general techniques must be specifically adapted / optimized to allow the removal / reduction of conformational variants.

[0354] Steps ii) and iii) can be achieved by specific analytical chromatography techniques, as described in Section 5.4.5 below. For example, if there is no detectable post-peak shoulder and / or discrete post-peak in (analytical) SE-HPLC, the chromatographic fraction contains only multivalent ISVD polypeptides but no conformational variants. The presence of conformational variants can also be excluded if there is no detectable pre-peak shoulder and / or discrete pre-peak, or no post-peak shoulder and / or discrete post-peak in analytical IEX-HPLC.

[0355] In the prior art, those skilled in the art were unaware of the existence of conformational variants of polypeptides comprising or consisting of at least three or at least four ISVDs when produced in lower eukaryotic hosts as provided herein. Only based on this knowledge provided by the present application, those skilled in the art can adjust / optimize the above steps i) to iii) so that specific elimination / reduction of conformational variants can be achieved.

[0356] Fractions containing conformational variants may be discarded or may be treated according to the conversion methods described herein (Section 5.4.3 "Conversion of Conformation Variants to Desired Polypeptide Products") to convert the conformational variants to the desired polypeptide product. Successful conversion may be assessed as described herein, for example, by analytical chromatographic techniques described in Section 5.4.5 below.

[0357] The fractions containing only multivalent ISVD polypeptides obtained after step iii) may optionally be subjected to purification or filtration steps known in the art.

[0358] A fraction is considered to "contain only multivalent ISVD polypeptides (but not conformational variants)" if there is essentially no detectable post-peak shoulder and / or discrete post-peaks in (analytical) SE-HPLC. Alternatively, a fraction is considered to "contain only multivalent ISVD polypeptides (but not conformational variants)" if there is essentially no detectable pre-peak shoulder and / or discrete pre-peak, or if there is essentially no detectable post-peak shoulder and / or discrete post-peak in analytical IEX-HPLC. "Essentially free of pre-peak shoulders and / or separated pre-peaks" or "essentially free of post-peak shoulders and / or separated post-peaks" means that in an SE-HPLC or IEX-HPLC chromatogram, respectively, the ratio of the area under the curve (AUC) for the pre-peak / post-peak (shoulder) to the total area under the curve for the main peak and pre-peak / post-peak (shoulder) is less than 5%, e.g., 4.5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. In one embodiment, there are no detectable pre-peaks / post-peaks (shoulders) in an SE-HPLC or IEX-HPLC chromatogram, respectively.

[0359] In another aspect, conformational variants are removed or reduced by applying a composition comprising a multivalent ISVD polypeptide and conformational variants to a chromatography column using a loading rate of at least 20 mg protein per ml of resin. In one embodiment of this aspect, the loading rate is at least 30 mg protein per ml of resin, or at least 45 mg protein per ml of resin. In one embodiment, the chromatography column is a Protein A column. Thus, conformational variants are removed or reduced by applying a composition comprising a multivalent ISVD polypeptide and conformational variants to a Protein A column using a loading rate of at least 20 mg protein per ml of resin. In another embodiment, conformational variants are removed or reduced by applying a composition comprising a multivalent ISVD polypeptide and conformational variants to a Protein A column using a loading rate of at least 45 mg protein per ml of resin.

[0360] Chromatographic techniques used to remove (or reduce) conformational variants from compositions comprising ISVD polypeptides and their conformational variants can be applied to culture supernatants containing multivalent ISVD polypeptides. For example, a capture step can be used for removal or reduction. Chromatographic techniques used to remove (or reduce) conformational variants can also be applied to partially or highly purified preparations of multivalent ISVD polypeptides. For example, chromatographic techniques used to remove (or reduce) conformational variants can be applied after the capture step but before or during the first polishing step, or during one or more additional polishing steps, or after a polishing step.

[0361] 5.4.5 Analysis method Analytical methods used to observe conformational variants Conformational variants of polypeptides comprising or consisting of at least three or at least four ISVDs can be identified by specific analytical chromatographic techniques provided herein. Analytical chromatographic methods such as analytical SE-HPLC and IEX-HPLC are known to those skilled in the art. However, these methods need to be adapted / optimized to the problem of identifying conformational variants. Therefore, the requirement for the adaptation / optimization of such analytical chromatographic techniques is that at least three or fewer ISVDs are required. It is known that the production in lower eukaryotes of polypeptides comprising or consisting of at least four ISVDs may (partially) give rise to conformational variants as described herein.

[0362] As provided herein, conformational variants can be distinguished from the desired polypeptide product based on their reduced hydrodynamic volume. Thus, the presence of conformational variants can be detected by analytical SE-HPLC. Using appropriate conditions, the presence of conformational variants is evidenced in the SE-HPLC chromatogram by a post-peak shoulder or a separate post-peak. Therefore, SE-HPLC adapted / optimized for identifying conformational variants can be used to confirm conditions that convert conformational variants into the desired polypeptide product as described herein. Furthermore, SE-HPLC adapted / optimized for identifying conformational variants can be used to confirm the removal or reduction of conformational variants from a composition containing the desired polypeptide product and its conformational variants.

[0363] As further provided herein, conformational variants can be distinguished from desired polypeptide products based on altered surface charge and / or surface hydrophobicity. Therefore, using appropriate conditions, the presence of conformational variants can be detected by (specifically developed) analytical IEX-HPLC. Depending on the nature of the alteration in surface charge and / or surface hydrophobicity, the presence of conformational variants can be indicated in the IEX-HPLC chromatogram by pre- / post-peak shoulders or separate pre- / post-peaks. Therefore, IEX-HPLC adapted / optimized for identifying conformational variants can be used to confirm conditions that convert conformational variants into desired polypeptide products. Furthermore, IEX-HPLC adapted / optimized for identifying conformational variants can be used to confirm the removal and reduction of conformational variants from compositions containing the desired polypeptide product and its conformational variants.

[0364] Based on the present disclosure, one skilled in the art will be able to find suitable chromatographic conditions for identifying conformational variants of multivalent ISVD polypeptides. For example, but not limited to, the chromatographic conditions used in the Examples herein can be used for detecting conformational variants of multivalent ISVD polypeptides comprising at least three or at least four ISVDs. The chromatographic conditions used in the Examples herein can at least serve as a reference point for developing chromatographic conditions suitable for detecting conformational variants for a particular multivalent ISVD polypeptide comprising at least three or at least four ISVDs. Basic exemplary conditions are shown in Table C.

[0365] Further analytical methods used for characterization of conformational variants The following analytical techniques are known to those skilled in the art. For example, but not limited to, suitable conditions are shown in Table C.

[0366] [Table 3]

[0367] Assay methods used to monitor efficacy of ISVD polypeptides Conformational variants can also be distinguished from the desired polypeptide product by a change in potency, where the conformational variant has a decreased potency compared to the desired polypeptide product. Furthermore, the (successful) conversion of the conformational variant to the desired polypeptide product can be achieved. Conversion may be demonstrated by partial or complete restoration of potency relative to the potency of the respective desired polypeptide product, or relative to a reference ISVD polypeptide that has not been enriched or depleted of conformational variants.

[0368] In this context, potency refers to the binding ability (to a particular target), functional activity, and / or amount of a polypeptide required to produce a particular effect due to one or more of the at least three or at least four ISVDs present in the polypeptide. Potency can be measured in vitro (e.g., competitive ligand binding assays or cell-based assays) or in vivo (e.g., in animal models). Without limitation, potency can refer to inhibition of TNFα-induced expression of a luciferase reporter gene, inhibition of IL-23-induced expression of a luciferase reporter gene, inhibition of OX40L-induced expression of a luciferase reporter gene, or ability to bind to human serum albumin. Suitable exemplary assays for determining potency differences between a desired polypeptide product and its conformational variants include (without limitation): Cell-based reporter assay for potency testing of TNF-alpha binding moieties is.

[0369] Global response TMHEK293_NFkB-NLucP cells are TNF receptor-expressing cells stably transfected with a reporter construct encoding nano-luciferase under the control of an NFκB-dependent promoter. Incubation of these cells with soluble human TNFα results in NFκB-mediated nano-luciferase expression.

[0370] The assay may generally be performed as follows: Glo response TM HEK293_NFkB-NLucP cells should be seeded in standard growth medium at appropriate cell numbers in appropriate tissue culture plates. A dilution series of the ISVD construct to be tested is added to an appropriate and sufficient amount of human TNFα and incubated with the cells for a sufficient time (e.g., approximately 5 hours) at 37°C and 5% CO2. During this incubation, TNF-induced expression of the luciferase reporter gene is inhibited by the ISVD construct. After incubation, the plate is cooled (e.g., for 10 minutes), and then Nano-Glo luciferase substrate is added to quantify luciferase activity. Five minutes after substrate addition, luminescence can be measured, for example, using a Tecan Infinite F-plex plate reader. Luminescence, expressed as relative light units (RLU), is directly proportional to the luciferase concentration.

[0371] Cell-based reporter assay for potency testing of IL-23 binding moieties Global response TM HEK293_human IL-23R / IL-12Rb1-Luc2P cells are stably transfected with a reporter construct containing a luciferase gene under the control of a sis-inducible element (SIE)-responsive promoter. Furthermore, these cells constitutively overexpress both subunits of the human IL-23 receptor, i.e., IL-12Rb1 and IL-23R. Stimulation of these cells with human IL-23 induces expression of the luciferase reporter gene.

[0372] The assay may generally be performed as follows: Glo response TM HEK293_human IL-23R / IL-12Rb1-Luc2P cells should be seeded in appropriate tissue culture plates in normal growth medium at the appropriate cell number. A dilution series of the ISVD construct to be tested is added to the cells, followed by an appropriate amount of recombinant hIL-23 (e.g., 3 pM). The cells should be incubated at 37°C for a sufficient time (e.g., about 6 hours). After the incubation step, a cooling period (e.g., 10 minutes) of the plate is required, after which the luciferase substrate 5'-fluoroluciferin is added. Bio-Glo TM Luciferase activity is quantified by adding a luciferase assay system (Luciferase Assay System). Five minutes after adding the substrate, luminescence can be measured, for example, in a Tecan Infinite F-plex plate reader. Luminescence (expressed as relative light units, RLU) is directly proportional to luciferase concentration.

[0373] Cell-based reporter assay for potency testing of OX40L binding moieties The efficacy of OX40L inhibition can be assessed using cell-based reporter assays, e.g., Glo Response TM NFkB-luc2 / OX40 Jurkat suspension cells should be seeded in appropriate tissue culture plates at appropriate cell numbers in normal growth medium. A dilution series of the ISVD construct is added to the cells, followed by a fixed concentration of 700 pM OX40L. The plates should then be incubated in an incubator at 37°C and 5% CO2 for a sufficient time (e.g., 3 hours) to allow activation of the NF-kB promoter by OX40L / OX40 signaling, which in turn results in transcription of the luciferase gene. After the incubation step, a cooling period (e.g., 10 minutes) of the plates is required, after which the luciferase substrate 5'-fluoroluciferin (Bio-Glo) is added. TMLuciferase activity is quantified by adding a luciferase assay system. Five minutes after adding the substrate, luminescence can be measured, for example, in a Tecan Infinite F200 plate reader. Luminescence (expressed as relative light units, RLU) is directly proportional to the concentration of luciferase.

