Purification of protein q by ion exchange chromatography

Abstract

The current invention provides an improved process of the manufacture of pharmaceutical products comprising proteins, useful for the prevention or treatment of leishmaniasis. The pharmaceutical composition comprises a protein chimera Q, which is the product of a chimeric gene encoding the antigenic determinants of four proteins of Leishmania infantum.

Description

[0001] Improved purification process

[0002] Field

[0003] This invention is of utility within the industry dedicated to the manufacturing process of pharmaceutical products in general. The invention relates to a process of producing proteins. The invention in particular relates to an improved process of purifying proteins useful for the prevention or treatment of leishmaniasis, further defined herein.

[0004] Background of the invention

[0005] Proteins are widely utilised in the pharmaceutical and medical industries as hormones, therapeutic medications, vaccinations, and / or clinical research. Today proteins are being increasingly used in many industrial processes such as food processing, cosmetics and / or pharmaceutical industries, as well as in organic chemistry field. It is further well known that major developments in protein purification have also taken place in the area of therapeutic proteins and / or enzymes production. Upscaling of the manufacturing and / or purifying processes was required to meet the needs of the industries. Such upscaling could only have been made possible with optimization of these processes. The yields of protein production and protein purification have been steadily improved over the years.

[0006] Proteins are typically required in purified and stable forms for their use in industry, research and organic chemistry. Henceforth, the purification of proteins is an important step in their production process. Non-chromatographic approaches and chromatographic processes may be used to achieve this objective. Examples of non-chromatographic processes are for instance, selective precipitation, cohn fractionation process, centrifugation-based method, or self-cleaving aggregating tag systems, among others. While non-chromatographic processes can be performed on bulk proteins, chromatographic processes are recognized as the gold standard in the downstream processing of therapeutic proteins. This is mainly due to the robustness, selectivity, and high resolution of chromatographic processes (Sanchez-T rasvina C, et al. Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies. Front Bioeng Biotechnol.

[0007] 2021 Aug 20;9:717326. doi: 10.3389 / fbioe.2021.717326. PMID: 34490225; PMCID: PMC8417561).

[0008] This invention in particular focuses on the process of producing, or at least purifying, proteins used for the prevention or treatment of leishmaniasis, in particular canine leishmaniasis. The parasite protozoa belonging to the Leishmania genus are the causative agents of Leishmaniasis, a group of diseases with a global distribution and a broad spectrum of clinical symptoms manifestations. Humans are regarded as secondary hosts for the primary zoonotic types of Leishmaniasis. The species known as L. infantum is the source of visceral leishmaniasis (LV) in canines and humans. It is extensively dispersed over various Mediterranean regions. Dogs infected by Leishmania infantum are actually the primary host of this parasite, especially during the protracted incubation period that precedes the manifestation of clinical symptoms. The prevalence of canine Leishmaniasis and the parasite's ability to spread to people are directly correlated, according to epidemiological research. Because of this, early detection of the illness or infection is essential in initiatives aimed at halting its spread.

[0009] W02000039298 relates to a pharmaceutical composition for the prevention and treatment, human or animal, of leishmaniasis. The pharmaceutical composition contains the protein chimera Q (named herein protein Q), which comprises antigenic determinants of four proteins of L. infantum (LiP2a, LiP2b, LiPO, and H2A).

[0010] Last years, there was a wish to further improve the global yield of protein Q. Hence, it is the object of the invention to design an improved manufacturing and / or purifying process of such a protein. It is further the object of the invention to design an improved manufacturing and / or purifying process that may be implemented in producing or at least purifying other proteins, especially other proteins that are highly homologous with protein Q. It is further an object of the invention to improve the manufacturing or at least the purifying process of such a protein, wherein said process comprises chromatography columns.

[0011] Purification or downstream processing can cost as much as 80% of the total production cost when producing recombinant proteins on a large scale for applications in industry and medicine (Pradeep Kumar, SM Sharma. An overview of purification methods for proteins. Int J Appl Res 2015;1 (12):450-459). An optimised purifying technique may reduce this expense. Furthermore, the characteristics of the specific protein being purified may determine or at least influence which purification method is the best adapted. The main object of this invention is to provide an optimised purification process for a protein such as Protein Q using a chromatography column. It is further an object of the invention to provide an optimised purification process for a protein such as Protein Q using a chromatography column in both laboratory (or lab) scale and industrial (or plant) scale.

[0012] Summary of the invention

[0013] In a first aspect of the invention, there is provided a process for purifying a protein comprising a step of purifying said protein from a mixture by a chromatography column, using at least one column comprising a resin, wherein the total elution time is ranged from 80 minutes to 24 hours; preferably from 160 to 210 minutes.

[0014] In one embodiment of this first aspect, wherein the mixture to be loaded to the column (also called load mixture) comprising said protein is characterized as follows:

[0015] the load density, is ranged from 20 to 247 mg TP / cm2; and optionally

[0016] the load ratio of total protein (TP) to the resin is ranged from 5.0 to 12 mg TP / mL resin; and / or

[0017] the load concentration is ranged from 0.15 to 0.60 mg / mL.

[0018] In a second aspect of the invention, there is provided a process for purifying a protein, preferably represented by an amino acid sequence having at least 60 or at least 80% identity or similarity with SEQ ID NO:1, comprising a step of purifying said protein from a mixture by a chromatography column, using at least one column comprising a resin, wherein the total elution time is ranged from 5 to 220 minutes; preferably from 160 to 210 minutes and / or wherein the mixture to be loaded to the column comprising said protein is characterized as follows:

[0019] the load density, is ranged from 20 to 247 mg TP / cm2;

[0020] and optionally

[0021] the load ratio of total protein (TP) to the resin is ranged from 5.0 to 12 mg TP / mL resin; and / or

[0022] the load concentration is ranged from 0.15 to 0.60 mg / mL.

[0023] In one embodiment of this first or second aspect, there is provided a process wherein the isoelectric point of the protein is from 1 to 3 units lower than the pH of the load mixture.

[0024] In one embodiment, the protein is soluble and / or stable in the presence of a chaotropic agent like urea.

[0025] In one embodiment of this first or second aspect, there is provided a process wherein the elution linear flow rate of the column is ranged from 10 to 127 cm / h, preferably from 15 to 127 cm / h, more preferably said linear flow rate is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 125, 126, 127 cm / h.

[0026] In one embodiment of this first or second aspect, there is provided a process wherein a flow stop of at least 0.5, 1, 1.5, 2, 2.5 and up to 24 hour is carried out when eluting the column, preferably said flow stop is 2 hour.

[0027] In one embodiment of this first or second aspect, there is provided a process wherein the flow stop is carried out during the elution phase, preferably in the middle of the elution phase.

[0028] In one embodiment of this first or second aspect, there is provided a process wherein the load ratio is higher than 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, and / or lower than 12, 11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11.0, 10.9, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, 10.0, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6 mg TP / mL resin; preferably the load ratio is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, or 6.1 mg TP / mL resin. In one embodiment of this first or second aspect, there is provided a process wherein the load density is higher than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 mg TP / cm2, and / or lowerthan 247, 160, 155, 150, 145, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95 TP / cm2; preferably the load density is ranged from 20-150, 30-140, 35-135, 40-130, 45-125, 50-120, 55-115, 60-110, 65-110, 70-105 mg TP / cm2, preferably ranged from 20-55, 20-50, 25-40, 28-35, 29-32 mg TP / cm2, preferably 85-86 mg TP / cm2. In one preferred embodiment, the load density is ranged from 20 to 247 mg TP / cm2, preferably ranged from 20 to 55, from 20 to 50, from 25 to 65, from 25 to 60, from 25 to 40, from 28 to 58, from 28 to 35, from 29 to 32 or from 31 to 55, or from 40-65, or from 45-60, or from 50-56 mg TP / cm2, more preferably 31 and / or 55 mg TP / cm2.

[0029] In a preferred laboratory scale embodiment, the load density is ranged from 31 to 55 mg TP / cm2, more preferably is 31 and / or 55 mg TP / cm2. In this invention, the preferred ranges of the laboratory scale are extrapolable to any scale, in particular to an industrial scale.

[0030] In an industrial scale preferred embodiment, the load density is ranged from 31 to 55 mg TP / cm2, more preferably is 31 and / or 55 mg TP / cm2.

[0031] In one embodiment of this first or second aspect, there is provided a process wherein the load concentration is higher than 0.2, 0.21, 0.22, 0.23, 0.24 and / or lowerthan 0.55, 0.54, 0.53, 0.52, 0.51 mg / mL; preferably the load concentration is 0.38, 0.39, 0.40, or 0.41 mg / mL.

[0032] In one embodiment of this first or second aspect, there is provided a process wherein:

[0033] the total elution time is ranged from 5 to 220 minutes; preferably from 160 to 210 minutes, even more preferably from 168 to 202 minutes; and / or

[0034] the load density is ranged from 20-247 mg TP / cm2;

[0035] and optionally

[0036] the load ratio is ranged from 5.5 to 11.5 mg TP / mL resin;

[0037] the load concentration is ranged from 0.20 to 0.55 mg / mL;

[0038] the flow rate is ranged from 10 to 127 cm / h; and / or

[0039] the flow stop is ranged from 1.5 to 2.5 hour.

[0040] In one embodiment of this first or second aspect, there is provided a process wherein:

[0041] the total elution time is ranged from 5 to 220 minutes; preferably from 160 to 210 minutes, even more preferably from 168 to 202 minutes; and / or

[0042] the load density is ranged from 30-55 mg TP / cm2;

[0043] and optionally

[0044] the load ratio is ranged from 5.9 to 6.1 mg TP / mL resin; the load concentration is ranged from 0.35 to 0.45 mg / mL;

[0045] the flow rate is ranged from 10 to 127 cm / h; and / or

[0046] the flow stop is ranged from 1.5 to 2.5 hour.

[0047] In one embodiment of this first or second aspect, there is provided a process wherein:

[0048] the total elution time is ranged from 5 to 220 minutes; preferably from 160 to 210 minutes, even more preferably from 168 to 202 minutes; and / or

[0049] the load density is ranged from 20-247 mg TP / cm2;

[0050] and optionally

[0051] the load ratio is ranged from 5.5 to 11.5 mg TP / mL resin;

[0052] the load concentration is ranged from 0.20 to 0.55 mg / mL;

[0053] the flow rate is ranged from 10 to 127 cm / h; and / or

[0054] the flow stop is ranged from 1.5 to 2.5 hour.

[0055] In one embodiment of this first or second aspect, there is provided a process wherein:

[0056] the total elution time is ranged from 5 to 220 minutes; preferably from 160 to 210 minutes, even more preferably from 168 to 202 minutes; and / or

[0057] the load density is ranged from 30-55 mg TP / cm2;

[0058] and optionally

[0059] the load ratio is ranged from 5.9 to 6.1 mg TP / mL resin;

[0060] the load concentration is ranged from 0.35 to 0.45 mg / mL;

[0061] the flow rate is ranged from 10 to 127 cm / h; and / or

[0062] the flow stop is ranged from 1.5 to 2.5 hour.

[0063] In one embodiment of this first or second aspect, there is provided a process wherein the column is an IEC (Ion Exchange Chromatography) column. Preferably, the column is for anion exchange chromatography technique. It is also viable that the column holder is suitable for other chromatography techniques.

[0064] In one embodiment of this first or second aspect, there is provided a process wherein the column, preferably IEC column, is made of a polymer, glass, stainless steel, inert plastic, or metal.

[0065] In one embodiment of this first or second aspect, there is provided a process wherein the column, preferably the IEC column, comprises the resin having quaternary amine (Q) functional group. The chemical formula of the resin may be represented by [-CH2-N+(CH3)3].

[0066] The column or resin is preferably an anion exchanger, more preferably a strong anion exchanger.

[0067] In one embodiment of this first or second aspect, there is provided a process wherein:

[0068] the pH of the load mixture is 10.0±0.5, 10.0±0.2 or 10.0±0.1; preferably 10.0;

[0069] the IEC process is performed with the equilibrium pH at 9.0±0.5, preferably 9.0±0.2, and the elution pH at 8.0±0.5, preferably 8.0±0.2.

[0070] In one embodiment of this first or second aspect, there is provided a process wherein the protein is represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1, or preferably having 100% identity of SEQ ID NO:1.

[0071] Detailed description of the invention

[0072] Various features of the aspects and embodiments of this invention are further described below. It is noted that headings used throughout this specification are to assist navigation only and should not be interpreted as definitive, and that features described in different sections may be relevant for all aspects and embodiments described herein and may thus be combined as appropriate.

[0073] The inventors surprisingly discovered that the purification process of a protein of interest, such as preferably the one having at least 60 or 80% identity or similarity with SEQ ID NO:1, through a chromatography column may be further optimized by increasing the elution time (about 180 min) optionally combined with a flow stop to further increase the elution time (1 to 2 hours) and / or a relatively slow elution rate (about 15 cm / h) to further increase the elution time as demonstrated in the experimental part.

[0074] The inventors surprisingly discovered that the purification process of a protein of interest, such as preferably the one having at least 60 or 80% identity or similarity with SEQ ID NO:1, through a chromatography column may be further optimized by increasing the elution time (up to 24 hours) optionally combined with a flow stop to further increase the elution time (up to 24 hours) and / or a relatively slow elution rate (about 15 cm / h) to further increase the elution time.

[0075] Moreover, and surprisingly, such a purification process may also be further optimised by adjusting the characteristics of the mixture to be loaded to the column. The characteristics of the mixture to be loaded to the column include the load ratio, the load density and / or the load concentration and are all defined later herein.

[0076] The inventors surprisingly found that adjusting the load density of the mixture allows an improvement of the method. For example, using a relatively low load density of the mixture to be loaded to the column (such as 20-60 mg TP / cm2) is an improvement as demonstrated in the experimental part. In one embodiment, the advantage of using a relatively low load density of this invention is that it prevents column head overload. By decreasing the quantity of TP per column section, the contact surface with the resin is increased, facilitating TP uptake into the resin and so preventing protein precipitation at the top of the column.

[0077] In some embodiments, improvements are obtained by increasing the elution time (about 180 min) and / or using a relatively low load density of the mixture to be loaded to the column (such as 20-60 mg TP / cm2). Optionally, a flow stop is used to further increase the elution time (flow stop of 1 to 2 hours) and / or a relatively slow elution rate (about 15 cm / h) is used to further increase the elution time as demonstrated in the experimental part.

[0078] These improvements contribute to an increase of the interaction time of the protein of interest with the elution buffer in the column.

[0079] In an embodiment, adding a flow stop allows to keep the elution buffer in contact with the protein for a longer period of time. This is a technical solution specific for proteins with low solubility and that have low detachment and solubilization kinetics such as the one represented by SEQ ID NO:1. In an embodiment, adding a flow stop allows to keep the elution buffer in contact with the protein for a longer period of time also allows to upscale the IEC to industrial scales. It also allows to reduce buffer consumption, energy and process supervision. In an embodiment, the flow stop may be up to 24 hours.

[0080] In an embodiment, increasing the elution time and / or using a relatively low density (such as 20-60 mg TP / cm2) allows to better solubilize the protein remaining in the column.

[0081] Each of these improvements are further described in details herein.

[0082] Optimising the elution time and / or the characteristics of the mixture to be loaded to the column lead to the following advantages:

[0083] an improved yield (or percentage of protein of interest recovery): yield up to 90% (see example 1, figure 1),

[0084] an increase of the yield of the protein of interest compared to the yield obtained using process with no optimisation of the elution time and / or of the characteristics of the mixture to be loaded to the column (up to 40% increase, see example 1),

[0085] an increase in the amount of protein of interest produced (grams of final purified drug substance, protein Q) obtained per batch (up to 41 % increase, see example 2), a more reproducible process (see lower coefficient of variation in example 2 compared to coefficient de variation obtained with control process),

[0086] a process which is easier and more flexible to handle (see example 1) and

[0087] a process which can be conducted either at laboratory scale or at industrial scale obtaining the same benefits in terms of yield (see example 2).

[0088] It is to pointed out that the quality of the protein of interest is not affected by these optimisations of the purification process as demonstrated in the experimental part.

[0089] The inventors demonstrated in example 3 that this process could be applied with success to a protein having at least 60% identity to Protein Q. Two distinct proteins (Protein 2 having 59.8% identity with Protein Q, and Protein 3 having 87.4% identity with Protein Q) have been successfully purified in example 3. An increase in the amount of protein of interest produced (grams of final purified drug substance, protein 2) obtained per batch (up to 70% increase, see example 3), An increase in the amount of protein of interest produced (grams of final purified drug substance, protein 3) obtained per batch (up to 31% increase, see example 3),

[0090] In a first aspect of the invention, there is provided a process for purifying a protein comprising a step of purifying said protein from a mixture by a chromatography column, using at least one column comprising a resin, wherein the total elution time is ranged from 80 minutes to 24 hours; preferably from 160 to 210 minutes.

[0091] In one embodiment of this first aspect, wherein the mixture to be loaded to the column (also called load mixture) comprising said protein is characterized as follows:

[0092] the load density, is ranged from 20 to 247 mg TP / cm2;

[0093] and optionally

[0094] the load ratio of total protein (TP) to the resin is ranged from 5.0 to 12 mg TP / mL resin; and / or

[0095] the load concentration is ranged from 0.15 to 0.60 mg / mL.

[0096] In a second aspect, there is provided a process for purifying a protein, preferably represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1, comprising a step of purifying said protein from a mixture by a chromatography column, using at least one column comprising a resin, wherein the total elution time is ranged from 5 to 220 minutes; preferably from 160 to 210 minutes and / or wherein the mixture to be loaded to the column comprising said protein is characterized by a load density ranged from 20 to 247 mg / TP / cm2.

[0097] Optionally, the mixture to be loaded to the column comprising said protein is further characterized as follows:

[0098] the load ratio of total protein (TP) to the resin, ranged from 5.0 to 12 mg TP / mL resin; and / or

[0099] the load concentration, ranged from 0.15 to 0.60 mg / mL.

[0100] Chromatography techniques

[0101] The invention relates to a manufacturing process, especially a purification process comprising a chromatography technique. The basic principle of chromatography is as follows: mixtures of molecules are deposited on solid surfaces or into fluid stationary phases (stable phases) and molecules will separate from one another as they move with the aid of a mobile phase (an eluent). Molecular characteristics linked to adsorption (liquid-solid), partition (liquid-solid), affinity, or variations in their molecular weights are among the factors that have an impact on this separation process. Based on this approach three components form the basis of chromatography technique. Firstly, a stationary phase which is a phase that is composed of a “solid” phase or “a layer of a liquid adsorbed on the surface of a solid support”. Secondly, a mobile phase which is a phase that is composed of “liquid” or a “gaseous component.” Lastly, separated molecules are the result of the separation process. The type of interaction between the stationary phase, mobile phase, and molecules contained in the mixture is the driver for the separation of molecules from each other. Chromatography methods based on partition are very effective on separation, and identification of small molecules as amino acids, carbohydrates, and fatty acids. However, ion-exchange chromatography, hereafter “IEC” or “IEX chromatography” are more effective in the separation of macromolecules as nucleic acids, and proteins. Typically, IEX chromatography exploits the charged nature of macromolecules, making it particularly well-suited for nucleic acids and proteins. For example, nucleic acids are negatively charged due to their phosphate backbone, allowing them to interact strongly with positively charged resins. Proteins have varying charge distributions based on their amino acid composition, enabling separation based on their overall charge at a given pH. (Coskun O. Separation techniques: Chromatography. North Clin Istanb. 2016 Nov 11;3(2):156-160. doi: 10.14744 / nci.2016.32757. PMID: 28058406; PMCID: PMC5206469.)

[0102] Column chromatography

[0103] Since a wide range of molecules, in particular proteins, have different characteristic features as size, shape, net charge, stationary phase used, and binding capacity, each molecule or protein can be purified using chromatographic methods. Among these methods, most frequently column chromatography is applied. This technique is used for the purification of biomolecules.

[0104] A stationary phase is an immobile material inside the chromatography column that interacts with the sample mixture or the load mixture as they pass through. It typically comprises of solid particles or beads (most common); and / or liquid coating on a solid support; and / or a porous matrix. Common materials used for stationary phases may include silica, cross-linked polyacrylamide, agarose (e.g., Sepharose), or dextran (e.g., Sephadex).

[0105] The load mixture refers to the sample or solution that is applied or loaded to the column. The mixture components comprise the various (bio)molecules or substances present in the sample that is applied to the chromatography column. The mixture components may comprise the protein of interest and impurities. The protein of interest is the protein to be separated or purified. Impurities can be any unwanted molecules present in the mixture, such as other proteins, salts, or small molecules that may interfere with the separation process. It is also possible that the mixture components include the salts or pH-adjusting agents in the buffer solution used to prepare the mixture, which ensure that the sample environment is compatible with the column chromatography process.

[0106] A biomolecule refers to any molecule that is produced by living organisms. As such, most of them are organic molecules. The four major groups of biomolecules include polysaccharides, proteins, nucleic acids (DNA and RNA), and lipids. They are found in and produced by living organisms. Thus, a biomolecule could be a (bio)polymer. A polymer is a compound made up of several repeating units (monomers) or protomers and produced by polymerization. Biomolecules can contain carbon atoms covalently bound to other atoms, for instance Carbon-Carbon (C-C) and Carbon-Hydrogen (C-H). The four major element constituents are carbon, hydrogen, oxygen, and nitrogen.