[0374] ELISA-based albumin binding assay for potency testing of albumin-binding moieties Binding potency to human serum albumin (HSA) can be measured by direct binding ELISA. For example, a 96-well microtiter plate can be coated overnight with an appropriate amount of HSA in bicarbonate buffer at pH 9.6. Nonspecific binding sites on the plate can be blocked using Superblock T20 at room temperature (RT) for approximately 30 minutes. Serial dilutions of ISVD constructs can be prepared in PBS + 10% Superblock T20 and transferred to the HSA-coated plate, followed by an incubation step of approximately 75 minutes at RT with shaking at 600 rpm. For example, bound ISVD constructs can be detected using 1 μg / mL mouse anti-ISVD construct antibody for 90 minutes at RT with shaking at 600 rpm, followed by incubation with 0.2 μg / mL horseradish peroxidase (HRP)-labeled polyclonal rabbit anti-mouse antibody for 50 minutes at RT with shaking at 600 rpm. The bound HRP-labeled polyclonal antibody can be measured by adding 1 / 3 diluted 3,5,3'5'-tetramethylbenzidine (TMB) one-part solution. 1M HCl is added to stop the color reaction between HRP and the substrate. The optical density can be measured using, for example, a plate-spectrophotometer at a wavelength of 450 nm and a reference wavelength of 620 nm. This OD is directly proportional to the amount of ISVD construct bound to the coated HSA.

[0375] 5.5 Multivalent ISVD Polypeptide Products Obtainable by Production and / or Isolation or Purification Methods The present application also describes improved compositions comprising multivalent ISVD polypeptide products obtainable by the methods described herein, characterized by a reduced level or complete absence of product-associated conformational variants. For example, ISVD polypeptides obtainable by the methods described herein contain less than 5%, e.g., 0-4.9%, 0-4%, 0-3%, 0-2%, or 0-1% of product-associated conformational variants. In another embodiment, ISVD polypeptides obtainable by the methods described herein contain less than 1%, less than 0.5%, or less than 0.01% of product-associated conformational variants. In one embodiment, multivalent ISVD polypeptide products obtainable by the methods described herein are free of product-associated conformational variants. For example, ISVD polypeptides obtainable by the methods described herein contain less than 5%, e.g., 0-4.9%, 0-4%, 0-3%, 0-2%, or 0-1% of product-associated conformational variants. A composition comprising an ISVD polypeptide obtainable by the methods described herein contains less than 5%, e.g., 0-4.9%, 0-4%, 0-3%, 0-2%, or 0-1% of product-associated conformational variants. In another embodiment, a composition comprising an ISVD polypeptide obtainable by the methods described herein contains less than 1%, less than 0.5%, or less than 0.01% of product-associated conformational variants. In one embodiment, a composition comprising a multivalent ISVD polypeptide product obtainable by the methods described herein is free of product-associated conformational variants. One skilled in the art can readily determine the proportion of product-associated conformational variants as a % of total polypeptide (i.e., by determining the AUC of the pre-peak or post-peak (shoulder) / total AUC of both the main peak and the pre-peak or post-peak (shoulder)), for example, by SE-HPLC and IEX-HPLC as described herein.

[0376] In other words, the multivalent ISVD polypeptide products obtainable by the methods described herein are characterized by improved structural homogeneity compared to prior art preparations, which may contain 5% or more of product-associated conformational variants, e.g., 5-15%, 5-20%, 5-25% or more of product-associated conformational variants.

[0377] In view of the improved structural homogeneity, the multivalent ISVD polypeptide products obtainable by the present methods are advantageous compared to prior art preparations. For example, the multivalent ISVD polypeptide products obtainable by the methods of the present invention are advantageous for therapeutic applications. In the context of therapeutic antibody uses, structural homogeneity is of paramount clinical and regulatory importance.

[0378] Accordingly, the present application also describes pharmaceutical formulations and other compositions comprising the multivalent ISVD polypeptide products obtainable by the methods described herein. The multivalent ISVD polypeptide products obtainable by the methods described herein may also be used in therapy (i.e., medical applications).

[0379] Those skilled in the art can easily formulate, based on their well-known general knowledge, pharmaceutically suitable preparations of the multivalent ISVD polypeptide products obtainable by the methods described herein. Furthermore, references cited herein that specifically deal with multivalent ISVD polypeptides are expressly incorporated by reference. Formulations for standard routes of application can be prepared, including, without limitation, formulations for nasal, oral, intravenous, subcutaneous, intramuscular, intraperitoneal, intravaginal, rectal, topical, or inhalation administration.

[0380] Those skilled in the art can also readily devise suitable methods of treatment featuring the use of therapeutically effective amounts of multivalent ISVD polypeptides obtainable by the methods described herein. [Example]

[0381] 6. Working Example The following examples describe the identification of the presence of conformational variants of multivalent ISVD constructs during the production and purification process. It was shown that such conformational variants exhibit characteristic biochemical / biophysical behavior, allowing their separation by chromatographic methods. It was also found that, apart from differences in biochemical / biophysical properties, conformational variants exhibit differences in the potency of one or more ISVD building blocks against their respective targets. Finally, such undesired conformational variants can be converted into intact ISVD polypeptides by appropriate treatment conditions during the purification process of the ISVD constructs and / or can be isolated as intact ISVD polypeptides. It could be shown that both morphological as well as undesired conformational variants can be specifically reduced / removed from the containing composition.

[0382] 6.1 Example 1: Identification of Conformational Variants of Compound A Conformational variants could be identified during the steps of the capture process of multivalent ISVD constructs. Conformational variants of the multivalent ISVD construct were identified during the first step of the purification of the multivalent ISVD construct (i.e., the capture step), which was performed to recover the maximum ISVD product from the clarified supernatant.

[0383] During the capture purification process of Compound A (SEQ ID NO: 1), it was observed that the analytical size exclusion profile (SE-HPLC; conditions shown in Table C) differed depending on the resin and elution buffer used during the chromatographic process.

[0384] Compound A (SEQ ID NO: 1) is a multivalent ISVD construct containing three different sequences optimized variable domains of heavy chain llama antibodies that bind to three different targets. The ISVD building blocks are fused head-to-tail (N- to C-terminus) with a G / S linker in the following format: OX40L-linked ISVD - 9GS linker - OX40L Conjugated ISVD - 9GS linker - TNFα conjugated ISVD - 9GS linker - human serum albumin conjugated ISVD - 9GS linker - TNFα conjugated ISVD, and and has the following sequence:

[0385] [Table 4]

[0386] Figure 1 shows the SE-HPLC profiles for the eluates after chromatographic purification with Protein A or non-Protein A capture resin. The SE-HPLC profiles of the eluates showed a less pronounced post-peak shoulder (denoted as Post-Peak 1 in Figure 1) when Protein A was used as the capture resin compared to non-Protein A. It was concluded that the presence of the post-peak shoulder (Post-Peak 1) depends on the conditions / resin used during chromatographic purification. In contrast to non-Protein A resin, elution with Protein A resin is at a low pH. Based on these observations, the effect of elution buffer pH on the SE-HPLC profile was investigated. Therefore, buffers A to D (listed in Table 2) with different acidic pHs were compared for elution of Compound A from Protein A capture resin.

[0387] [Table 5]

[0388] Figure 2 shows the SE-HPLC profiles for Protein A capture and eluates after elution (without neutralization) using different elution buffers A, B, C, and D. Post-peak 1 was less pronounced in elution buffer A compared with buffers B, C, and D. Figure 3 shows the SE-HPLC profiles of capture eluates immediately after elution using elution buffer A (Figure 3(1)) and elution buffer B (Figure 3(2)), as well as capture eluates neutralized to at least pH 6.7 using 1 M HEPES pH 7.0. As seen in Figure 2 and Figures 3(1) and 3(2), the post-peak shoulder (denoted post-peak 1) in the SE-HPLC profile was lower for the eluate at pH 2.9 (buffer A) compared with the eluates at pH 3.6–4.7 (buffers B–D). However, post-peak 1 did not decrease when the eluate was immediately neutralized (compare the eluate and neutralized eluate in Figure 3(1)). Therefore, "pH maintenance" may have an effect on post-peak 1. For elution buffer B, post-peak 1 was observed after elution independent of subsequent neutralization of the resulting eluate (Figure 3(2)).

[0389] Based on these observations, it was concluded that there is an effect of pH on the detectability of post-peak 1 in SE-HPLC. Furthermore, it was speculated that post-peak 1 may represent a conformational variant of the ISVD construct. The slightly increased retention time may indicate a more compact conformation compared to the intact form of the ISVD construct represented by the main peak.

[0390] Conformational variants could be identified during the polishing process steps of multivalent ISVD constructs Conformational variants of the multivalent ISVD constructs could also be identified during the polishing process step, which was performed after the capture step to improve the purity of the multivalent ISVD-containing composition.

[0391] For the polishing step of the ISVD construct, cation exchange chromatography (CEX) was performed. Thus, a linear salt gradient from 0 to 350 mM NaCl in 25 mM citrate pH 6.0 was applied to the polishing CEX resin at room temperature over 20 column volumes (CV). The chromatographic profile is shown in Figure 4.

[0392] The top fraction (referred to as fraction 2A1 in Figure 4) and the side (front) fraction (referred to as fraction 1C2 in Figure 4) eluted during the linear gradient were further analyzed by SE-HPLC and compared with the load material (Figure 5). Post-peak 1, observed by SE-HPLC for the load material, was absent from the top fraction of this gradient on the CEX resin. In contrast, significant post-peak 1 (approximately 60%) by SE-HPLC was observed for the side (front) fraction.

[0393] Therefore, conformational variants of the ISVD construct could also be identified during the polishing step. Different eluate fractions of the CEX polishing step were shown to contain different ratios of the intact form (main peak) and conformational variants (post-peak 1) by SE-HPLC (Figure 5). The top fractions of the CEX polishing step were found to be depleted in conformational variants, whereas the side fractions were rather enriched.

[0394] These results were similar for various cation exchange resins such as Capto SP Impres (GE Healthcare) and Capto S ImpAct (GE Healthcare), which were tested for polishing procedures using a gradient from 0 to 350 mM NaCl in 25 mM citrate pH 6.0 over 20 CV, as well as for other CEX resins tested for polishing procedures, e.g., using a gradient from 0 to 400 mM NaCl in 25 mM citrate pH 6.0 over 20 CV (data not shown), and using 25 mM histidine pH 6.0 and a gradient from 0 to 400 mM NaCl over 20 CV (data not shown).

[0395] These observations further reinforced the conclusion that post-peak 1 observed by SE-HPLC may represent a conformational variant of the ISVD construct. While the slightly increased retention time in SE-HPLC indicates a more compact morphology (i.e., a reduced hydrodynamic volume), the slight difference in retention time observed in preparative CEX indicates an altered surface charge compared to the intact ISVD product. Therefore, it is possible to separate the conformational variant and the intact ISVD product using appropriate chromatographic techniques, such as preparative SEC or CEX.

[0396] 6.2 Example 2: Identification and Characterization of Conformational Variants of Compound A In Example 1, compound A was shown to elute as a main peak and post-peak 1 (post-peak shoulder) during analytical SE-HPLC. Due to the slightly longer retention time, it was concluded that post-peak 1 may refer to a more compact form of the multivalent ISVD construct. Furthermore, Protein A affinity chromatography using an elution buffer at pH 2.5 may result in a decreased post-peak 1 / main peak ratio. However, the post-peak 1 / main peak ratio remained unchanged when the capture eluate was immediately neutralized. Therefore, it was concluded that the conformational variants could be converted to the intact ISVD product, and therefore, the molecular size is not different.

[0397] To further characterize the nature of the conformational variants and to exclude the presence of mass variants, the conformational variant-depleted top fraction and the conformational variant-enriched fraction from the CEX polishing of Example 1 were subjected to analytical ion exchange-high resolution chromatography. The samples were analyzed by high performance liquid chromatography (IEX-HPLC; conditions shown in Table C, Protocol I), capillary isoelectric focusing (CE-IEF) and reversed phase ultra-high performance liquid chromatography (RP-UHPLC).

[0398] Analytical IEX-HPLC behavior Similar to analytical SE-HPLC, the IEX-HPLC chromatogram showed a significant post-peak 1 (approximately 46%) for the conformational variant-enriched side fraction, which was absent in the conformational variant-depleted top fraction (Figure 6).

[0399] Behavior in CE-IEF / RP-UHPLC In CE-IEF analytical studies, the results obtained in preparative CEX (data not shown) Little difference was observed between the side ("enriched") and top ("depleted") fractions. Similarly, no difference was observed between both fractions by RP-UHPLC (data not shown).