[0107] On a column (stationary phase), the load mixture comprising a protein of interest to be separated is loaded, also referred to as a load mixture. Subsequently, the wash buffer is applied.

[0108] Said load mixture can flow or eluate through inside column material placed on a support, such as fiberglass support. The load mixture or the eluates (comprising the analytes) are going or passing through of the column in a time-, and volume-dependent manner.

[0109] The term "wash buffer" refers to the buffer that is applied to the ion exchange material or matrix (stationary phase) after the loading of the mixture and before the elution of the protein of interest. The wash buffer may eliminate one or more contaminants or impurities from the column, without significant elution of the target protein.

[0110] The term "washing" the ion exchange material or matrix refers to the process of traversing a suitable buffer through or over the column in this invention.

[0111] An eluate refers to the mixture or the solution that flows from chromatography's elution process. In particular, it is the mixture that has been treated or extracted from a chromatography column, typically by an elution buffer. The eluate may contain the analytes (the compounds or proteins of interest being separated) and / or any co-eluting solutes that pass through the column. In a typical chromatography process, as the mobile phase (buffer or eluent) passes through the column, it carries the separated components out of the column. The sample that comes out of the column containing the separated components is the eluate. The eluate may be collected in fractions for further analysis to identify and quantify the separated compounds.

[0112] The term "elution buffer" denotes the buffer utilised to elute the target protein from the column. The term “mixture” or “solution” as utilised in this context denotes either a buffered or non-buffered mixture, inclusive of water.

[0113] The term "eluting" a (bio)molecule (e.g., a target protein or contaminant) from a column refers to the process of extracting the biomolecule from the column, usually by running an elution buffer over it.

[0114] The term “contaminant” or “impurities” denotes any unwanted or undesirable molecule, especially a biological macromolecule such as DNA, RNA, or a protein, that is present in a mixture of the protein undergoing purification, except the protein itself. Contaminants encompass, for instance, additional proteins from cells that release the protein undergoing purification, as well as other proteins.

[0115] The terms “separate” in the context of protein purification denote the extraction of a target protein from a secondary protein or other contaminants within a mixture comprising both, ensuring that a significant proportion of the target protein molecules are extracted from the fraction predominantly comprising the secondary protein or contaminants.

[0116] Column chromatography is a highly effective method for separation. It has the ability to divide mixture components according to: size (gel filtration / size exclusion), charge (ion exchange), and / or affinity (a specific binding affinity). The utilisation of a resin (may be referred to as “solid or stationary phase”) with specific chemical properties contained in a cylinder — referred to as a "column" — unites the three different types of chromatography techniques. As a buffered solution (may be referred to as “mobile phase”) passes along the column, and at for instance a laboratory scale, it collects in tubes (may be referred to as "fractions") as it comes to an end of the column. Whereas at an industrial scale, tanks or bags may be used and the whole eluate may be collected together therein. A mixture comprising proteins, or hereafter a load mixture, is applied in the mobile phase and percolates through the column. Depending on these characteristics as well as those of the resin, different proteins migrate in different ways, and therefore a given protein may be separated and thus purified from a complex mixture.

[0117] The main types of column chromatography may include, but not limited to Flash Column Chromatography; Normal Phase Chromatography; Reverse Phase Chromatography; High-Performance Liquid Chromatography (HPLC); Ion Exchange Chromatography (IEC); Size Exclusion Chromatography (SEC); Affinity Chromatography; Gas Chromatography (GC); Adsorption Chromatography; Partition Chromatography; Gel Chromatography; Hydrophobic Interaction; Chromatography (HIC); Pseudoaffinity Chromatography; and Chiral Chromatography. These techniques can be broadly categorized into liquid chromatography (LC) and gas chromatography (GC) methods. Each type has specific applications and principles based on the stationary phase, mobile phase, and separation mechanism used. The choice of method depends on the properties of the compounds to be separated and the specific requirements of the analysis.

[0118] The core working principle of a chromatography column is based on the differential interactions between the components of a sample and the stationary phase within the column. As the sample passes through the stationary phase, components that interact more strongly with the stationary phase take longer to elute, while those with weaker interactions move through more quickly. This differential interaction leads to the separation of the sample components. As the separated components leave the column at different times, it can form peaks on a chromatogram. A detector may record these signals, and the separated components can be collected individually for further analysis and / or steps. As used herein, the load mixture also called the mixture to be loaded or the sample mixture is at least partly introduced into the column, either at the top (in liquid chromatography) or carried by the mobile phase (in gas chromatography). In some embodiments, the load mixture may be injected into the column under high pressure or using automated injectors. In some embodiments for IEX chromatography, during loading the mixture comprising biomolecules, preferably comprising a protein of interest, molecules with opposite charges to the functional groups on the resin bind to the stationary phase of the resin. Neutral molecules or those with the same charge as the stationary phase will pass through without binding.

[0119] As used herein, stationary phase in chromatography is a solid phase, or a liquid phase coated on the surface of a solid phase. Mobile phase flowing over the stationary phase is a gaseous or liquid phase. If mobile phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC). In this invention, it is preferred that the mobile phase is a liquid phase.

[0120] As used herein, mobile phase in chromatography, is in a form of eluents or buffers or aqueous solutions comprising salt such as sodium chloride, potassium chloride, sodium sulfate, and / or potassium sulfate. Salts may also be added to the buffers.

[0121] The buffer used in this invention, such as the elution buffer used in this invention, preferably comprises sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O), urea (CH4N2O), sodium chloride (NaCI), and / or sodium hydroxide (NaOH), and optionally a non-ionic detergent (or a nonionic surfactant).

[0122] In an embodiment, Triton, in particular Triton X-100 (C14H21(C2H4O)nOH), is a non-limiting example of a non-ionic detergent (or a non-ionic surfactant) that contributes to the solubilization and purification of protein of interest, such as Protein Q represented by SEQ ID NO:1 or having at least 60 or 80% identity or similarity with SEQ ID NO:1. In an embodiment, Triton, in particular Triton X-100, may be switched to at least another alternative detergent and / or surfactant, such as, but not limited to, n-Dodecyl-beta-D-glucopyranoside, n-Nonyl-beta-D-glucopyranoside, Octyl p-D-glucopyranoside, Triton CG-110, Triton X100RS, Triton X-114, Triton X-45, Triton CG-50, Triton CG-600, Triton CG-650, Methyl 6-O-(N-heptylcarbamoyl)-a-Dglucopyranoside, Lauryldimethylamine oxide, Saponin, Digitonin, IGEPAL® CA-630, Deviron® C16, BRIJ C10, Tween 20, Tween 80, Poloxamer 188, Pluronic F127, Ecosurf SA-9, Ecosurf EH-6 / EH-9, Tergitol 15-S-9, Tergitol 15-S-7, Tergitol TMN-100X, Brij 35 and / or Brij L23. In an example, all the detergent candidates mentioned herein may solubilize the protein of interest, in particular Protein Q represented by SEQ ID NO:1 or having at least 60 or 80% identity or similarity with SEQ ID NO:1, with at least similar performance than Triton and demonstrated an optimal yield. In another embodiment, it is also possible that no detergent and / or surfactant is used in the buffer. As mentioned herein, each step of the process defined by the invention may be suitable for any type of chromatography, in particular column chromatography, more in particular liquid chromatography, even more in particular Ion Exchange Chromatography (IEC).

[0123] As used herein, a column or a chromatography column is referred to as a device through which at least a part of the sample mixture or the load mixture and a mobile phase (for instance, an eluent or a buffer solution) pass. The shape of the column is typically, but not limited to, cylinder or tube. The column may comprise a stationary phase (for instance, a resin), which interacts with the sample or the load mixture components, resulting in their separation based on various physicochemical properties such as size, charge, polarity, or affinity. In an embodiment, the column may comprise a resin having quaternary amine (Q) functional group. In this embodiment, separation of molecules may be based on their charge. For example, the column containing resin with quaternary amine (Q) functional groups would have a positive charge. Resins with quaternary amine (Q) functional groups are commonly used in anion exchange chromatography.

[0124] In a further embodiment, the column of the invention may comprise at least a frit. Frits are porous filters located at the ends of the column to hold the stationary phase in place while allowing the mobile phase and the sample or the load mixture to pass through.

[0125] In a further embodiment, the column of the invention may be connected to a detector, such as a UV-visible spectrophotometer, mass spectrometer, or a fluorescence detector, which records the separated compounds as they exit the column. In a preferred embodiment, a UV detector can be used to control the absorbance during elution phase.

[0126] Ion exchange chromatography (IEC)

[0127] Ion exchange chromatography is a potent liquid chromatography method that is essential for the separation and purification of charged molecules in a variety of disciplines, such as environmental science, biochemistry, and medicine. In numerous analytical and preparative applications, its capacity to segregate molecules, for instance proteins, according to their ionic characteristics renders it an essential instrument.

[0128] Ion exchange chromatography (IEC) is a type of column chromatography and considered as a type of liquid chromatography. It is based on electrostatic interactions between charged molecules or protein groups in the load mixture and the oppositely charged functional groups on the stationary phase, or the solid support material (matrix). Matrix of the stationary phase has an ion load opposite to that of the protein to be separated, and the affinity of the protein to the column is achieved with ionic bonds. Proteins are separated from the column either by changing pH, concentration of ion salts or ionic strength of the buffer solution. Within liquid chromatography field, ion exchange chromatography (IEC) is a widely recognised method. By using a (liquid) mobile phase, typically an ion-containing aqueous buffer solution, this technique separates molecules according to how their ions interact with a charged stationary phase. In IEC, the stationary phase is made up of charged functional groups attached to a solid support, typically in the form of resins, such as resin beads.

[0129] The differential attraction between the oppositely charged groups on the stationary phase and the charged molecules in the load mixture is the basis for the IEC separation mechanism. Stronger ionic contacts cause molecules to be held longer in the liquid mobile phase while weaker interactions elute more quickly when the sample passes through the column. Effective separation of proteins, peptides, nucleic acids, and other charged biomolecules in solution is made possible by this technique. The stationary phase is typically packed inside a column, which is referred to as an Ion Exchange Chromatography column or IEC column in this invention.

[0130] The elution process of IEC is one of its main characteristics. Usually, the ionic strength or pH of the liquid mobile phase is gradually adjusted in order to elute the analytes from the column. By adjusting the ionic connections' strength, this modification permits the bound molecules to be released under regulated conditions. In an embodiment, the process of this invention is characterized by the pH of the load mixture is 10.0±0.5, preferably 10.0±0.2; wherein the IEC process is performed with the equilibrium pH at 9.0±0.5, preferably 9.0±0.2, and the elution pH at 8.0±0.5, preferably 8.0±0.2.

[0131] Proteins possess an isoelectric point (pl) at which their net charge is neutral. Proteins exhibit a positive charge below their isoelectric point (pl) and a negative charge above it. Modifying the buffer pH can alter the interaction strength between the analyte and the stationary phase, hence affecting how strong the protein is bound to the column. In an embodiment, when the protein's pl is above the buffer pH, the column is for cation exchange chromatography. In an embodiment, when the protein's pl is below the buffer pH, the column is for anion exchange chromatography. The protein of the invention preferably carries a net positive charge. The buffer pH should typically be at least 1-2 or 1-3 o 2-3 units away from the protein's pl to ensure it is fully charged. The pH of the buffer dictates the ionisation state of the analyte or protein of interest.

[0132] In one embodiment, there is provided a process wherein the isoelectric point of the protein is at least from 1-2 or 1-3 or 2-3 units lower than the pH of the load mixture.

[0133] In an embodiment, the protein is soluble and / or stable in the presence of a chaotropic agent like urea. Examples of chaotropic agents include n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea.

[0134] In one embodiment, there is provided a process wherein the isoelectric point of the protein is at least from 1-2 or 1-3 or 2-3 units lower than the pH of the load mixture and the protein is soluble and / or stable in the presence of a chaotropic agent like urea. To assess solubility we may check that there is no precipitation or turdidity by visual inspection and / or by means of an spectrophotometer by comparing the UV spectra to Protein Q and specifically, the absorbance at 350 nm. The equipment utilised in IEC is typical of liquid chromatography systems; it comprises of detectors appropriate for liquid phase analysis, pumps for providing the mobile phase, and injection devices for introducing the sample or the load mixture.

[0135] It is significant to note that IEC differs greatly from gas chromatography methods. Ionic interactions, which are essential to IEC, cannot occur in gas-phase separations and require a liquid medium. Furthermore, the analytes that are commonly separated by IEC are not suited for gas chromatography because they are frequently non-volatile and may even be thermally unstable. IEC stands out as a liquid chromatography technique because its stationary phases are made expressly for interactions in a liquid environment. Hence, in this invention, the load mixture to be treated by the IEC steps as described in this specification is preferably liquid.

[0136] Separation and IEC typically depends upon the reversible adsorption of charged solute molecules to immobilized groups of opposite charge. Generally, ion exchange (I EX) experiments are performed in four main stages: equilibration, sample (or load mixture of the invention) application and wash, elution buffer, and regeneration. The first step comprises the equilibration of the stationary phase to the desired start conditions. When equilibrium is reached, all stationary phase charged groups are associated with exchangeable counter ions, such as chloride (Cl ) or sodium chloride. The second step comprises loading the mixture (also called load mixture) comprising proteins of the invention and wash. The goal in this step is to bind the target molecule (i.e. the protein as earlier defined herein) and wash out all unbound material. The sample buffer should have the same pH and ionic strength as the starting buffer, in order to bind all appropriately charged proteins. In the third step, elution, biomolecules are released from the ion exchanger by a change in the buffer composition. A common way is to increase the ionic strength with sodium chloride or another simple salt, in order to desorb the bound proteins. Proteins are desorbed relative to the number of charged groups on their surface. The final step comprises regeneration, removes all molecules still bound. This ensures that the full capacity of the stationary phase is available for the next run.

[0137] It is also possible that in some embodiments, the process of the invention may further comprise washing or equilibrating the resin or matrix with a wash or equilibration buffer between the loading and eluting steps. In a non-limitative process according to the invention, the column chromatography process is performed with the equilibrium pH at 9.0±0.5, preferably 9±0.2 and the elution pH at 8.0±0.5, preferably 8±0.2; wherein the pH of the load mixture is 10.0±0.5, preferably 10±0.2. The skilled person understands that the equilibrium pH and elution pH may be adapted depending on the identity of the protein. Values mentioned here are particularly suited for Protein Q (SEQ ID NO:1).

[0138] The two main types of IEC columns are anion exchange columns and cation exchange columns. Ion exchange chromatography (IEC) is an effective technique for separating charged molecules, in particular proteins, according to their ionic interactions with a stationary phase. Its foundational columns are utilised in IEC. The purpose of anion exchange columns is to separate anions, or negatively charged molecules. Positively charged functional groups covalently bound to inert support materials, like resins, in particular resin beads, make up the stationary phase in these columns. Quaternary ammonium is a common functional group utilised in strong anion exchangers, while diethylaminoethyl (DEAE) is employed in weak anion exchangers. Negatively charged molecules in at least a part of a sample of load mixtures are drawn to and bond with the positively charged groups on the stationary phase when at least a part of the sample of load mixtures is added to the column. When the ionic strength or pH of the mobile phase is changed, various anions elute from the column at different rates, allowing for separation. The intensity of this binding is dependent on the charge density of the molecules.

[0139] Conversely, positively charged molecules, or cations, are separated using cation exchange columns. These columns comprise functional groups that are negatively charged in a stationary phase. Sulfopropyl (SP) groups are commonly used by strong cation exchangers, whereas carboxymethyl (CM) groups are frequently used by weak cation exchangers. Positively charged molecules in the sample bind to the negatively charged groups on the stationary phase, following a similar concept to that of anion exchange but in reverse. This interaction's strength fluctuates according to the charge properties or characteristics of the molecules, allowing for separation as the conditions of the mobile phase are modified to elute the bonded cations.

[0140] Ion exchange chromatography columns have extensive application in many domains such as pharmaceutical development, water analysis, and protein purification. They are a valuable tool in analytical and preparative applications, enhancing other chromatographic techniques in the arsenal of contemporary separation research due to their capacity to separate molecules depending on charge.

[0141] Depending on the kind of molecules that need to be separated, anion or cation exchange columns could be used.

[0142] Ion exchanger

[0143] In an embodiment, an ion exchanger of a column comprises a resin. As described herein, the columns are filled with either non-porous or porous resin beads composed of cellulose, agarose, or dextran. The beads are made to endure flow rates, fluctuating pH levels, and different concentrations and / or densities of the load mixture. They also offer a sizable surface area for interactions. The resolution of the separation may be influenced by the size and porosity of these beads. In an embodiment of the invention, the column cylinder is made of a polymer, glass, stainless steel, inert plastic, or metal; and the column comprises the resin having quaternary amine (Q) functional group. Strong and weak ion exchangers are further classifications for both kinds of columns. Strong ion exchangers can be adaptable to a variety of applications because they retain their charge throughout a broad pH range. On the other hand, weak ion exchangers have a charge that changes with pH, which can be useful for some separations but necessitates more cautious buffer condition management. The charge may be provided by attaching one or more charged ligands to the solid phase, e.g. by covalent linking. Alternatively, or in addition, the charge may be an inherent property of the solid phase.

[0144] The terms “strong" and "weak" in ion exchange chromatography refer to the extent of ionization with pH, and not to the binding strength of the functional group to the target species. A “weak” exchanger is ionized over only a limited pH range, while a “strong” exchanger shows no variation in ion exchange capacity with changes in pH. Weak exchange resins can gain or lose protons with changes in buffer pH, and that added variation in charge offers an additional dimension of selectivity for binding and elution. Strong exchangers do not vary and remain fully charged over a broad pH range, which can make optimizing separation simplerthan with weak exchangers. Table 1 below summarizes the most common ion-exchange chromatography resins.

[0145] Resin Weak or Strong

[0146] Functional Group Functional pH Range Abbreviation Exchanger

[0147] DEAE Diethylaminoethyl [-N+(C2H5)2H+] Weak anion pH 2 - 9

[0148] ANX Diethylaminopropyl [-N+(C2H5)2H+] Weak anion pH 2 - 9

[0149] Q Quaternary amine [-N+(CH3)3] Strong anion pH 1 - 14

[0150] CM Carboxymethyl [-O-CH2-COO ] Weak cation pH 5 - 10

[0151] Methyl sulfonate [O-CH2-CHOH-CH2- S Strong cation pH 2 - 12

[0152] O-CH2-CHOH-CH2-SO3-]

[0153] SP Sulfonyl [-CH2-CH2-CH2-SO3-] Strong cation pH 2 -14

[0154]

[0155] Table 1. Summary of ion-exchange chromatography resins and their properties (McSweeney, E. 5 July 2023.

[0156]

[0157] https: / / bitesizebio.com / 31744 / basics-ion-exchange-chromatography).

[0158] As used herein, the column of the invention comprises resin configured to be the ion exchanger. Preferably, the resin of the invention comprises a quaternary amine (Q) functional group [-CH2-N+(CH3)3]. Quaternary amines (Q) comprise characteristics such as: they contain a nitrogen atom bonded to four alkyl or aryl groups; the nitrogen atom in a quaternary amine has a permanent positive charge, regardless of the pH of the solution; and / or Quaternary ammonium compounds are also known as "quats" and have the general structure [NR4]+, where R represents alkyl or aryl groups. In a preferred embodiment, the column or resin comprises Q Sepharose® Fast Flow.

[0159] Q Sepharose® Fast Flow is an ion exchange chromatography resin with a quaternary amine (Q) functional group [-CH2-N+(CH3)3] attached to the resin. The Q group serves as a strong anion exchanger, which is completely ionized over a broad pH range. It is comprised of crosslinked 6% agarose beads, with quaternary ammonium (Q) strong anion exchange groups. The parent Sepharose® Fast Flow is a cross-linked derivative of Sepharose®. The particle size range is 45-165 pm. The average bead diameter is ~90 pm. The counterion in the product is sulfate (SO42-). Recommended cation buffers to use with Q Sepharose® Fast Flow include alkylamines, ammonium, ethylenediamine, imidazole, pyridine, or Tris. In terms of pH, it is suggested to operate within 0.5 pH unit of the buffer's pKa. With proteins, it is suggested to operate at least 1 pH unit above the pl of the protein, to facilitate binding. Oxidizing agents, and anionic detergents and buffers, should not be used with Q Sepharose® Fast Flow. Likewise, extended exposure of Q1126 to pH < 4 should be avoided. Q Sepharose® Fast Flow is offered as a suspension in 20% ethanol.