[0400] In contrast to CE-IEF, IEX-HPLC showed different chromatographic profiles between the conformational variant-enriched side CEX fraction and the conformational variant-depleted top CEX fraction. The main difference between the two charge-based methods, CE-IEF and IEX-HPLC, is that CE-IEF is performed under denaturing conditions (3 M urea). The lack of difference in CE-IEF indicates the absence of chemical modifications that result in an overall charge difference between the intact ISVD product and the conformational variant. However, the difference in IEX-HPLC suggests a slightly altered surface charge of the conformational variant compared to the intact ISVD product. In other words, only the surface charge changed due to the conformational change, but the total charge of the molecule remained unchanged. These findings also suggested the hypothesis that conformational variants could be removed by denaturing conditions.

[0401] The similar behavior of both CEX fractions in RP-UHPLC ruled out that the elaborate conformational variant was due to scrambled disulfide bridges compared to the intact form of the ISVD construct.

[0402] Differential potency of the intact ISVD construct and its conformational variants To further investigate whether the conformational variants differ to any extent in their potency in target binding, the following assays were performed on the conformational variant-enriched side fractions and conformational variant-depleted top fractions obtained from the preparative CEX described above.

[0403] The potency of the ISVDs against their respective targets was determined using the following assays (as described in Section 5.4.5 above): - Cell-based reporter assays for potency testing of TNF-alpha binding moieties; - Cell-based reporter assays for potency testing of OX40L binding moieties; - ELISA-based albumin binding assay for potency testing of albumin-binding moieties.

[0404] The results for the potency of the side ("enriched") and top ("depleted") fractions are shown in Table 3.

[0405] [Table 6]

[0406] A significant decrease in potency was observed in the control compared to the depleted fraction in the TNFα potency assay. This was observed for the conformational mutant enriched fraction. Thus, the conformational change of Compound A affects its TNFα binding potency.

[0407] 6.3 Example 3: Determination of Conditions Affecting the Conformation of Compound A Based on the findings from Examples 1 and 2, additional experiments were designed to evaluate the influence of specific experimental conditions that may affect the conformation of multivalent ISVD constructs. The conditions tested were mild denaturation, stress, or the presence of chaotropic agents. The conditions tested are summarized in Table 4.

[0408] [Table 7]

[0409] Low pH treatment For the low-pH treatment, compact variant-enriched and depleted materials from preparative CEX (described above) were treated with pH 2.5, pH 3.0, or pH 3.5 to reach a final concentration of 100 mM glycine, or with formulation buffer pH 6.5 (control). Samples were incubated at each pH for 4 hours and then analyzed either directly or neutralized with 0.1 M NaOH. The effect of treatment at pH 2.5 on compact variant-enriched and depleted materials was analyzed by SE-HPLC and IEX-HPLC (conditions shown in Table C; IEX-HPLC protocol I) and is shown in Figures 7(1) and (2) (SE-HPLC) and 8 (IEX-HPLC; compact variant-enriched fraction only).

[0410] For the conformational variant-enriched material incubated at pH 2.5, SE-HPLC and IEX-HPLC post-peak 1 were significantly reduced. Because this reduction was accompanied by an increase in the main peak in both analyses, this demonstrated that the conformational variant had been converted to the intact form. Furthermore, when the eluate was incubated at pH 2.5 for 4 h (data not shown), this conversion was maintained after neutralization. No changes were observed for either the control sample or the conformational variant-depleted material (Figure 7(2); data not shown for IEX-HPLC). For the material incubated at pH 3.0 and 3.5, only a slight decrease in the SE-HPLC and IEX-HPLC post-peak was observed, suggesting that the pH was not low enough to allow conversion of the conformational variant to the intact form (data not shown).

[0411] The stability of the compact mutant converted to the intact form was then examined after low pH treatment at pH 2.5 and subsequent neutralization. After storage of this compact mutant converted to the intact form at 25°C for up to 2 weeks, the SE-HPLC profile remained unchanged, demonstrating that the conversion of the compact mutant-enriched material to the intact form upon pH treatment was maintained (similar to the compact mutant-depleted material). The same results were obtained for 2 weeks of storage at 5°C (data not shown).

[0412] Treatment with chaotropic agents To assess the effect of chaotropic agents, conformational variant-enriched and -depleted materials were incubated with or without 1 M, 2 M, or 3 M guanidinium chloride (GuHCl) for 0.5 h and analyzed by SE-HPLC (conditions shown in Table C; SE-HPLC) and IEX-HPLC (conditions shown in Table C; IEX-HPLC protocol II). The results of the effect of treatment with 2 M and 3 M GuHCl denaturant on the conformational variant-enriched material are shown in Figure 9 (SE-HPLC) and Figure 10 (IEX-HPLC).

[0413] For the compact variant-enriched material incubated with GuHCl, post-peak 1 in SE-HPLC and IEX-HPLC was significantly reduced when a 2 M GuHCl concentration was applied. Furthermore, the reduction in post-peak 1 was accompanied by an increase in the main peak for both analyses, demonstrating that the conformational variants were converted to intact forms. No changes were observed for the conformational variant-depleted control sample (data not shown).

[0414] The concentration of 3M GuHCl was too high for the Compound A tested, resulting in decomposition of the product as evidenced by the formation of high molecular weight (HMW) species (pre-peak in SE-HPLC).

[0415] There was only a slight decrease in post-peak area in IEX-HPLC and SE-HPLC analyses upon application of 1 M GuHCl. For compound A, these conditions appeared to be insufficiently denaturing to completely convert the conformational mutant to the intact form (data not shown).

[0416] Heat stress treatment For heat treatment, conformational variant enriched and depleted materials were incubated for 1 or 4 hours at 50°C or 60°C and then re-equilibrated to room temperature (RT). The effects of heat stress at 50°C for 1 hour are shown in Figure 11 (SE-HPLC) and Figure 12 (IEX-HPLC).

[0417] For the conformational variant-enriched material, the post-peaks in SE-HPLC and IEX-HPLC significantly decreased when the material was heated at 50 °C for 1 and 4 h of incubation (data for the 4 h incubation not shown). This decrease was accompanied by an increase in the main peak for both analyses, demonstrating that the conformational variants were converted to the intact form. No change was observed for the conformational variant-depleted sample (data not shown).

[0418] Incubation at 60° C. appears to be too high for Compound A, resulting in a decrease in total area (loss of product) in SE-HPLC and IEX-HPLC (data not shown) and decomposition of the product.

[0419] Efficacy recovery upon pH or GuHCl treatment The potency of Compound A (as described in Example 2) against TNFα present in the conformational variant-enriched and -depleted fractions after 4 hours of pH 2.5 treatment or 0.5 hours of 2 M GuHCl treatment was determined compared to the untreated sample. The results are shown in Table 5.

[0420] [Table 8]

[0421] A reduction in potency was observed for the untreated conformational variant-enriched fraction compared to the conformational variant-depleted fraction. Low pH treatment of the conformational variant-enriched fraction resulted in a restoration of TNFα potency to the level observed for the conformational variant-depleted fraction. For the GuHCl-treated sample, potency was lower but comparable to the enriched and depleted fractions after treatment.

[0422] Abstract Overall, these experiments confirmed the existence of conformational variants that could be converted to the intact form under specific mild denaturing conditions or when electrostatic interactions (pH) were altered. It was also shown that conversion of the conformational variants to the intact ISVD product was maintained after removal of the denaturing conditions or pH adjustment. Furthermore, potency after conversion was restored and maintained for 2 weeks at 25°C or 5°C (data not shown).

[0423] 6.4 Example 4: Separation of Compound A Conformational Variants by Protein A Affinity Chromatography Use of alternative elution buffers or removal of conformational variants of Compound A during Protein A affinity chromatography Based on the results obtained during the characterization of the conformational variants (Examples 1 and 2), alternative elution buffer conditions were tested during compound capture.

[0424] The elution conditions and results are shown in Table 6, Figure 13 (SE-HPLC) and Figure 14 (IEX-HPLC) (conditions shown in Table C, SE-HPLC and IEX-HPLC protocol I).

[0425] [Table 9]

[0426] pH adjustment of the eluate to a pH of at least 7.0 was performed using 0.1 M NaOH.

[0427] For runs performed using an elution buffer at pH 2.2 in 0.1 M glycine, one portion of the eluate material was immediately adjusted to pH 7.1 using 0.1 M NaOH, and for another portion of the eluate material, the pH was adjusted to pH 2.5, incubated for 1.5 hours, and then the pH was readjusted to pH 7.0 using 0.1 M NaOH.

[0428] In SE-HPLC, post-peak 1 was found to be significantly reduced for elution buffers containing GuHCl. However, the presence of GuHCl resulted in product degradation, as indicated by the formation of HMW species (pre-peak in SE-HPLC) in the eluate compared to elution using a pH 2.2 buffer. For elution at pH 2.2, SE-HPLC post-peak 1 was higher when the eluate was immediately neutralized compared to the non-neutralized eluate or the eluate adjusted to pH 2.5 and incubated for 1.5 hours before neutralization (Figure 13). This was supported by IEX-HPLC, where the post-peak shoulder disappeared for the eluate adjusted to pH 2.5 and incubated before neutralization compared to the eluate immediately neutralized (Figure 14).

[0429] For both analyses, the decrease in the post-peak (conformational variant) was accompanied by an increase in the main peak (intact form), suggesting the conversion of the compact variant to the intact form.

[0430] Use of low pH incubation after protein A affinity chromatography for conversion of conformational variants of compound A Based on the results obtained above, low pH treatment was investigated for compound A as a means of converting conformational mutants.

[0431] The effect of low pH treatment and length of incubation was investigated at pH 2.1, pH 2.3, pH 2.5, and pH 2.7, and at 0, 1, 2, 4, 6, and 24 hours of incubation. The pH of the capture eluate was adjusted to the appropriate pH (2.1, 2.3, 2.4, 2.6, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7) using 0.1 M HCl. The pH of the eluate was either lowered to pH 6.0 (0.5 or 2.7) and immediately adjusted to pH 6.0 with 0.1 M NaOH (T0), or incubated at low pH for 1, 2, 4, 6, or 24 hours and then adjusted to pH 6.0 with 0.1 M NaOH (T1h, T2h, T4h, T6h, or T24h). The product quality of the various low-pH treated samples was compared with the capture eluate (control; T0) immediately adjusted to pH 6.0 with 0.1 M NaOH and analyzed by IEX-HPLC, SE-HPLC, RP-UHPLC, and capillary gel electrophoresis (CGE) (IEX-HPLC conditions, Protocol I, as shown in Table C and ). The SE-HPLC results are shown in Figures 15(1) and (2) (for T0) and Figures 15(3) and (4) (for T1h).

[0432] At TO, the post-peaks observed in SE-HPLC were lower at pH 2.1 and pH 2.7 compared to the control. This indicates that the conversion of conformational variants of Compound A occurs instantaneously in this pH range. However, the post-peaks observed in SE-HPLC were already lower at pH 2.1, 2.3, and 2.5 at TO, implying that the conversion of conformational variants occurs instantaneously at pHs equal to or lower than pH 2.5. This was confirmed by IEX-HPLC (data not shown), where the post-peaks were lower at pH 2.3 and 2.5 compared to pH 2.7 at TO.

[0433] From the 1 hour incubation onwards, the post-peak shoulder in SE-HPLC was similar for all pH treatments.

[0434] Thus, the above data indicated that the conversion of the conformational mutants of Compound A to the intact form was effective for all treatments for at least 1 hour at pHs ranging from 2.1 to 2.7.

[0435] No changes were observed by RP-UHPLC and CGE (data not shown), indicating that the compact variants do not differ in molecular weight (not LMW), chemical composition, or disulfide bridges (scrambled SS).