[0160] In other embodiments, the IEX resin can comprise another anion exchange (AEX) resin, e.g., Sartobind Q, Sartobind Q Nano, DEAE Sepharose Fast Flow, ANX Sepharose 4 Fast Flow, Q Sepharose XL, Q Sepharose big beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A-25, QAE Sephadex A-50, Q sepharose high performance, Q sepharose XL, Sourse 15Q, Sourse 30Q, Resourse Q, Capto Q, Capto DEAE, Mono Q, Toyopearl Super Q, Toyo- pearl DEAE, Toyopearl QAE, Toyopearl Q, Toyopearl Giga-Cap Q, TSKgel SuperQ, TSKgel DEAE, Fractogel EMD TMAE, Fractogel EMD TMAE HiCap, Fractogel EMD DEAE, Fractogel EMD DMAE, Macroprep High Q, Macro-prep-DEAE, Unosphere Q, Nuvia Q, POROS HQ, POROS PI, DEAE Ceramic HyperD, or Q Ceramic HyperD.

[0161] In other embodiments, the IEX column or resin can comprise a cation exchange (CEX) column or resin, e.g., SP Sepharose, CM Sepharose, Sartobind S, Toyopearl SP 650M, and Fractogel SO3“, Fractogel SO3“ SE HiCap (M), Fractogel COO“ (M), YMC- BioPro S75, Capto S, SP Sepharose XL / FF, CM Sepahrose FF, SP / CM Toyopearl 650m, Toyopearl SP 550c, Toyopearl GigaCap, UNOsphere S, Eshmuno S, Macroprep High S, or POROS HS 50.

[0162] Elution

[0163] The process of extracting or removing bound molecules from a chromatographic column's stationary phase is known as elution in chromatography. It is possible to separate and gather various components in a mixture due to this phase. The extracting process can be performed by washing of loaded ion-exchange resins to remove captured ions, or eluting proteins or other biomolecules from a gel electrophoresis or chromatography column. Generally, there are two main types of elution used in chromatography according to the invention: gradient elution and isocratic elution. In gradient elution, the composition or characteristics of the load mixture can be modified while the chromatographic run is ongoing. In this method, the ionic strength of the mobile phase is progressively amplified to successively elute analytes with varying charges and binding affinities. Contrary, in isocratic elution, the composition or characteristics of the load mixture is consistent while the chromatographic run is ongoing. The order in which compounds elute from the column is typically determined by their affinity for the stationary phase. Molecules with weaker interactions usually elute first, while those with stronger interactions elute later. This principle is used to separate and identify different components in the load mixture. In an embodiment, the process of the invention is isocratic or comprises an isocratic elution method.

[0164] In a liquid chromatography experiment, an analyte is typically adsorbed by an adsorbent within a liquid chromatography column. The adsorbent, referred known as the "stationary phase," is a typically powdered substance applied on a solid support. The composition of an adsorbent determines its various affinities to retain other molecules, resulting in the formation of a thin film on the surface of its particles. Elution is the process of extracting analytes from the adsorbent by passing a solvent, commonly referred to as a “mobile phase” or “elution buffer” or “buffer” or "eluent," across the adsorbent-analyte combination. As the solvent molecules elute through the chromatography column, they may either bypass the adsorbent-analyte combination or displace the analyte by binding to the adsorbent in its substitute. Once the solvent molecules displace the analyte, the sample being analysed can be eluted from the column for analysis. The "eluate" (the solution containing the analyte material that emerges from a chromatographic column during the elution process) is collected by a fraction collector or flows into a detector as it exits the column for compositional analysis.

[0165] The elution volume, or retention volume, refers to the quantity of eluent necessary to achieve elution. Under typical conditions for a specific load mixture in a particular technique, the elution volume may suffice to identify the solutes. A combination of amino acids can be separated using column chromatography, such as IEC. Under specific conditions, the amino acids will elute in a consistent sequence and at identical elution volumes. Elution volume (Ve), which is the volume of mobile phase to elute a compound, can be calculated by multiplying an elution / retention time (tR) by a flow rate (F).

[0166] wherein: Ve = Elution volume (mL); tR = Retention time (min); F = Flow rate (mL / min).

[0167] Ve = tR x F

[0168] The definition of retention volume (and / or time) provided by International Union of Pure and Applied Chemistry (IUPAC): “The volume of mobile phase entering the column between sample injection and the emergence of the peak maximum of the sample component of interest, or the corresponding time. It includes the hold-up volume (or time).” As described herein, the elution volume is the amount of mobile phase (or buffer) required to elute a particular compound. These parameters are used to identify and quantify analytes in chromatography.

[0169] As described herein, the eluent or eluant is the "carrier" portion of the mobile phase. It moves the analytes through the chromatograph column. In liquid chromatography, the eluent is the liquid solvent; in gas chromatography, it is the carrier gas. For instance, the eluent or eluant is the buffer. As described herein, the eluate contains the analyte material that emerges from the chromatograph. It specifically includes both the analytes and coeluting solutes passing through the column, while the eluent is only the carrier.

[0170] Elution time

[0171] Elution time is also called the duration of the elution phase (Elution volume (mL) / elution flow rate (mL / min). It typically starts when an elution buffer is applied to the column (typically after product injection and column washes phases). Retention time, which is included in the total elution time, is the time interval between the injection of the elution buffer into a chromatographic column and the detection of a specific analyte or protein of interest as it emerges from said column. Elution time is directly related to the retention time, which describes how long an analyte stays bound to the stationary phase. The stronger the interaction between the analyte and the stationary phase, the longer its retention time, resulting in a longer elution time. Generally, the nature and strength of the mobile phase may affect the elution time. Furthermore, the length or height, diameter, and packing material of the column may also impact elution times. Finally, the modulation of the flow rate is also a way of modifying the elution time. A flow stop during the elution time is also a way of increasing the elution time. The elution time may also be defined as the duration of the elution phase.

[0172] The total elution time comprises the retention time. The retention time is usually measured from the moment of a molecule is introduced into the column to the appearance of the peak apex on a chromatogram, preferably at any rate. It is usually expressed in minutes or seconds. In an embodiment, retention times can be automatically recorded and displayed alongside the chromatogram. In an embodiment, (total) elution times can be automatically recorded and displayed alongside the chromatogram.

[0173] Multiple parameters may relate the elution time in column chromatography, which can be modified to regulate the separation or the purification, such as the characteristics of the load mixture. In this invention, these characteristics are for instance load ratio of Total Protein (TP) to the resin (expressed in mg TP / ml resin), load density (expressed in TP / cm2) and / or load concentration (expressed in mg / mL).

[0174] Further, the protein of interest’s net charge at a specified pH considerably influences its binding affinity to the stationary phase. Highly charged molecules will exhibit more intense electrostatic interactions and consequently longer retention time, hence longer elution time. Conversely, weakly charged molecules may exhibit reduced retention time, hence shorter elution time. The ionic strength of the mobile phase, for example, represented by the concentration of salts like NaCI, may affect the elution. For instance, in the gradient elution method, as the concentration of competing ions in the mobile phase increases, these mobile phase ions can compete with the bound analytes for interaction with the stationary phase. Typically, a high salt concentration can displace the bound analytes, resulting in their expedited elution from the column.

[0175] In the context of IEC, elution time is the period during which the protein of interest is eventually released or displaced from the stationary phase by buffer ions. The characteristics of the stationary phase may influence the binding affinity of the protein of interest. Various IEC resins (such as beads or matrices) may possess distinct charge densities and binding capabilities, affecting the elution time of protein of interest within the column. Increased charge densities on the stationary phase generally lead to longer retention periods (hence longer elution time) for strongly charged protein of interest in this invention.

[0176] In an embodiment of the process in this invention, the total elution time is ranged from 5 to 220 minutes; preferably from 50 to 220 or from 70 to 220 or from 90 to 210 or from 150 to 210 or from 160 to 210 minutes. More preferably, the total elution time is ranged from 168 to 202 minutes. In an embodiment of the process in this invention, the total elution time is ranged from 80 minutes to 72 hours; preferably from 100 minutes to 72 hours or from 150 minutes to 72 hours.

[0177] In an embodiment, the total elution time is at least 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 minutes or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours.

[0178] In a preferred embodiment, the total elution time is ranged from 80 minutes to 72 hours; preferably from 100 minutes to 70 hours or from 150 minutes to 60 hours or from 160 minutes to 50 hours or from 160 minutes to 36 hours or from 160 minutes to 24 hours. In example 5, attractive results were obtained with an elution time of 24 hours. The skilled person knows that depending on the protein used the optimal elution time will not be the same.

[0179] In an embodiment, the increase of the elution time is preferably from 21 minutes to 176 minutes in the “Narrow” column. In another embodiment, the increase of elution time is preferably from 8 minutes to 168 minutes for the “Wide” column. Advantageously, such increase may be obtained by applying a flow stop, ranged from 1,5 to 2,5 hours, preferably about 2 hours flow stop in the process. The inventors surprisingly discovered that a relatively long elution time (for example ranged from 100 to 220 minutes) is beneficial for the purification yield of the protein of interest. For example increasing the elution time of at least 10%, 50%, 100% is expected to lead to an increase in the yield of the protein of interest of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%. The total elution time in this invention may include a flow stop. A flow stop is a period introduced when the load mixture is within the column and it does not flow or it stays within the column temporarily. In an embodiment of this invention, a flow stop of at least 0.5, 1, 1.5, 2, 2.5 hour is carried out when eluting the column, preferably said flow stop is 2 hour. In an embodiment of this invention, a flow stop of at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24 hour is carried out when eluting the column. However, depending on the protein, the flow stop may be of at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours. In a preferred embodiment, the flow stop is 2 hour. The advantageous effect of introducing the flow stop is that it may retain the target protein or protein of interest within the column longer, hence increasing the elution time and therefore further increasing the yield of the protein of interest. Moreover, introducing the stop increases contact time of elution buffer with the target protein so it promotes the target protein unbounding from the resin, increasing its recovery. The increased elution time, in combination with the reduced flow rate, is beneficial for the yield improvement of the protein of interest in this invention, in particular a protein having at least 60 or 80% identity with SEQ ID NO:1, preferably SEQ ID NO:1 which is protein Q. It is concluded that reducing the flow rate through the chromatography column would lead to an improvement of the yield because the contact time (hence total elution time) between the elution buffer and the protein of interest is further increased. In an embodiment, the flow stop is carried out during the elution phase, preferably in the middle of the elution phase.

[0180] In a preferred embodiment suitable for laboratory and / or industrial scales, there is provided a process according to the invention, wherein the total elution time is ranged from 165, 166, 167, 168, 169, 170 to 200, 201, 202, 203, 204, 205 minutes, more preferably from 168-202 minutes, preferably including a two-hour flow stop. In this embodiment, the load density is ranged from 20 to 247 mg TP / cm2, preferably ranged from 25 to 65 or from 25 to 60 or from 28 to 58, or from 40-65, or from 45-60, or from 50-56, or even more preferably ranged from 31 to 55 mg TP / cm2, even more preferably is 31 and / or 55 mg TP / cm2.