[0436] Low pH incubation for conversion of conformational mutants of compound A is independent of the concentration of the pH-adjusted stock solution To examine the effect of the concentration of the pH adjusting solution, two sets of pH adjusting solutions were tested: 0.1 M HCl to lower the pH to 2.6, and 0.1 M HCl to adjust the pH to 6.0. The first set used NaOH, and the second set used 2.7 M HCl (equivalent to 10% HCl) to lower the pH to 2.6 and 1 M NaOH to adjust the pH to 6.0. The samples were incubated at pH 2.6 for 1 hour and then adjusted to pH 6.0. The SE-HPLC results are shown in Figure 16A.

[0437] The use of the two sets of pH-adjusted solutions gave comparable results, with a decrease in the SE-HPLC post-peak accompanied by an increase in the main peak, associated with the conversion of the conformational variant to the intact form.

[0438] To assess intermediate scalability, a low pH incubation step was then introduced into the process for the intermediate scale runs: the pH was lowered to pH 2.6 using 0.1 M HCl, and then adjusted to pH 6.0 after 1 hour by adding 0.1 M NaOH.

[0439] The SE-HPLC (conditions shown in Table C and ) results showed a decrease in post-peak 1 accompanied by an increase in the main peak for the capture filtrate (low pH incubation) compared to the capture eluate (before low pH treatment), confirming the conversion of the conformational variant to the intact form. (data not shown).

[0440] Effects of other low pH treatments on the conformational mutants of compound A After expression of compound A in Pichia pastoris and clarification using tangential flow filtration, compound A was isolated from other impurities using capture chromatography using Amsphere A3 resin.

[0441] The column was first equilibrated with PBS buffer, pH 7.5, and then loaded with the clarified cell-free harvest material containing the compound of interest. Compound A bound to the Amsphere A3 resin, and impurities flowed through the column. The loaded resin was then washed with the same PBS buffer as in the equilibration step, followed by a wash with Tris buffer. The Tris buffer contained 100 mM Tris and 1 M NaCl at pH 8.5. The resin was further washed with a second 100 mM Tris buffer, pH 5.5. Compound A was eluted from the column using a low-pH glycine buffer. The low-pH glycine elution buffer contained 100 mM glycine at pH 3.0. Finally, the resin was sanitized with 100 mM NaOH and then stored in the same PBS buffer used for equilibration. All buffers were run at 183 cm / h.

[0442] In the first experiment, the pH of the Compound A capture eluate material was lowered to pH 2.6, pH 2.8, pH 2.9, and pH 3.0 using 1 M HCl. After 1 and 2 hours of incubation at low pH, the sample was adjusted to pH 6.0 using 0.2 M NaOH. The TO sample, or control sample, was a capture chromatography run that was frozen immediately after elution. This sample had a pH of 4.3.

[0443] In the second experiment, the pH of the product eluted from the chromatography column was 4.1 and 3.7 in the two capture chromatography runs. The pH of the capture eluate was lowered to pH 3.2 or pH 3.6 using 1 M HCl. After 2 and 4 hours of incubation at low pH, the sample was adjusted to pH 6.0 using 0.2 M NaOH. TO was generated by lowering Compound A to the target low pH (i.e., pH 3.2 or 3.6) using 1 M HCl and immediately adjusting to pH 6.0 using 0.2 M NaOH (TO).

[0444] The effect of pH on product quality was analyzed as a function of time by IEX-HPLC, see Tables 6-1 and 6-2 and Figures 16B and C.

[0445] [Table 10]

[0446] [Table 11]

[0447] The IEX-HPLC results show a positive effect of low pH treatment on the presence of conformational variants in the samples over time. In the first set of experiments (pH 2.6, pH 2.8, pH 2.9, and pH 3.0), the level of conformational variants in the control sample was 3.5%. In the second set of experiments (pH 3.2 and 3.6), the level of conformational variants was 3.1%.

[0448] After 2 hours of incubation at low pH, the level of conformational variants was reduced at all pH values ​​tested. The positive effect of low pH treatment on conformational variants increased with decreasing pH. The best reduction was observed between pH 3.0 and pH 2.6.

[0449] 6.5 Example 5: Scale-up of Low pH Treatment of Compound A (10 L and 100 L) Based on previous examples, the conditions selected for the low pH incubation of Compound A were a target pH of 2.6, at room temperature for ≥ 60 and ≤ 120 minutes. The pH of the capture eluate was lowered using 0.1 M HCl, and then adjusted to pH 6.0 after ≥ 60 and ≤ 120 minutes by adding 0.1 M NaOH. The fermentation process was scaled to 10 L and 100 L. The product quality of the capture eluate before the low pH treatment (referred to as "capture eluate") and the capture eluate after the above low pH treatment, subsequent pH adjustment to 6.0, and filtration (referred to as "capture filtrate") was determined by analytical methods such as SE-HPLC, CGE, and IEX-HPLC (conditions shown in Table C, IEX-HPLC protocol I). Three cycles of the capture step were performed for each scale to process all the starting material. The results for the different scales are shown in Table 7.

[0450] [Table 12]

[0451] Regardless of the scale of fermentation and purification, the low pH treatment and filtration steps had no effect on product purity in terms of the % main peak in CGE analysis. These results were within the range of method variability. Surprisingly, however, a decrease in the % HMW species by SE-HPLC (see Figure 17(1) (10 L) and Figure 17(2) (100 L)) was observed in both the fermentation (10 L and 100 L, respectively) and purification scale-ups (7 cm and 20 cm column diameter, respectively) when comparing the capture filtrate and capture eluate; this decrease is a result of the low pH treatment and / or filtration steps. Furthermore, as previously observed at small scale, a significant increase in the % main peak purity, as well as a decrease in the % post-peak (conformational variant) was observed in IEX-HPLC after low pH treatment when comparing the capture filtrate with the capture eluate (Table 7 and Figures 18 (10 L) and 19 (100 L)). Furthermore, the decrease in post-peak 1 (shoulder) was accompanied by an increase in the main peak for the capture filtrate profile. This correlates with the IEX data and supports the conversion of the conformational mutant of Compound A to the intact form.

[0452] Overall, these results demonstrated that low pH treatment is a scalable process and is effective for converting conformational mutants of Compound A to the intact form.

[0453] 6.6 Example 6: Separation of conformational variants of Compound A by other chromatographic techniques Use of mixed-mode chromatography (MMC) for the removal of conformational variants of compound A In the above examples, it was demonstrated that conformational variants of Compound A can be reliably separated using IEX-based chromatographic methods. To determine whether removal of less potent conformational variants from a mixture of conformational variants and the intact form can also be achieved by other chromatographic methods, mixed-mode chromatography (MMC) was performed using CHT ceramic hydroxyapatite type II (40 μm) resin (BioRad). The chromatographic conditions are summarized in Table 8.

[0454] [Table 13]

[0455] The chromatographic profile on hydroxyapatite resin is shown in Figure 20. Similar to CEX, a side (front) fraction (F8) and a top fraction (F11) were isolated and used for further SE-HPLC and IEX-HPLC analysis. The results of both analyses are shown in Figures 21(1) / (2) (SE-HPLC) and 22(1) / (2) (IEX-HPLC). A significant post-peak 1 was observed for fraction F8 (a fraction taken from the peak preceding the main / top peak) in SE-HPLC and IEX-HPLC (conditions shown in Table C; IEX-HPLC protocol I), indicating that this fraction contained a large amount of conformational variants. For fraction F11, SE-HPLC and IEX-HPLC post-peak 1 were significantly reduced compared to the load material, indicating that this fraction was depleted of conformational variants.

[0456] In conclusion, the results with hydroxyapatite resin were similar to those obtained with cation exchange resin, thus demonstrating that hydroxyapatite resin is suitable for removing less potent conformational variants from a mixture of conformational variants and intact forms of Compound A.

[0457] Use of Hydrophobic Interaction Chromatography (HIC) for the Removal of Conformational Variants of Compound A Because separation of the tight conformational variants from the intact form of Compound A was observed for a variety of chromatographic techniques and resin types, another chromatographic method, hydrophobic chromatography (HIC), was tested. First, a gradient using HIC TSK Phenyl Gel 5 PW(30) (Tosoh) resin was run using the conditions shown in Table 9.

[0458] [Table 14]

[0459] The corresponding HIC chromatograms are shown in Figure 23. As can be seen in the CEX and MMC chromatograms, the tested gradients produced HIC profiles containing two distinct peaks: a first (main) peak followed by a second (side) peak. One representative fraction from each peak was further analyzed. SE-HPLC data (conditions shown in Table C) from selected fractions of the main peak (F26; top fraction) and the side peak (F41; side fraction) are shown in Figure 24(1) / (2). The corresponding SE-HPLC profiles revealed that the top fraction consisted exclusively of the earlier-eluting intact form, since post-peak 1 was not observed in the SE-HPLC. In contrast, the SE-HPLC data indicated that the major species in the side fraction was almost entirely the later-eluting conformational variant (almost 100% post-peak 1).

[0460] Thus, using a gradient in HIC, good separation of the conformational variants from the desired intact form could be achieved, demonstrating that this HIC resin is suitable for removing the conformational variants of compound A from a mixture of both the conformational variants and the intact form.

[0461] Because the first HIC resin tested (TSK Phenyl Gel 5 PW(30) resin) was a high-resolution resin, other HIC resins that are more suitable for large-scale processing were tested: Capto phenyl High Sub (GE Healthcare), Capto phenyl ImpRes (GE Healthcare), and Capto butyl ImpRes (GE Healthcare), Phenyl HP (GE Heal) (GE Healthcare) and Capto Butyl (GE Healthcare). Gradients using ammonium sulfate and sodium chloride were tested. The conditions used are listed in Table 10 below. The SE-HPLC profile of the top fraction and load for the resin Capto Butyl Impres used with the ammonium sulfate gradient is shown in Figure 25.

[0462] [Table 15]

[0463] The post-peak in SE-HPLC was significantly reduced for all resins tested, except for Capto Phenyl High sub, using both sodium chloride and ammonium sulfate gradients. The results confirmed that conformational variants can be removed by using a process suitable for HIC resins with either sodium chloride or ammonium sulfate gradients.

[0464] The HIC chromatogram of the resin Capto Butyl Impres and the ammonium sulfate gradient is shown in Figure 26. As can be seen in the chromatogram, the tested gradient produced two separate peaks: a first (main) peak followed by a smaller second (side) peak. Several fractions of the main peak (F15 and F20) and one fraction of the second (side) peak (F29) were further analyzed by SE-HPLC. The resulting chromatogram (Figure 27) showed that fraction F29 contained only the later-eluting conformational variant (almost 100% SE-HPLC post-peak 1; see the peak shift compared to the load peak). In contrast, fractions 15 and 20 of the main peak did not show the presence of SE-HPLC post-peak 1, demonstrating that these fractions were depleted of the later-eluting undesired conformational variant.

[0465] Thus, using Capto Butyl Impres resin, good separation of conformational variants of Compound A using a gradient of hydrophobic interactions was achieved, thus demonstrating that this resin can be used to remove conformational variants from mixtures of both conformational variants and intact forms of Compound A.

[0466] Use of membrane-based HIC for removal of conformational variants of compound A Since separation of the conformational variants and the intact form could be satisfactorily achieved using HIC resin in a column, a flow-through mode process was developed that contained the desired intact form in the flow-through. An additional HIC setup using Phenyl (Sartorius) was performed. The conditions for screening on Sartobind phenyl membrane (filter plate) are listed in Table 11.

[0467] [Table 16]

[0468] [Table 17]

[0469] The SE-HPLC profile of a representative condition (condition C2) is shown in Figure 28. SE-HPLC post-peak 1 was significantly reduced compared to a reference sample containing conformational variants. The reference used capture eluate (from Protein A affinity chromatography) that was not subjected to low pH treatment but was neutralized directly to pH 7.4. The reference was not subjected to HIC. Therefore, the conformational variants were neither removed nor converted from the reference sample.

[0470] Further optimization was performed using 3 mL Sartobind phenyl membrane, and the conditions are listed in Table 12. Ammonium sulfate and sodium chloride were used at various concentrations to optimize recovery of the intact form in the flow-through.

[0471] [Table 18]

[0472] The HIC chromatogram for the optimal conditions is shown in Figure 29. SE-HPLC data from the load, fraction pool 2, and stripping fractions are shown in Figure 30. SE-HPLC post-peak 1 was significantly reduced from the membrane flow-through for pool 2. Stripping was enriched in the SE-HPLC post-peak shoulder, i.e., undesired conformational variants. Therefore, conformational variants were removed from the desired intact form of compound A in flow-through mode using the HIC phenyl membrane. Recoveries were 74% (pool 2) using ammonium sulfate and 63% (pool 2) using sodium chloride.

[0473] 6.7 Example 7: Identification and Initial Characterization of Compact Variants of Compound B Further investigations were performed on compound B to confirm that compact variants also emerge for other multivalent ISVD constructs.

[0474] Compound B (SEQ ID NO: 2) is a multivalent ISVD construct containing four different sequence-optimized variable domains of a heavy chain llama antibody that binds to three different targets. The ISVD binding blocks are fused head-to-tail (N- to C-terminal) using a G / S linker in the following format: TNFα-binding ISVD - 9GS linker - IL23p19-binding ISVD. VD - 9GS linker - human serum albumin-binding ISVD - 9GS linker - IL23p19-binding ISVD, and has the following sequence:

[0475] [Table 19]

[0476] The quality of Compound B protein was assessed by, among other techniques, analytical IEX-HPLC (conditions shown in Table C, IEX-HPLC protocol II).

[0477] For the purified Compound B protein, several distinct side peaks were observed in the IEX-HPLC profile (Figure 31). 2D-LC multiple heart-cutting analysis coupled with mass spectrometry (MS) was performed to identify the variants. By 2D-LC-MS, the top fractions of all peaks observed in the IEX-HPLC (1D) profile were separately collected and, after a desalting step (2D), analyzed by a Q-TOF mass spectrometer to determine the molecular weights of the proteins represented by the IEX peaks. 2D-LC-MS analysis showed that post-peak 1 had the same molecular weight as the product (main peak). It was concluded that post-peak 1 was an "intact mass variant" with an altered surface charge distribution compared to the product, potentially resulting in a more compact morphology (data not shown).

[0478] Furthermore, during the polishing step of Compound B, several CEX (cation exchange chromatography) resins exhibited chromatographic profiles (i.e., a main peak with a pre-peak "shoulder") similar to those previously observed for Compound A by CEX (see, e.g., Examples 1 and 2). Therefore, the material produced during the polishing step of Compound B was subsequently analyzed by IEX-HPLC. A gradient using the CEX resin was performed during the polishing process, and the run conditions are shown in Table 14, with the chromatogram shown in Figure 32.

[0479] [Table 20]

[0480] Fraction 2C4 and the pool of fractions 2C7 to 2C11 (Figure 32) were subjected to IEX-HPLC analysis and SE-HPLC analysis (conditions shown in Table C). The results are shown in Figures 33 and 34, respectively.

[0481] In IEX-HPLC analysis (Figure 33), fraction 2C4 contained 33.6% IEX-HPLC post-peak 1, while this variant was present at <1.0% in the pool of fractions 2C7-2C11. SE-HPLC results showed a chromatographic profile similar to that observed for compound A, with fraction 2C4 exhibiting a post-peak shoulder compared to fractions 2C7-2C11. Taken together, these results suggested that IEX-HPLC post-peak 1 may be a "compact" variant that could potentially have an impact on potency, as observed for compound A. Therefore, fraction 2C4 and the pool of fractions 2C7-2C11 were submitted for potency analysis.

[0482] The potency of Compound B against TNFα, IL-23 and HSA was determined as described in Section 5.4.5: - Cell-based reporter assays for potency testing of TNF-alpha binding moieties; - cell-based reporter assays for potency testing of IL-23 binding moieties; - ELISA-based albumin binding assay for potency testing of albumin-binding moieties.

[0483] The results of the efficacy analysis are shown in Table 15.

[0484] [Table 21]

[0485] A significant decrease in potency, at least against TNFα, was observed for concentrated fraction 2C4, containing 33.6% IEX-HPLC post-peak 1, compared to pooled fractions 2C7-2C11. It was concluded that in addition to affecting the hydrodynamic volume and charge of compound B, the conformational change also affects binding to at least TNFα.

[0486] Therefore, means to remove / convert compact mutants were investigated.

[0487] 6.8 Example 8: Determination of Conditions Affecting the Conformation of Compound B Low pH treatment of compound B Based on the observations made with Compound A, low pH incubation of the capture eluate material with Compound B was tested. The pH of the capture eluate was lowered to pH 2.1, pH 2.3, or pH 2.5 using 1 M HCl. After 1 hour of incubation at low pH, The samples were adjusted to pH 5.5 with 1 M sodium acetate. Product quality of the samples treated at various low pHs was compared to the capture eluate (control) immediately adjusted to pH 5.5 with 1 M sodium acetate and analyzed by IEX-HPLC (Table 16 and Figure 35) and SE-HPLC (Figure 36) (conditions also shown in Example 7 and Table C; IEX-HPLC protocol II).

[0488] [Table 22]

[0489] The results of the IEX-HPLC analysis indicate that the low pH treatment not only increased the product (% main peak purity) but also decreased the compact variant (% IEX-HPLC post-peak 1). Furthermore, and similar to compound A, the main peak observed in SE-HPLC became "sharpened" after the low pH treatment, suggesting the presence of a variant in the capture eluate, which was immediately adjusted to pH 5.5. Taken together, these results demonstrate the presence of a compact variant that can be converted to product (main peak in IEX-HPLC and / or SE-HPLC), and thus to the active product observed for compound A.

[0490] Based on the observations made with compound A, and to assess whether IEX-HPLC post-peak 1 could be transformed, conditions based on chaotropic agents, heat, or low pH were tested on compound B. Samples were then analyzed by RP-UHPLC, SE-HPLC, and IEX-HPLC. Only results with changes related to IEX-HPLC post-peak 1 are shown here.

[0491] Low pH treatment For the low pH treatment, Compound B was treated with 100 mM final glycine pH 2.5, pH 3.0, or pH 3.5, or formulation buffer pH 6.5 (control). After 4 hours of incubation at room temperature, samples were analyzed or neutralized with 0.1 M NaOH before analysis. IEX-HPLC and SE-HPLC results for the unneutralized samples are shown in Figures 37 and 38, respectively; all results are summarized in Table 17.

[0492] [Table 23]

[0493] When the sample was treated with 100 mM glycine, final pH 2.5, and incubated at room temperature for 4 hours with or without neutralization, the IEX-HPLC main peak percentage of compound B increased, while the IEX-HPLC post-peak percentage decreased compared to the control (Table 17 and Figure 37), suggesting that IEX-HPLC post-peak 1 is a conformational variant. IEX-HPLC post-peak 1 may be converted to the main peak and thus the active product. Furthermore, when the sample was treated with 100 mM glycine, final pH 3.5, and incubated at room temperature for 4 hours with or without neutralization, no significant changes were observed in the IEX-HPLC results compared to the control, and only a limited decrease in IEX-HPLC post-peak 1 could be observed after pH 3.0 treatment. Regarding the SE-HPLC results (Table 17 and Figure 38), no increase in HMW species was observed, indicating that IEX-HPLC post-peak 1 was not converted to HMW species (e.g., soluble aggregates). Furthermore, the SE-HPLC results indicated that pH 2.5 treatment affected the shape of the main peak. The main peak became "sharpened" after pH 2.5 treatment, which correlated with the IEX-HPLC results and those produced with Compound A.

[0494] Treatment with chaotropic agents For treatment with chaotropic agents, Compound B was treated with either 3 M final guanidine hydrochloride, 2 M final guanidine hydrochloride, 1 M final guanidine hydrochloride, or Milli Q (control), followed by incubation for 0.5 hours at room temperature. The IEX-HPLC results are shown in Figure 39.

[0495] The presence of GuHCl in the samples interfered with the IEX-HPLC method conditions, resulting in a decrease in the UV signal of the treated samples compared to the control. Although the integrated data is due to a lack of signal fidelity (and therefore not shown), the chromatogram overlay indicates that the addition of GuHCl can reduce the peak of the compact variant (IEX-HPLC post-peak 1). These results are consistent with those obtained for compound A.

[0496] Heat stress treatment For heat treatment, Compound B was incubated at 50°C for 1 hour, 50°C for 4 hours, 60°C for 1 hour, 60°C for 4 hours (followed by re-equilibration to room temperature), room temperature for 4 hours, or not incubated (control). The IEX-HPLC and SE-HPLC results are shown in Figures 40 and 41, respectively (for 50°C for 1 hour) and summarized in Table 18.

[0497] [Table 24]

[0498] When compound B was heat-treated at 50°C for 1 hour, 50°C for 4 hours, 60°C for 1 hour, or 60°C for 4 hours, the IEX-HPLC main peak % increased, while the IEX-HPLC post-peak 1 % decreased compared to the control, suggesting that IEX-HPLC post-peak 1 is a conformational variant (Table 18 and Figure 40). IEX-HPLC post-peak 1 could potentially be converted to the main peak and thus the active product. Furthermore, when incubated at room temperature for 4 hours, no significant changes were observed compared to the control. When these samples were analyzed by SE-HPLC, no increase in HMW species was observed, indicating that IEX-HPLC post-peak 1 was not converted to HMW species (e.g., soluble aggregates). Furthermore, the SE-HPLC results (Table 18 and Figure 41) indicated that heat treatment affected the shape of the main peak. The main peak became "sharper" upon heat treatment, which correlates with the IEX-HPLC results and those obtained with compound A.

[0499] Abstract Taken together, these results confirmed that IEX-HPLC post-peak 1 is a conformational variant of compound B (referred to herein as the less potent "compact variant") that can be converted to the more potent intact form of the main peak in IEX-HPLC and SE-HPLC (referred to herein as the "intact product") by low pH treatment at pH 2.5, GuHCl treatment, and / or heat treatment.

[0500] 6.9 Example 9: Optimization of Low pH Treatment for Compound B Based on the results of the Compound A and Compound B treatments described in Example 8 above, the low pH treatment was optimized for Compound B as a means of converting compact mutants.

[0501] After expression and harvest of compound B in Pichia pastoris, capture chromatography using Amsphere A3 resin was used to isolate compound B from other impurities.

[0502] The column was first equilibrated with PBS buffer pH 7.5 and loaded with the clarified cell-free harvest material containing the compound of interest. Compound B was attached to Amsphere A3 resin. The resin bound to the column, and impurities flowed through. The loaded resin was then washed with the same PBS buffer as used in the equilibration step, followed by a Tris buffer. The Tris buffer contained 100 mM Tris and 1 M NaCl at pH 8.5. The resin was further washed with a second 100 mM Tris buffer, pH 5.5. Compound B was eluted from the column using a low pH glycine buffer. The low pH glycine elution buffer contained 100 mM glycine at pH 3. Finally, the resin was sanitized with 100 mM NaOH and then stored in the same PBS buffer used in the equilibration step. All buffers were run at 183 cm / hr.

[0503] After capture chromatography, the pH of the product eluting from the chromatography column was pH 3.8. A low pH incubation step was then applied to compound B.

[0504] Low pH incubation time (1) Initial Experiments: Initially, the effects of low-pH treatment at pH 2.3 and pH 2.5 (see Example 1) were confirmed in subsequent experiments, and the length of incubation at low pH was further evaluated. The pH of the capture eluate was either lowered to pH 2.3 or pH 2.5 using 1 M HCl and immediately adjusted to pH 5.5 using 1 M sodium acetate (T0), incubated at low pH for 1 hour and then adjusted with 1 M sodium acetate (T1), incubated at low pH for 2 hours and then adjusted with 1 M sodium acetate (T2), or incubated at low pH for 4 hours and then adjusted with 1 M sodium acetate (T4). The product quality of the various low-pH-treated samples was compared with that of the capture eluate immediately adjusted to pH 5.5 using 1 M sodium acetate (control) and analyzed by IEX-HPLC, SE-HPLC, and CGE (conditions shown in Table C; IEX-HPLC protocol II). The SE-HPLC results are shown in Figures 42A and 42B and summarized in Table 19.