[0181] In a preferred laboratory scale embodiment, the total elution time is ranged from 168 to 202 minutes and the load density is ranged from 31 to 55 mg TP / cm2, more preferably is 31 mg TP / cm2. In this invention, the preferred ranges of the laboratory scale are extrapolable to any scale, in particular to an industrial scale.

[0182] In a preferred industrial scale embodiment, the total elution time is ranged from 168 to 202 minutes and the load density is ranged from 31 to 55 mg TP / cm2, more preferably is 31 and / or 55 mg TP / cm2.

[0183] The optimization of the total elution time in combination with the load density may lead to the yields of the protein of interest of at least 70%, up until 90% both in laboratory and industrial scales. Moreover, in an embodiment, by carrying out a flow stop of 1,5-2 hours and / or a total elution time of at least 160 up until 220 minutes, preferably from 165, 166, 167, 168, 169, 170 to 200, 201, 202, 203, 204, 205 minutes, more preferably from 168-202 minutes, it allows optimization of the yields of the protein of interest. In the preferred embodiment, the flow rate is 21 cm / h, preferably in the wide column. In another preferred embodiment, the flow rate is 48 cm / h, preferably in the narrow column. Also, this optimized process may reduce buffer consumption, energy and process supervision.

[0184] Flow rate

[0185] In column chromatography, flow rate indicates the velocity at which the mobile phase (solvent or buffer or eluent) traverses the column. This rate determines the velocity at which the sample and mobile phase move through the stationary phase, influencing the separation process. The optimal flow rate balances the velocity of the analysis with the necessity for sufficient separation (resolution). The elution flow rate of the invention is thought to optimize the upscaling process for increasing the yield of the protein of interest, in particular a protein having at least 60 or 80% identity with SEQ ID NO:1, preferably SEQ ID NO:1 which is protein Q. In this invention, slower flow rate impacts the yield positively. There are two types of flow rate in general within the chromatography technology, a linear flow rate and a volumetric flow rate. Volumetric flow rate is the volume of the mobile phase passing through the column per unit time. It is typically expressed in units of volume per unit time (e.g., mL / min or L / min). Volume flow rate is a more direct measure of the amount of mobile phase being delivered to the column per unit time, hence less suitable for comparison experiments in at different scales. This invention henceforth focuses on the linear flow rate. Linear flow rate denotes the speed or velocity at which the mobile phase travels through the column. It is typically expressed in units of distance per unit time, for instance cm / h. Linear flow rate calculation formula:

[0186] F

[0187] U~ A

[0188] wherein u = Linear flow rate (cm / h); F = Volumetric flow rate (mL / h); A = Cross-sectional area of the column (cm2).

[0189] Linear flow rate is particularly useful when comparing the performance of different columns, as it takes into account the column dimensions. Linear flow rate may be efficiently used to scale up and / or down, both in laboratory and industrial settings. Furthermore, it directly influences the separation efficiency and resolution in chromatography, among other characteristics. Elevated linear flow rates may expedite separations but potentially diminish resolution, whereas reduced linear flow rates often enhance resolution at the expense of increased elution time. On the other hand, faster flow rates typically result in shorter elution times, which may result in reduced yield, resolution and / or separation efficacy.

[0190] In an embodiment, the elution linear flow rate of the column is ranged from 10 to 127 cm / h, preferably from 15 to 127 cm / h, preferably from 20 to 60 cm / h, or from 21 to 48 cm / h, more preferably said linear flow rate is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 125, 126, 127 cm / h. In lab trials using an IEC, it was observed that a slow elution rate (approximatively 15 cm / h) (which means longer elution time of at least 180 min) increased the IEC yield by means of increasing elution contact times of the protein of interest with the column resin. A 15 cm / h elution flow rate is not possible in industrial scale, hence, to allow a longer elution time in industrial scale, the lowest flow rate possible in industrial columns was combined with a 2h flow stop, specifically an elution flow rate of 21 cm / h flow rate (for column d=30 cm) and 2h flow stop; and 48 cm / h flow rate (for column d=20 cm) and 2h flow stop. These combinations are equivalent to 15 cm / h per 180 min conditions in the downscaled lab trials, leading to similar improvement of IEC yields.

[0191] The reference flow rate of the experiment is approximatively 127 cm / h. In one embodiment of the process of the invention wherein the chromatography columns comprises the same flowrate 127 cm / h for both lab scale “Wide” (d=2.6 cm) test and “Narrow” (d=1.6 cm) reference columns, the “Wide” test column surprisingly exhibits better yield. This condition can be extrapolated to the industrial process by adjusting the dimensions of the column accordingly.

[0192] In practice, flow rates are frequently optimised via trial and error or according to established guidelines for the individual column type and chromatography technique. Hence, it is the surprising effect of the invention that the flow rate of the invention increases the yield of protein of interest described herein.

[0193] In a preferred embodiment, there is provided a process according to the invention, wherein the flow rate is ranged from 15 to 22 cm / h. This embodiment may not need a flow stop to increase the elution time. This embodiment relates to both a lab scale and a plant or industrial scale.

[0194] In a further embodiment, there is provided a process according to the invention, wherein the flow rate is ranged from 10 to 127 cm / h or from 20 to 110 cm / h or 30 to 100 cm / h or 20 to 60 cm / h or 21 to 48 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour or is about 2 hour.

[0195] In a further embodiment, there is provided a process according to the invention, wherein the load density is ranged from 20 to 250 or from 25 to 248 or from 30 to 247 mg TP / cm2; the flow rate is ranged from 5 to 130 or from 10 to 127 cm / h, preferably from 20 to 60 cm / h or 21 to 48 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour. This embodiment relates to both a preferred lab scale and a preferred plant or industrial scale.

[0196] In a further embodiment, there is provided a process according to the invention, wherein the load ratio is ranged from 4.5 to 13.5 or from 5.0 to 12.5 or from 5.5 to 11.5 mg TP / mL resin; the load density is ranged from 20 to 250 or from 25 to 248 or from 30 to 247 mg TP / cm2; the load concentration is ranged from 0.10 to 0.45 or from 0.15 to 0.50 or from 0.20 to 0.55 mg / mL; the flow rate is ranged from 5 to 130 or from 10 to 127 cm / h, preferably from 20 to 60 cm / h or 21 to 48 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour. This embodiment relates to both a preferred lab scale and a preferred plant or industrial scale.

[0197] In a further embodiment, there is provided a process according to the invention, wherein the load density is ranged from 20 to 247 mg TP / cm2; the flow rate is ranged from 5 to 130 or from 10 to 127 cm / h, preferably from 20 to 60 cm / h or 21 to 48 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour. This embodiment relates to both a preferred lab scale and a preferred plant or industrial scale. In a further embodiment, there is provided a process according to the invention, wherein the load ratio is ranged from 5.5 to 11.5 mg TP / mL resin; the load density is ranged from 20 to 247 mg TP / cm2; the load concentration is ranged from 0.20 to 0.55 mg / mL; the flow rate is ranged from 5 to 130 or from 10 to 127 cm / h, preferably from 20 to 60 cm / h or 21 to 48 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour. This embodiment relates to both a preferred lab scale and a preferred plant or industrial scale.

[0198] In a further embodiment, there is provided a process according to the invention, wherein the load density is ranged from 20 to 65 or from 25 to 60 or from 30 to 55mg TP / cm2; the flow rate is ranged from 8 to 140 or from 9 to 135 or from 10 to 127 cm / h, preferably from 20 to 60 cm / h or 21 to 48 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour. This embodiment relates to a preferred lab scale. In a further embodiment, there is provided a process according to the invention, wherein the load ratio is ranged from 5.7 to 6.5 or from 5.8 to 6.3 or from 5.9 to 6.1 mg TP / mL resin; the load density is ranged from 20 to 65 or from 25 to 60 or from 30 to 55mg TP / cm2; the load concentration is ranged from 0.25 to 0.55 or from 0.30 to 0.50 or from 0.35 to 0.45 mg / mL; the flow rate is ranged from 8 to 140 or from 9 to 135 or from 10 to 127 cm / h, preferably from 20 to 60 cm / h or 21 to 48 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour. This embodiment relates to a preferred lab scale.

[0199] In a further embodiment, there is provided a process according to the invention, wherein the load density is ranged from 30 to 55mg TP / cm2; the flow rate is ranged from 10 to 127 cm / h, preferably from 20 to 60 cm / h or 21 to 58 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour. This embodiment relates to a preferred lab scale. In a further embodiment, there is provided a process according to the invention, wherein the load ratio is ranged from 5.9 to 6.1 mg TP / mL resin; the load density is ranged from 30 to 55mg TP / cm2; the load concentration is ranged from 0.35 to 0.45 mg / mL; the flow rate is ranged from 10 to 127 cm / h, preferably from 20 to 60 cm / h or 21 to 58 cm / h; and optionally the flow stop is ranged from 1.5 to 2.5 hour. This embodiment relates to a preferred lab scale.

[0200] Load mixture

[0201] The load mixture refers to a sample or solution that contains multiple components or compounds that is introduced into a chromatography column or other separation system for analysis or purification. In this invention, the load mixture comprises a protein target or protein of interest, preferably a protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1. In an embodiment, the protein has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity or similarity with SEQ ID NO:1. The load mixture is introduced to the column in order to separate and analyze its individual components based on their distinct physical or chemical properties. The load mixture typically further comprises impurities or contaminants, which are unwanted components that need to be separated from the protein target, such as byproducts, degradation products, or other similar molecules.

[0202] The load mixture represents the starting material that needs to be separated into individual components. Types of load mixtures could include biological load mixtures, such as fermentation broth, cell lysates, plasma, or tissue extracts containing proteins, DNA, lipids, and / or other biomolecules; chemical load mixtures, such as desired reaction products along with unreacted starting materials, side products, or catalysts in a chemical synthesis; and / or environmental load mixtures, such as water samples containing pollutants, metals, or organic compounds that need to be separated and analyzed. In an embodiment, the load mixture comprises biomass.

[0203] A load mixture may be characterised by its load density (expressed in TP / cm2), its load ratio of Total Protein (TP) to the resin (expressed in mg TP / ml resin), and / or its load concentration (expressed in mg / mL).

[0204] It has been discovered that the key parameter characterising the load mixture is the load density (expressed in TP / cm2).

[0205] In an aspect, there is provided a process for purifying a protein, preferably represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1, comprising a step of purifying said protein from a mixture by a chromatography column, using at least one column comprising a resin, wherein the total elution time is ranged from 5 to 220 minutes; preferably from 160 to 210 minutes and / or wherein the mixture to be loaded to the column comprising said protein is characterized by a load density ranged from 20 to 247 mg / TP / cm2.