[0505] [Table 25]

[0506] Regarding the IEX-HPLC results (Table 19), the control, pH 2.3 TO and pH 2.5 No differences were observed between TO. A significant increase in the % main peak purity as well as a decrease in the % IEX-HPLC post-peak 1 (compact variant) was observed for 1 hour, 2 hours, and 4 hours incubation at low pH. Furthermore, reduction of IEX-HPLC post-peak 1 was most effective at the longest incubation times. Regarding the SE-HPLC results (Table 19, Figures 42A and 42B), lowering the pH of the capture eluate to pH 2.3 or pH 2.5 resulted in a slight increase in the HMW species (pre-peak), but primarily in a narrower main peak, as previously observed. CGE profiles (Table 19) showed no significant differences in main peak purity between different samples, confirming the initial 2D-LC results (Example 7) that the compact variant did not have a molecular weight different from that of the intact product. Taken together, these results confirmed that IEX-HPLC post-peak 1 is a compact variant that can be converted to the main peak in IEX-HPLC and SE-HPLC by low pH 2.3 and pH 2.5 treatment for 1, 2, and 4 hours.

[0507] (2) Further experiments: The low pH treatment of the initial experiment was then expanded. The pH of the Compound B capture material was lowered to pH 2.7, pH 2.9, pH 3.1, pH 3.3, pH 3.5, and pH 3.9 using 1 M HCl. After 2 and 4 hours of incubation at low pH, the samples were adjusted to pH 5.5 with 1 M sodium acetate.

[0508] The pH of the Compound B capture eluate was lowered to the target low pH (i.e., pH 2.7-3.9 as shown above) using 1 M HCl and immediately adjusted to pH 5.5 using 1 M sodium acetate to generate TO (TO).

[0509] The effect of low pH treatment as a function of time on product quality was analyzed by IEX-HPLC, see Table 20 and Figures 43A and B.

[0510] [Table 26]

[0511] The IEX-HPLC results show a positive impact of low pH treatment on the presence of conformational variants in the samples. The level of conformational variants in the TO samples was similar in all samples tested. In the initial set of experiments, i.e., at pH 2.3 and 2.5, this level was approximately 4.5%. In further experiments, the level of conformational variants in the control samples at TO (pH 2.7, 2.9, 3.1, 3.3, 3.5, and pH 3.7) was approximately 3%.

[0512] After 2 hours of incubation at low pH, the level of conformational mutants was At all pH levels tested The positive effect of low pH on the conformational mutants increased at lower pH, i.e., below pH 3.0.

[0513] After 4 hours of incubation at low pH, the level of conformational variants was further reduced for all pHs tested, with the best reduction occurring at pH 2.3 and no significant reduction at pH 2.9. was observed.

[0514] All results obtained in this example indicate a positive effect of low pH, especially pH 3 or below, on conformational mutants.

[0515] Further low pH treatment Next, to examine the breadth of the operating range of the low pH treatment, 2-hour low pH incubations at pH 2.4 and pH 2.6 were investigated. The pH of the capture eluate was lowered to pH 2.4 or pH 2.6 using 1 M HCl, and the samples were incubated at room temperature for 2 hours. The samples were then adjusted to pH 5.5 using 1 M sodium acetate. The product quality of the various low pH treated samples was compared to a capture eluate (control) immediately adjusted to pH 5.5 using 1 M sodium acetate and analyzed by IEX-HPLC, SE-HPLC, and GE. The results are shown in Figure 44 and summarized in Table 21.

[0516] [Table 27]

[0517] The HPLC results (Table 21) show a significant increase in the % main peak purity, as well as a decrease in the % IEX-HPLC post-peak 1 (compact variant) after 2 hours of incubation at pH 2.4 and pH 2.6. The SE-HPLC results (Table 21 and Figure 44) show that lowering the pH of the capture eluate to pH 2.4 or pH 2.6 resulted in a slight increase in the HMW species, but a narrower main peak, as previously observed. The CGE profiles (Table 21) showed no significant differences between the control and low-pH-treated samples, confirming the earlier 2D-LC results (Example 7) that the compact variant does not have a molecular weight different from the intact product. Taken together, these results confirmed that IEX-HPLC post-peak 1 is a conformational variant that can be converted to the intact form of the main peak in IEX-HPLC by 2 hours of treatment at pH 2.4 and 2.6.

[0518] Low pH Adjustment Procedure Finally, a pH adaptation procedure was investigated to examine its impact on the subsequent purification steps of the method. Indeed, increasing the pH with 1 M sodium acetate after the low pH treatment until it reached pH 5.5 significantly increased the conductivity of the sample. The sample then had to be highly diluted with water to a conductivity suitable for the subsequent chromatographic step (≦6.0 mS / cm). This significantly increased the load volume and, consequently, the process time.

[0519] Different approaches for pH adjustment after low pH treatment were performed in two independent experiments (Table 22). In experiment 1, the capture eluate was either immediately adjusted to pH 5.5 and conductivity ≦6.0 mS / cm with 1 M sodium acetate pH 9 (control 1), or the capture eluate was first adjusted to pH 2.4 with 1 M HCl for 2 hours and then adjusted to pH 5.5 with 1 M sodium acetate (control 2). The pH was adjusted to 0.5 and diluted with MilliQ water until a constant conductivity (≦6.0 mS / cm) was reached.

[0520] In Experiment 2, the capture eluate was either immediately adjusted to pH 5.5 and a conductivity of ≦6.0 mS / cm with 1 M sodium acetate pH 9 (Control 2), or the capture eluate was first adjusted to pH 2.6 with 1 M HCl for 2 hours, and then adjusted to pH 5.5 and a conductivity of ≦6.0 mS / cm by (i) adding a volume of 1 M sodium acetate pH 5.5 to reach approximately 50 mM sodium acetate, (ii) adjusting to pH 5.5 with 0.1 M NaOH, and (iii) adjusting to a conductivity of ≦6.0 mS / cm with water, if necessary.

[0521] [Table 28]

[0522] A similar decrease in the % of IEX-HPLC post-peak 1 was observed in IEX-HPLC, independent of the approach taken to increase the pH to pH 5.5 after the low pH treatment (Tables 21 and 22). Surprisingly, compared to the previous results for Compound B, there was no increase in HMW species in SE-HPLC using the new pH adjustment approach (a mixture of 1 M sodium acetate pH 5.5 and 0.1 M NaOH) (Table 22 and Figure 45). Furthermore, a narrowing of the SE-HPLC main peak was still observed after the low pH treatment at pH 2.6 and the new pH adjustment approach (Figure 45). Finally, the dilution factor (volume of eluate adjusted to pH 5.5 / volume of capture eluate) was significantly lower with the new pH adjustment approach (Table 22), thus facilitating the process by reducing the volume to be processed in the next purification step. Overall turnaround time has been improved.

[0523] 6.10 Example 10: Effect of Low pH Treatment on Compound B Because initial characterization showed a decrease in potency for fractions enriched in IEX-HPLC post-peak 1 (i.e., the compact variant), and because low pH treatment converts the compact variant of Compound B to a more active intact product, the effect of low pH treatment on the conformational variant of Compound B was investigated below to assess whether potency could be restored. A gradient was run using CEX resin using the run conditions shown in Table 23. The chromatogram is shown in Figure 46.

[0524] [Table 29]

[0525] The CEX chromatogram showed the expected main peak shoulder containing the compact variant. A pool of fractions 10-14 (Figure 46) was subjected to IEX-HPLC analysis (conditions shown in Table C; IEX-HPLC protocol II) without low pH treatment or after low pH treatment at pH 2.5. A summary of the IEX-HPLC results is shown in Table 24.

[0526] [Table 30]

[0527] The low pH treatment converted the compact variant of IEX-HPLC post-peak 1 to the intact product of the main peak, as evidenced by a decrease in IEX-HPLC post-peak 1 from 19.5% to 8.0%. The low pH treated samples were subjected to potency analysis and compared to previously obtained results (Table 25). The low pH treatment restored potency, particularly against TNFα, by converting the compact variant to an active product. Thus, the low pH treatment is a means of converting the compact variant of Compound B to an active intact product.

[0528] [Table 31]

[0529] 6.11 Example 11: Use of HIC to remove less potent compact variants of Compound B Having successfully removed / enriched the compact variants of compound A by hydrophobic interaction chromatography (HIC), HIC was also tested for the removal of the compact variants of compound B. A gradient using Capto Butyl ImpRes resin (GE Healthcare) was performed using the run conditions shown in Table 26. The chromatogram is shown in Figure 47. The HIC load (polishing eluate buffer exchanged at the appropriate loading conditions) and elution fractions 14, 19, 20, 24, and 28 were analyzed by SDS-PAGE (Figure 48). Fraction 14 and fractions 18-26 were analyzed by IEX-HPLC (Table 27).

[0530] [Table 32]

[0531] [Table 33]

[0532] As observed in the chromatographic HIC profile (Figure 47), this gradient produced two distinct peaks. SDS-PAGE analysis (Figure 48) showed that the main bands in the various fractions had similar molecular weights, as expected for the compact variant. Interestingly, IEX-HPLC analysis (Table 27) revealed that only the first peak (fraction 14) in the HIC profile contained the active product and the less active compact variant, with 47.9% "intact product" and 52.1% "compact variant," respectively. Furthermore, IEX-HPLC analysis (Table 27) showed that the compact variant was absent in the second peak (fractions 19-26) in the HIC profile. Thus, the conformational variants of compound B could be completely removed and / or enriched by hydrophobic interaction chromatography.

[0533] 6.12 Example 12: Removal / reduction of less potent compact variants by increasing the loading rate on the capture column.

[0534] To optimize the capture step for compound B, various parameters (i.e., factors), such as loading rate (mg product / ml resin), load flow rate (cm / hr), elution buffer pH, load pH, and purification process wash, were evaluated using a design of experiments (DOE) approach using the Definitive Screening Design (DSD) approach from JMP (SAS Institute) software. Various outputs (i.e., responses) were measured to evaluate the impact of these factors on the response. Responses include, but are not limited to, IEX-HPLC analysis to evaluate whether IEX-HPLC post-peak 1 could be reduced / eliminated during the capture step. DOE results were analyzed using JMP software according to the DSD approach. Interestingly, of the various factors tested, only loading rate had an effect on IEX-HPLC post-peak 1 (Figure 49). Surprisingly, IEX-HPLC post-peak 1 of the compact variant could be significantly eliminated / reduced by increasing the loading rate (Table 28). Therefore, increasing the loading rate on the capture column with ISVD products can be used as a means to reduce / remove undesired lower potency compact variants.

[0535] [Table 34]

[0536] 6.13 Example 13: Scale-up of Compound B Low pH Treatment (10 L and 100 L) Based on the above example, the conditions selected for the low-pH incubation of compound B were a target pH of 2.5 at room temperature for 2 hours. The pH of the capture eluate was lowered using 1 M HCl, and then after 2 hours, it was adjusted to pH 5.5 and conductivity ≦6.0 mS / cm by (i) adding a predetermined volume of 1 M sodium acetate pH 5.5 to reach approximately 50 mM sodium acetate, (ii) adjusting to pH 5.5 with 0.1 M NaOH, and (iii) adjusting to a conductivity ≦6.0 mS / cm with water, if necessary. The compound B production process was then scaled up to 10 L and 100 L fermentation scales for further purification. Analytical methods, such as SE-HPLC, IEX-HPLC, and CGE, were used to analyze the product quality of the capture eluate before low-pH treatment (i.e., capture eluate) and the capture eluate after low-pH treatment, which had been adjusted to pH 5.5 and filtered as described above (i.e., capture filtrate). Two cycles of the capture step were performed for each scale. The results for the various scales are shown in Table 29.

[0537] [Table 35]

[0538] First, regardless of fermentation and purification scale, the low pH treatment and filtration steps had no effect on product quality in terms of the % main peak in CGE analysis and CGE profiles, and these results were within the range of method variability (Table 29). Surprisingly, a decrease in the % HMW species was observed at both scales when comparing the capture filtrate and capture eluate, and therefore could be attributed to the low pH treatment and / or filtration steps (Table 29). Furthermore, SE-HPLC results (Figures 50 and 51) confirmed that low pH treatment affected the shape of the main peak. The main peak became "sharper" after low pH treatment (e.g., in the capture filtrate), which correlated with the IEX-HPLC results and those obtained for Compound A and Compound B. Finally, as previously observed at smaller scales, a significant increase in the % main peak purity, as well as a decrease in the % IEX-HPLC post-peak 1 (compact variant), was also observed by IEX-HPLC after low pH treatment when comparing the capture filtrate with the capture eluate (Table 29).

[0539] Taken together, these results demonstrate that low pH treatment is a scalable process and is effective in converting less potent, undesirable compact variants of multivalent ISVD product constructs into potent, intact products.

[0540] 6.14 Example 14: Identification and Initial Characterization of Compact Variants of Compound C Further investigations were carried out on compound C to confirm that compact mutants also emerge for other multivalent ISVD constructs.

[0541] Compound C (SEQ ID NO: 69) is a multivalent ISVD construct containing three immunoglobulin single variable domains of a heavy chain llama antibody that binds to two different targets. The lock is fused head-to-tail (N-terminus to C-terminus) with a G / S linker in the following format: TNFα binding ISVD - 9GS linker - human serum albumin binding ISVD - 9GS linker - TNFα binding ISVD, and has the following sequence:

[0542] [Table 36]

[0543] After expression of compound C in Pichia pastoris and harvesting the compound by tangential flow filtration, capture chromatography using Amsphere A3 resin was used to isolate compound C from other impurities.

[0544] The column was first equilibrated with PBS buffer, pH 7.3, and loaded with the clarified cell-free harvest material containing compound C. Compound C bound to the Amsphere A3 resin, and impurities flowed through the column. The loaded resin was then washed with the same PBS buffer as in the equilibration step. Compound C was eluted from the column using a low-pH glycine buffer. The low-pH glycine elution buffer contained 100 mM glycine, pH 3.0. Finally, the resin was sanitized with 100 mM NaOH and then stored in the same PBS buffer used for equilibration. All buffers were run at 183 cm / h.

[0545] After capture chromatography, the pH of the product eluted from the chromatography column was pH 3.5. Compound C was then incubated at low pH. The pH of the capture eluate was lowered to pH 2.5 or pH 3.0 using 1 M HCl. After 2 and 4 hours of incubation at low pH, the sample was adjusted to pH 5.5 using 1 M sodium acetate pH 6.0. TO was generated by lowering Compound C to the target low pH (i.e., pH 2.5 or 3.0) using 1 M HCl and immediately adjusting to pH 5.5 using 1 M sodium acetate (TO).

[0546] The quality of the compound C protein was assessed by SE-HPLC. A clear post-peak was also observed in SE-HPLC for compound C (Figures 53A and B).

[0547] The effect of pH on product quality was analyzed as a function of time by SE-HPLC (see Table 31 and Figure 54).

[0548] [Table 37]

[0549] The SE-HPLC results show a positive impact of low pH treatment on the presence of conformational variants in the samples. The levels of conformational variants in the TO sample were similar in the two samples tested: 6.7% compact variants for the pH 2.5 sample and 6.8% conformational variants for the pH 3.0 sample. These two values ​​are similar to the initial sample, i.e., the capture eluate not treated at low pH, in which the level of conformational variants was 6.9%. After 2 hours of incubation at low pH, a decrease in conformational variants was observed for all pHs tested. This decrease continued for up to 4 hours of further incubation at low pH.

[0550] All the results obtained in this example show a positive effect of low pH on the percentage of conformational variants.

[0551] 6.15 Example 15: Absence of Compact Mutants during ISVD Production in CHO Cells After expression of Compound C (SEQ ID NO: 69) in CHO cells, capture chromatography using MabSelect Xtra resin was used to isolate Compound C from other impurities.

[0552] The column was first equilibrated with Tris buffer and loaded with the clarified cell-free harvest material containing the compound of interest. The equilibration buffer contained 50 mM Tris, 150 mM NaCl at pH 7.5. Compound C bound to the MabSelect Xtra resin, and impurities flowed through the column. The loaded resin was then washed with the same Tris buffer as in the equilibration step, followed by a second wash with Tris wash buffer. The wash buffer contained 10 mM Tris, 10 mM NaCl at pH 7.5. Compound C was eluted from the column using a low-pH glycine buffer. The low-pH glycine elution buffer contained 50 mM glycine at pH 3.0. Finally, the resin was regenerated with 100 mM glycine buffer, pH 2.5, and sanitized with 50 mM NaOH, 1 M NaCl before being stored in Et-OH. All buffers were run at 191 cm / h.

[0553] After capture chromatography, the product eluting from the chromatography column had a pH of 3.4. Compound C was then incubated at low pH. The pH of the capture eluate was lowered to pH 2.5 or pH 3.0 using 1 M HCl. After 2 hours of incubation at low pH, the sample was lowered to pH 5.5 using 1 M HEPES pH 7.0. The capture eluate, which was immediately adjusted to pH 5.5, served as the control sample in this experiment.

[0554] The quality of Compound C protein was assessed by SE-HPLC. When Compound C was produced in CHO cells, no post-peak was observed in SE-HPLC (Figure 55).

[0555] The SE-HPLC results showed that no conformational variants were present in the samples.

[0556] 6.16 Example 16: Identification and Initial Characterization of Conformational Variants of Compound D Compound D (SEQ ID NO: 70) is a multivalent ISVD construct containing four immunoglobulin single variable domains of heavy chain llama antibodies that bind to three different targets. The ISVD building blocks are fused head-to-tail (N- to C-terminus) with G / S linkers in the following format: TNFα-binding ISVD - 9GS linker - IL-6-binding ISVD - 9GS linker - human serum albumin-binding ISVD - 9GS linker. - an IL-6 binding ISVD and has the following sequence:

[0557] [Table 38]

[0558] After expression and harvesting of Compound D in Pichia, capture chromatography using Amsphere A3 resin was used to isolate Compound D from other impurities.

[0559] The column was first equilibrated with PBS buffer, pH 7.5, and loaded with the clarified cell-free harvest material containing the compound of interest. Compound D bound to the Amsphere A3 resin, and impurities flowed through the column. The loaded resin was then washed with the same PBS buffer as in the equilibration step. Compound D was eluted from the column using a low-pH glycine buffer. The low-pH glycine elution buffer contained 100 mM glycine at pH 3.0. Finally, the resin was sanitized with 100 mM NaOH and then stored in the same PBS buffer used for equilibration. All buffers were run at 233 cm / h.

[0560] Compound D was incubated at low pH. The pH of the capture eluate was lowered to pH 2.5, pH 2.7, pH 2.9, pH 3.1, pH 3.2, pH 3.4, and pH 3.6 using 1 M HCl. After 2 and 4 hours of incubation at low pH, the sample was adjusted to pH 5.5 using 0.1 M sodium acetate pH 5.6. Compound D was lowered to the target low pH (i.e., pH 2.3, pH 2.7, pH 2.9, pH 3.1, pH 3.2, pH 3.4, and pH 3.6) using 1 M HCl and immediately diluted with 1 M sodium acetate. To was generated by adjusting the pH to 5.5 using ethanol (To).

[0561] The effect of pH on product quality as a function of time was analyzed by SE-HPLC (Table 33 and Figure 56).

[0562] [Table 39]

[0563] The SE-HPLC results show the positive effect of low pH treatment on the presence of conformational variants in the samples. The level of conformational variants in the TO sample was similar in all samples tested. The level of conformational variants in the control samples at TO, pH 2.9, 3.1, 3.2, 3.4, and pH 3.6 was approximately 8.7%. At lower pHs, i.e., pH 2.5 and pH 2.7, the starting amount was lower (pH 7.6 and pH 8.2) due to the positive effect of pH.

[0564] After 2 hours of incubation at low pH, the level of conformational variants decreased. The positive effect of low pH on conformational variants increased at lower pH.

[0565] After 4 hours of incubation at low pH, the level of conformational variants was further reduced, with the best reduction occurring at pH 2.3 and continuing up to pH 3.1.

[0566] All the results obtained in this example show a positive effect of low pH on the conformational variants over time.

[0567] 6.17 Example 17: Identification and Initial Characterization of Conformational Variants of Compound E Compound E (SEQ ID NO: 71) is a multivalent ISVD construct containing four immunoglobulin single variable domains of a heavy chain llama antibody that bind to three different targets. The ISVD building blocks are fused head-to-tail (N- to C-terminus) using G / S linkers in the following format: TNFα-binding ISVD - 9GS linker - IL-6-binding ISVD - 9GS linker - human serum albumin-binding ISVD - 9GS linker - IL-6-binding ISVD, and has the following sequence:

[0568] [Table 40]

[0569] After expression and harvesting of Compound E in Pichia, capture chromatography using Amsphere A3 resin was used to isolate Compound E from other impurities.

[0570] The column was first equilibrated with PBS buffer, pH 7.5, and then loaded with the clarified cell-free harvest material containing the compound of interest. Compound E bound to the Amsphere A3 resin, and impurities flowed through the column. The loaded resin was then washed with the same PBS buffer used in the equilibration step. Compound E was then eluted from the column using a low-pH glycine buffer. The low-pH glycine elution buffer contained 100 mM glycine at pH 3.0. Finally, the resin was sanitized with 100 mM NaOH and then stored in the same PBS buffer used for equilibration. All buffers were run at 233 cm / h.

[0571] Compound E was incubated at low pH. The pH of the capture eluate was lowered to pH 2.5, pH 2.7, pH 2.9, pH 3.1, pH 3.2, pH 3.4, and pH 3.6 using 1 M HCl. After 2 hours of incubation at low pH, the sample was adjusted to pH 5.5 using 0.1 M sodium acetate pH 5.6. Compound E was lowered to the target low pH (i.e., pH 2.5, pH 2.7, pH 2.9, pH 3.1, pH 3.2, pH 3.4, and pH 3.6) using 1 M HCl and immediately adjusted to pH 5.5 using 1 M sodium acetate to generate TO (TO).

[0572] The effect of pH on product quality as a function of time was analyzed by SE-HPLC (Table 35 and Figure 57).

[0573] [Table 41]

[0574] The SE-HPLC results show the positive effect of low pH treatment on the presence of conformational variants in the samples. The level of conformational variants in the TO sample was similar in all tested samples. The level of conformational variants in the control samples at TO, pH 2.9, 3.1, 3.2, 3.4, and pH 3.6 was approximately 7.5% (or higher). At lower pHs, i.e., pH 2.5 and pH 2.7, the starting amount was lower (pH 7.2) due to the positive effect of pH.

[0575] After 2 hours of incubation at low pH, the level of conformational variants decreased. The positive effect of low pH on conformational variants increased at lower pH. All results obtained in this example show the positive effect of low pH on conformational variants over time. The best reduction was obtained at pH 2.5 up to pH 2.9.

Claims

1. 1. A method for isolating or purifying a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs) from a composition comprising said polypeptide and conformational variants thereof, the method comprising: a) applying conditions that convert a conformational variant into said polypeptide; b) removing conformational variants; or c) A combination of (a) and (b) The above method, comprising:

2. The method of claim 1, wherein the polypeptide to be isolated or purified is obtainable by expression in a host.

3. 3. The method of claim 2, wherein the polypeptide to be isolated or purified is obtainable by expression in a host that is not a CHO cell.

4. 4. The method of claim 2 or 3, wherein the polypeptide to be isolated or purified is obtainable by expression in a host which is a lower eukaryotic host.