[0206] The load density (TP / cm2) (or Protein load density) refers to the amount of total protein loaded onto the column relative to the surface area of the resin. It is typically expressed in milligrams of total protein (TP) per square centimeter of resin surface area (mg TP / cm2). This parameter is beneficial to be optimized since it affects the binding capacity, resolution, and overall performance of the chromatographic process. Increasing load density may improve yield up to an optimum point, after which overloading occurs, potentially reducing yield. It is an object of the invention to define optimum conditions wherein the load density results in optimised purity and yield for the protein to be separated. This parameter can be modified by means of increasing / decreasing the column diameter or by means of increasing / decreasing the amount of protein at which the product is loaded onto the chromatography column.

[0207] In an embodiment, the mixture to be loaded (or load mixture) to the column comprising a protein as earlier defined herein is characterized by a load density, ranged from 20 to 247mg TP / cm2.

[0208] In an embodiment, the mixture to be loaded to the column comprising the protein as earlier defined herein is characterized by a load density higher than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 mg TP / cm2, and / or lower than 247, 160, 155, 150, 145, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95 TP / cm2; preferably the load density is ranged from 20-150, 30-140, 35-135, 40-130, 45-125, 50-120, 55-115, 60-110, 65-110, 70-105 mg TP / cm2, preferably ranged from 20-55, 20-50, 25-40, 28-35, 29-32, 40-65, 45-60, 50-56 mg TP / cm2. More preferably, the load density is 31 mg TP / cm2according to the invention. In one preferred embodiment, the load density is ranged from 20 to 247 mg TP / cm2, preferably ranged from 31 to 55 mg TP / cm2, more preferably 31 and / or 55 mg TP / cm2. In a preferred laboratory scale embodiment, the load density is ranged from 25 to 65 or from 25 to 60 or from 28 to 58 or from 31 to 55 mg or from 31 to 55 mg TP / cm2, more preferably is 31 and / or 55 mg TP / cm2. In this invention, the preferred ranges of the laboratory scale are extrapolable to any scale, in particular to an industrial scale. In a preferred an industrial scale embodiment, the load density is ranged from 31 to 55 mg TP / cm2, more preferably is 31 and / or 55 mg TP / cm2. The optimization of the load density may lead to the yields of at least 70% both in laboratory and industrial scales.

[0209] In another preferred embodiment, the load density is ranged from 70-95 or 75-90 or 77-88 or 79-87 or 81-86 or 84-86 or 85.8-85.9 mg TP / cm2according to the invention.

[0210] Optionally, the mixture to be loaded to the column comprising said protein is further characterized by the load ratio of total protein (TP) to the resin and / or the load concentration, which are further described herein.

[0211] The load ratio of total protein (or load ratio) (mg TP / ml resin) refers to the total amount of protein loaded onto the column relative to the volume of the resin. The volume of the resin may also reflect the binding capacity of the resin. The total proteins may include the protein of interest (for instance, the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) and / or all other proteins present in the load mixture. This parameter can be modified by means of increasing / decreasing the amount of TP loaded onto the IEC or by means of increasing / decreasing the column volume. Therefore, when keeping the same column, this parameter is directly connected to protein load density. The load ratio of total protein to the resin is an important parameter that influences the yield of the purification process. The inventors understood that underloading can lead to inefficient column use and overloading can reduce yield. In an embodiment, the mixture to be loaded to the column comprising the protein of interest is characterized by a load ratio of total protein (TP) to the resin, ranged from 5.0 to 12 mg TP / mL resin.

[0212] In an embodiment, the mixture to be loaded to the column comprising the protein of interest is characterized by a load ratio higher than 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, and / or lower than 12, 11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11.0, 10.9, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, 10.0, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6 mg TP / mL resin; preferably the load ratio is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, or 6.1 mg TP / mL resin. More preferably, the load ratio is ranged from 5.5 to 6.5 mg TP / ml resin, or from 5.7 to 6.3 mg TP / ml resin or from 5.9 to 6.1 mg TP / ml resin or is 6.0 mg TP / mL resin according to the invention. In an embodiment, the load ratio of 6.0 mg TP / mL resin is preferable for use in the lab scale and / or the industrial scale.

[0213] For example, in the Load ratio in laboratory studies using an I EC performed according to the invention, the “Wide Column” (lab scale test: 5.4 cm bed height and 2.6 cm diameter) showed higher recoveries than the “Narrow Column” (lab scale reference / control: 14.3 cm bed height and 1.6 cm diameter) with both load ratios (8.9 and 6.0 mg TP / mL resin) tested. For a given column geometry, the value of the load ratio of total protein is negatively correlated with the protein recovery observed. Since the “Narrow” and “Wide” columns had the same volume, as wider is the column, greater is the cross section. Therefore, those results also suggest the existence of an inverse relationship between the amount of protein loaded per unit of cross section area ratio (protein / area ratio) and IEC yield. In this example of lab scale, the load ratio of total protein 6.0 mg TP / mL resin gave a better yield in comparison to the load ratio reference of 8.9 mg TP / mL resin.

[0214] The load concentration refers to the concentration of the TP being applied to the ion exchange column, typically expressed in milligrams per milliliter (mg / mL). This parameter can be modified by for instance, but not limited to, diluting the protein that comes from the previous purification step with more or less buffer volume. It may also depend on the yield achieved in the previous purification step, if any. If less grams of TP are obtained from the previous steps, since there is buffer addition to adjust pH and urea, the TP concentration to enter the column of the invention will be lower than before the buffer addition. In an embodiment, the mixture to be loaded (or load mixture) to the column comprising the protein as earlier defined herein is characterized by a load concentration, ranged from 0.15 to 0.60 mg / mL. In an embodiment, the load concentration is higher than 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 and / or lower than 0.55, 0.54, 0.53, 0.52, 0.51 mg / mL; preferably the load concentration is 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49 or 0.50 mg / mL. Preferably, the load concentration is from 0.20 and 0.50 mg / ml or 0.23 and 0.49 mg / ml or 0.25 and 0.47 mg / mL. More preferably, the load concentration is 0.40 mg / mL. More preferably, the load concentration of 0.40 mg / mL is preferable for use in the lab scale and / or the industrial scale, wherein the best results were obtained for both conditions.

[0215] In the experimental part of the invention, two IEC load concentrations were tested. The lower concentration (0.25 mg / mL) chosen was below the 0.4 mg / mL control concentration while the higher concentration (0.5 mg / mL) was above that value. The lower and higher concentrations were tested using both geometries of column (“Narrow” and “Wide”) already used in the previous example of this invention. When comparing columns with the same diameter (1.6 cm), but with different concentrations of the TP load (0.40; 0.25; 0.50 mg / mL), the highest yield was found using a TP concentration load of 0.40 mg / mL. In addition, comparing columns with the same diameter (2.6 cm), but with different concentrations of the TP load (0.40; 0.25; 0.50 mg / mL), the highest yield was found using a TP concentration load of 0.40 mg / mL. Higher concentrations may lead to overloading the column, which can result in less binding capacity (less functional ligands in the resin than target protein molecules) of the resin that may lead to washing out the target proteins instead, while lower concentrations can lead to underutilization of the column's capacity.

[0216] In a preferred embodiment, the mixture to be loaded to the column comprising the protein of interest can be characterized as follows:

[0217] the load density, ranged from 20 to 247, or 30 to 247 mg TP / cm2;

[0218] and optionally

[0219] the load ratio of total protein (TP) to the resin, ranged from 5.0 to 12 mg TP / mL resin; and / or

[0220] the load concentration, ranged from 0.15 to 0.60 mg / mL.

[0221] Preferably, the protein of interest is a protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1. It is encompassed by the present invention that the protein of interest may not exhibit at least 60 or 80% identity or similarity with SEQ ID NO:1. The skilled person would know how to adapt each of the parameters defined here to further optimize the purification process for said other protein.

[0222] In a further embodiment, the purification process of the invention is carried out using an optimised value of the load ratio (mg TP / mL resin) and the load TP concentration (mg / mL). In one embodiment, the highest throughput is achieved using a load ratio of 6.00 mg TP / mL resin at a load concentration of 0.40 mg / mL, giving better yields in “Wide” diameter test column (2.6 cm) than in “Narrow” diameter reference column (1.6 cm) at the lab scale. In a further embodiment, said combination can be extrapolated to the industrial scale column accordingly. Being comparable to the industrial column comprising a load ratio of 6.00 mg TP / mL resin combined with a load concentration of 0.40 mg / mL gives the best yield, a set of confirmation runs were run using these parameters but changing the column height to 9.2 cm (because the industrial process cannot accept such a low height of 5.4 cm). The results also surprisingly show higher yield than using the reference column (“Narrow” column).

[0223] In a further embodiment, the purification process of the invention is carried out using an optimized value of the load ratio (mg TP / mL resin) and of the load TP density (mg TP / cm2). In one embodiment, the highest throughput is achieved using a load ratio of 6.0 mg TP / mL resin and a load TP density ranged from 20 to 60 or from 30 to 55 mg TP / cm2giving better yields at the lab scale. Optionally in this embodiment, the load concentration is ranged from 0.35 to 0.55 or 0.38 to 0.53 or 0.39 to 0.50 or 0.40 to 0.50 or is 0.40 mg / ml.

[0224] In a further embodiment, said combination can be extrapolated to the industrial scale column accordingly.

[0225] In a further embodiment, it is provided a process according to the invention, wherein the load ratio is ranged from 5.5 to 11.5 mg TP / mL resin; the load density is ranged from 20-247 mg TP / cm2; and / or the load concentration is ranged from 0.20 to 0.55 mg / mL. This embodiment is applicable to both a lab scale and a plant or industrial scale.

[0226] In a further embodiment, it is provided a process according to the invention, wherein the load ratio is ranged from 5.9 to 6.1 mg TP / mL resin; the load density is ranged from 30-55mg TP / cm2; and the load concentration is ranged from 0.35 to 0.45 mg / mL. This embodiment is applicable to a lab scale or to a plant or industrial scale.

[0227] In an embodiment for a lab scale, it is provided a process according to the invention, wherein the load density is ranged from 30 to 32 or 54 to 56 mg TP / cm2.

[0228] In an embodiment for a lab scale, it is provided a process according to the invention, wherein the load ratio is ranged from 5.5 to 6.5 or 5.7 to 6.3 or 5.9 to 6.1 or 6.0 mg TP / mL resin; the load density is ranged from 30 to 32 or 54 to 56 mg TP / cm2; and the load concentration is ranged from 0.3 to 0.5 or 0.35 to 0.45 or 0.37 to 0.42 or 0.4 mg / mL. Preferably, the diameter of the column of this embodiment is 2.6 cm and the height is 5.4 cm. Preferably, the RD / H of the column of this embodiment is 0.48. This embodiment results in up to 90% yield, which is the best result obtained at lab scale. This may give an increase of up to 20% yield compared to the reference lab scale column (“Narrow” column). This optimized process steps are likely to be upscaled or implemented at plant or industrial scale to improve the yield of the protein purification process of the invention.