5. 5. The method of claim 4, wherein the lower eukaryotic host comprises a yeast such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Cytheromyces, Pachysolen, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, or Endomycopsis.

6. 6. The method of claim 5, wherein the yeast is a Pichia, such as Pichia pastoris.

7. The method of any one of claims 1 to 6, wherein the polypeptide comprises or consists of at least four immunoglobulin single variable domains (ISVDs).

8. The method of any one of claims 1 to 7, wherein the conformational variant is characterized by a more compact morphology compared to the polypeptide.

9. The method of any one of claims 1 to 8, wherein the conformational variant has a reduced hydrodynamic volume compared to the polypeptide.

10. The method of any one of claims 1 to 9, wherein the conformational variant is characterized by an increased retention time in SE-HPLC compared to the polypeptide.

11. The method of any one of claims 1 to 10, wherein the conformational variant is characterized by an altered retention time in IEX-HPLC compared to said polypeptide.

12. The conditions for converting the conformational mutant into the above polypeptide are: i) applying a low pH treatment in a step of the isolation or purification process, optionally wherein the low pH treatment comprises lowering the pH of the composition to about pH 3.2 or below, or to about pH 3.0 or below; ii) applying a chaotropic agent in a step of the isolation or purification method, optionally wherein the chaotropic agent is guanidinium chloride (GuHCl); iii) applying heat stress during an isolation or purification process step, optionally including incubating the conformational mutant at about 40°C to about 60°C; or iv) Any combination of i) to iii) The method according to any one of claims 1 to 11, wherein the compound is selected from the group consisting of:

13. 7. The method of any one of claims 1 to 6, wherein the polypeptide comprises or consists of at least four immunoglobulin single variable domains (ISVDs), and wherein the low pH treatment comprises lowering the pH of the composition to about pH 3.0 or below.

14. 14. The method of claim 12 or 13, wherein the pH is lowered to between about pH 3.2 and about 2.1, between about pH 3.0 and about 2.1, between about pH 2.9 and about pH 2.1, between about pH 2.7 and about pH 2.1, or between about pH 2.6 and about pH 2.

3.

15. The method of any one of claims 12 to 14, wherein the low pH treatment is applied for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

16. 16. The method of any one of claims 12 to 15, wherein the pH is lowered to between about pH 3.2 and about pH 2.1 for at least about 0.5 hours, such as at least about 1.0 hour.

17. 17. The method of any one of claims 12 to 16, wherein the pH is lowered to between about pH 3.0 and about pH 2.1 for at least about 0.5 hours, such as at least about 1.0 hour.

18. 18. The method of any one of claims 12 to 17, wherein the pH is lowered to between about pH 2.9 and about pH 2.1 for at least about 0.5 hours, such as at least about 1.0 hour.

19. 19. The method of any one of claims 12 to 18, wherein the pH is lowered to between about pH 2.7 and about pH 2.1 for at least about 0.5 hours, such as at least about 1.0 hour.

20. 20. The method according to any one of claims 12 to 19, wherein the low pH treatment is applied before, during or after a purification step based on chromatography techniques.

21. 21. The method of claim 20, wherein the low pH treatment is applied before applying the composition to a stationary phase of the chromatographic technique or after eluting the composition from the stationary phase of the chromatographic technique.

22. The method of any one of claims 12 to 21, wherein the chaotropic agent is guanidinium chloride (GuHCl) at a final concentration of at least about 1 M or at least about 2 M.

23. 23. The method of any one of claims 12 to 22, wherein GuHCl is applied for at least 0.5 hours, or at least 1 hour.

24. The method of any one of claims 12 to 23, wherein the heat stress is applied for at least about 1 hour.

25. 12. The method of any one of claims 1 to 11, wherein conformational variants are removed by one or more chromatographic techniques, optionally wherein the conformational variants have been identified by analytical chromatographic techniques such as SE-HPLC and IEX-HPLC before being removed by one or more chromatographic techniques.

26. Chromatography techniques involve the selection of chromatographic molecules based on hydrodynamic volume, surface charge, or surface hydrophobicity.

26. The method of claim 25, which is a lithography technique.

27. 27. The method of claim 26, wherein the chromatographic technique is selected from size exclusion chromatography (SEC), ion exchange chromatography (IEX), e.g., cation exchange chromatography (CEX), mixed mode chromatography (MMC), and hydrophobic interaction chromatography (HIC).

28. 28. The method of claim 27, wherein the HIC is based on a HIC column resin.

29. 28. The method of claim 27, wherein the HIC is based on a HIC membrane.

30. 30. The method of any one of claims 1 to 29, wherein isolating or purifying the polypeptide comprises applying the composition to a chromatography column, wherein the composition is applied to the column using a load factor of at least 20 mg protein / ml of resin, at least 30 mg protein / ml of resin, or at least 45 mg protein / ml of resin, optionally wherein the chromatography column is a Protein A column.

31. 31. The method of any one of claims 1 to 30, wherein one or more conditions that convert conformational variants into said polypeptide are applied alone or in combination with one or more techniques that remove conformational variants.

32. 1. A method for isolating or purifying a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs), the method comprising: i) applying a low pH treatment to a composition comprising the polypeptide in a step of an isolation or purification method, optionally wherein the low pH treatment comprises lowering the pH of the composition to about pH 3.2 or below, or to about pH 3.0 or below; ii) applying a chaotropic agent to a composition comprising said polypeptide in a step of the isolation or purification method, optionally wherein the chaotropic agent is GuHCl; iii) subjecting a composition comprising said polypeptide to heat stress during an isolation or purification process, optionally comprising incubating said composition at about 40°C to about 60°C; iv) applying the composition comprising the polypeptide to a chromatography column using a loading rate of at least 20 mg / ml, at least 30 mg / ml, at least 45 mg / ml, optionally wherein the chromatography column is a Protein A column; or v) Any combination of i) to iv). The above method, comprising one or more of the following:

33. 33. The method of claim 32, wherein the polypeptide to be isolated or purified is obtainable by expression in a host.

34. 34. The method of claim 33, wherein the polypeptide to be isolated or purified is obtainable by expression in a host that is not a CHO cell.

35. 35. The method of claim 33 or 34, wherein the polypeptide to be isolated or purified is obtainable by expression in a host which is a lower eukaryotic host.

36. Lower eukaryotic hosts include Pichia, Hansenula, Saccharomyces, Kluyveromyces, and Candida.

36. The method of claim 35, comprising yeast such as Da, Torulopsis, Torulaspora, Schizosaccharomyces, Cytheromyces, Pachysolenes, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, and Endomycopsis.

37. 37. The method of claim 36, wherein the yeast is a Pichia, such as Pichia pastoris.

38. 38. The method of any one of claims 32 to 37, wherein the polypeptide comprises or consists of at least four immunoglobulin single variable domains (ISVDs), optionally wherein the low pH treatment comprises lowering the pH of the composition to about pH 3.0 or below.

39. 39. The method of any one of claims 32 to 38, wherein the pH is lowered to between about pH 3.2 and about pH 2.1, between about pH 3.0 and about pH 2.1, between about pH 2.9 and about pH 2.1, between about pH 2.7 and about pH 2.1, or between about pH 2.6 and about pH 2.

3.

40. 40. The method of any one of claims 32 to 39, wherein the low pH treatment is applied for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

41. 41. The method of claim 39 or 40, wherein the pH is lowered to between about pH 3.2 and about pH 2.1 for at least about 0.5 hours, such as at least about 1.0 hour.

42. 42. The method of any one of claims 39 to 41, wherein the pH is lowered to between about pH 3.0 and about pH 2.1 for at least about 0.5 hours, such as at least about 1.0 hour.

43. 43. The method of any one of claims 39 to 42, wherein the pH is lowered to between about pH 2.9 and about pH 2.1 for at least about 0.5 hours, such as at least about 1.0 hour.

44. 44. The method of any one of claims 39 to 43, wherein the pH is lowered to between about pH 2.7 and about pH 2.1 for at least about 0.5 hours, such as at least about 1.0 hour.

45. 45. The method according to any one of claims 32 to 44, wherein the low pH treatment is applied before, during or after a purification step based on chromatography techniques.

46. 46. ​​The method of claim 45, wherein the low pH treatment is applied before applying the composition to a stationary phase of the chromatographic technique or after eluting the composition from a stationary phase of the chromatographic technique.

47. 47. The method of any one of claims 32 to 46, wherein the chaotropic agent is GuHCl at a final concentration of at least about 1 M, or at least about 2 M.

48. 48. The method of any one of claims 32 to 47, wherein GuHCl is applied for at least 0.5 hours, or at least 1 hour.

49. 49. The method of any one of claims 32 to 48, wherein the heat stress is applied for at least about 1 hour.

50. 50. A method for the preparation of a human immunoglobulin comprising: A method for producing a polypeptide.

51. The following steps: a) optionally culturing a host or host cells under conditions such that the host or host cells grow; b) maintaining the host or host cell under conditions such that the host or host cell expresses and / or produces the polypeptide; and c) isolating and / or purifying the secreted polypeptide from the culture medium, comprising one or more of the isolation or purification methods according to any one of claims 1 to 49.

51. The method of claim 50, comprising at least:

52. 52. The method of claim 50 or 51, wherein the host is not a CHO cell.

53. 53. The method of any one of claims 50 to 52, wherein the host is a lower eukaryotic host.

54. 54. The method of claim 53, wherein the lower eukaryotic host comprises a yeast such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Cytheromyces, Pachysolen, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, or Endomycopsis.

55. 55. The method of claim 54, wherein the yeast is a Pichia, such as Pichia pastoris.

56. 1. A method for isolating or purifying a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs) from a composition comprising said polypeptide and conformational variants thereof, said method comprising: (1) Identifying conformational variants by analytical chromatographic techniques such as SE-HPLC and IEX-HPLC; (2) adjusting the chromatographic conditions to allow for the specific removal of conformational variants; and (3) removing conformational variants from a composition comprising the polypeptide and its conformational variants by one or more chromatographic techniques. The above method, comprising:

57. 1. A method for optimizing one or more chromatographic techniques to allow the isolation or purification of a polypeptide comprising or consisting of at least three or at least four immunoglobulin single variable domains (ISVDs) from a composition comprising said polypeptide and conformational variants thereof, the method comprising: (1) Identifying conformational variants by analytical chromatographic techniques such as SE-HPLC and IEX-HPLC; (2) Optimizing chromatographic conditions to enable specific removal of conformational variants The above method, comprising:

58. 58. The method of claim 56 or 57, wherein the polypeptide to be isolated or purified is obtainable by expression in a host.

59. 59. The method of claim 58, wherein the polypeptide to be isolated or purified is obtainable by expression in a host that is not a CHO cell.

60. 60. The method of claim 58 or 59, wherein the polypeptide to be isolated or purified is obtainable by expression in a host which is a lower eukaryotic host.

61. 61. The method of claim 60, wherein the lower eukaryotic host comprises a yeast such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Cytheromyces, Pachysolen, Debaromyces, Metoscunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, or Endomycopsis.

62. 62. The method of claim 61 , wherein the yeast is a Pichia, such as Pichia pastoris.

63. The method of any one of claims 56 to 62, wherein the conformational variant is characterized as in claims 8 to 11.

64. 64. The method of any one of claims 56 to 63, wherein the chromatographic technique is a chromatographic technique based on hydrodynamic volume, surface charge or surface hydrophobicity.

65. 65. The method of claim 64, wherein the chromatographic technique is selected from size exclusion chromatography (SEC), ion exchange chromatography (IEX), mixed mode chromatography (MMC), and hydrophobic interaction chromatography (HIC).

66. 66. The method of claim 65, wherein the ion exchange chromatography (IEX) is cation exchange chromatography (CEX).

67. 66. The method of claim 65, wherein the HIC is based on a HIC column resin.

68. 68. The method of claim 67, wherein the HIC resin is selected from Capto Phenyl ImpRes, Capto Butyl ImpRes, Phenyl HP, and Capto Butyl.

69. 66. The method of claim 65, wherein the HIC is based on a HIC membrane.

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