[0229] In an embodiment for a lab scale, it is provided a process according to the invention, wherein the load density is 31 mg TP / cm2.

[0230] In an embodiment for a lab scale, it is provided a process according to the invention, wherein the load ratio is 6 mg TP / mL resin; the load density is 31 mg TP / cm2; and the load concentration is 0.4 mg / mL. Preferably, the diameter of the column of this embodiment is 2.6 cm and the height is 5.4 cm. Preferably, the RD / H of the column of this embodiment is 0.48. This embodiment results in up to 90% yield, which is the best result obtained at lab scale. This may give an increase of up to 20% yield compared to the reference lab scale column (“Narrow” column). This optimized process steps may be upscaled or implemented at plant or industrial scale to improve the yield of the protein purification process of the invention.

[0231] In a further embodiment, it is provided a process according to the invention, wherein the load density is ranged from 20 to 255 or 25 to 250 or 28 to 249 or 30-247 mg TP / cm2. In a further embodiment, it is provided a process according to the invention, wherein the load ratio is ranged from 5.0 to 20.0 or 5.2 to 15.0 or 5.4 to 12.0 or 5.5 to 11.5 mg TP / mL resin; the load density is ranged from 20 to 255 or 25 to 250 or 28 to 249 or 30-247 mg TP / cm2; and the load concentration is ranged from 0.10 to 0.65 or 0.15 to 0.60 or 0.20 to 0.55 mg / mL. Preferably, the diameter of the column of this embodiment is 20 or 30 cm and the height is 7.5 or 14.3 cm. Preferably, the RD / H of the column of this embodiment is 1.4 or 4. This embodiment relates to a preferred plant or industrial scale. The optimized process steps may likely be (further) upscaled or implemented at a plant or at industrial scale to (further) improve the yield of the protein purification process of the invention.

[0232] In a further embodiment, it is provided a process according to the invention, wherein the load density is ranged from 20-247 mg TP / cm2.

[0233] In a further embodiment, it is provided a process according to the invention, wherein the load ratio is ranged from 5.5 to 11.5 mg TP / mL resin; the load density is ranged from 20-247 mg TP / cm2; and the load concentration is ranged from 0.20 to 0.55 mg / mL. Preferably, the diameter of the column of this embodiment is 20 or 30 cm and the height is 7.5 or 14.3 cm. Preferably, the RD / H of the column of this embodiment is 1.4 or 4. This embodiment relates to a preferred plant or industrial scale. The optimized process steps may likely be (further) upscaled or implemented at a plant or at industrial scale to (further) improve the yield of the protein purification process of the invention.

[0234] In a further embodiment, it is provided a process according to the invention, wherein the load density is ranged from 20 to 65 or 25 to 60 or 29 to 58 or 30-55 mg TP / cm2.

[0235] In a further embodiment, it is provided a process according to the invention, wherein the load ratio is ranged from 5.0 to 7.0 or 5.5 to 6.5 or 5.9 to 6.1 mg TP / mL resin; the load density is ranged from 20 to 65 or 25 to 60 or 29 to 58 or 30-55 mg TP / cm2; and the load concentration is ranged from 0.25 to 0.55 or 0.35 to 0.50 or 0.35 to 0.45 mg / mL. Preferably, the diameter of the column of this embodiment is 20 or 30 cm and the height is 7.5 or 14.3 cm. Preferably, the RD / H of the column of this embodiment is 1.4 or 4. This embodiment relates to a preferred plant or industrial scale. The optimized process steps may likely be (further) upscaled or implemented at a plant or at industrial scale to (further) improve the yield of the protein purification process of the invention.

[0236] In a further embodiment, it is provided a process according to the invention, wherein the load density is ranged from 30-55 mg TP / cm2.

[0237] In a further embodiment, it is provided a process according to the invention, wherein the load ratio is ranged from 5.9 to 6.1. mg TP / mL resin; the load density is ranged from 30-55 mg TP / cm2; and the load concentration is ranged from 0.35 to 0.45 mg / mL. Preferably, the diameter of the column of this embodiment is 20 or 30 cm and the height is 7.5 or 14.3 cm. Preferably, the RD / H of the column of this embodiment is 1.4 or 4. This embodiment relates to a preferred plant or industrial scale. The optimized process steps may likely be (further) upscaled or implemented at a plant or at industrial scale to (further) improve the yield of the protein purification process of the invention. In a preferred embodiment, it is provided an improved process according to the invention, wherein load density is ranged from 84.5 to 86.5 or from 85.0 to 86.0 or from 85.5 to 85.9 or from 85.8 to 85.9 mg TP / cm2.

[0238] In a preferred embodiment for a plant or industrial scale, it is provided an improved process according to the invention, wherein load density is ranged from 25 to 135 or from 30 to 130 or from 37 to 128 TP / cm2.

[0239] In a preferred embodiment for a plant or industrial scale, it is provided an improved process according to the invention, wherein the load ratio is ranged from 5.5 to 6.5 or 5.7 to 6.3 or 5.9 to 6.1 or 6 mg TP / mL resin; the load density is ranged from 25 to 135 or from 30 to 130 or from 37 to 128 or from 31 to 55, or from 30-32, or from 40-65, or from 45-60, or from 50-56, or from 54-56 mg TP / cm2; and the load concentration is ranged from 0.3 to 0.5 or 0.35 to 0.45 or 0.37 to 0.42 or is 0.4 mg / mL. Preferably, the diameter of the column of this embodiment is 20 cm and the height is 14.3 cm. Preferably, the RD / H of the column of this embodiment is 1.4. This embodiment relates to a preferred plant or industrial scale. The optimized process steps may likely be (further) upscaled or implemented at a plant or at industrial scale to (further) improve the yield of the protein purification process of the invention.

[0240] In a preferred embodiment for a plant or industrial scale, it is provided an improved process according to the invention, wherein the load ratio is 6 mg TP / mL resin; the load density is ranged from 37 to 128 mg TP / cm2; and the load concentration is 0.4 mg / mL. Preferably, the diameter of the column of this embodiment is 20 cm and the height is 14.3 cm. Preferably, the RD / H of the column of this embodiment is 1.4. This embodiment relates to a preferred plant or industrial scale. The optimized process steps may likely be (further) upscaled or implemented at a plant or at industrial scale to (further) improve the yield of the protein purification process of the invention.

[0241] In another preferred embodiment for a plant or industrial scale, it is provided the improved process according to the invention, wherein the load ratio is ranged from 5.5 to 6.5 or 5.7 to 6.3 or 5.9 to 6.1 or is 6 mg TP / mL resin; the load density is ranged from 35 to 55 or from 39 to 50 or from 41 to 48 or is 45 mg TP / cm2; and the load concentration is ranged from 0.3 to 0.5 or 0.35 to 0.45 or 0.37 to 0.42 or is 0.4 mg / mL. Preferably, the diameter of the column of this embodiment may be 30 cm and the height may be 7.5 cm. Preferably, the RD / H of the column of this embodiment may be 4. This embodiment relates to a preferred plant or industrial scale. The optimized process steps may likely be (further) upscaled or implemented at a plant or at industrial scale to (further) improve the yield of the protein purification process of the invention.

[0242] In another preferred embodiment for a plant or industrial scale, it is provided the improved process according to the invention, wherein the load ratio is 6 mg TP / mL resin; the load density is 45 mg TP / cm2; and the load concentration is 0.4 mg / mL. Preferably, the diameter of the column of this embodiment may be 30 cm and the height may be 7.5 cm. Preferably, the RD / H of the column of this embodiment may be 4. This embodiment relates to a preferred plant or industrial scale. The optimized process steps may likely be (further) upscaled or implemented at a plant or at industrial scale to (further) improve the yield of the protein purification process of the invention.

[0243] In some embodiments of the invention, the column is preferably an IEC column.

[0244] Column chromatography dimensions

[0245] The physical column is typically made of materials such as glass, metal, stainless steel, polymer or inert plastics. The material and size of the column can vary depending on the specific type of chromatography and its application. In this invention, the column may be characterized by its diameter (D) and height (H), and by the ratio of the diameter and the height thereof (RD / H).

[0246] In an embodiment, it is possible that the diameter of the column is higher than 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, or 2.9 cm and / or lower than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 29, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 cm.

[0247] In an embodiment that is suitable for lab scale, it is possible that the diameter of the column is 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, or 2.9 cm. In this invention, the preferred diameter of the column suitable for lab scale is ranged from 1.5 to 2.9 cm, or is ranged from 1.6 to 2.6 cm, or is 1.6 or 2.6 cm.

[0248] In an embodiment that is suitable for plant or industrial scale, it is possible that the diameter of the column is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 cm. In this invention, the preferred diameter of the column suitable for plant or industrial scale is ranged from 15 to 34 or from 20 to 30 or is 20 or 30 cm.

[0249] In an embodiment, it is possible that the height of the column is at least 4 cm, preferably ranged from 4 to 15 cm, more preferably the height is at least 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 14, 14.1, 14.2, 14.3, 14.4, or 14.5 cm.

[0250] In an embodiment that is suitable for lab scale, it is possible that the height of the column is 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5 cm. In this invention, the preferred height of the column suitable for lab scale is ranged from 5.3 to 14.4, or ranged from 5.4 to 14.3, or is 5.4, or 9.2, or 14.3 cm.

[0251] In an embodiment that is suitable for plant or industrial scale, it is possible that the height of the column is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5 cm. In this invention, the preferred height of the column suitable for plant or industrial scale 7.5 cm or 14.3 cm.

[0252] In an embodiment, it is possible that the column comprises the diameter of 1.6 cm and the height of 14.3 cm; this embodiment may be used for lab scale. A lab scale experiment may be carried out as a control experiment. A control experiment may be carried out to test some process parameters in association with the column. In an embodiment, it is possible that the column comprises the diameter of 2.6 cm and the height of 5.4 cm; this embodiment may be used for lab scale. Such a lab scale experiment may be carried out as lab scale test. In an embodiment, it is possible that the column comprises the diameter of 2.6 cm and the height of 9.2 cm; this embodiment may be used for lab scale. Such a lab scale experiment may be carried out as lab scale test confirmation. In an embodiment, it is possible that the column comprises the diameter of 30 cm and the height of 7.5 cm; this embodiment may be used for industrial scale. In an embodiment, it is possible that the column comprises the diameter of 20 cm and the height of 14.3 cm; this embodiment may be used for plant or industrial scale.

[0253] In an embodiment, it is possible that the RD / H ratio is higher than 0.09 and / or lower than 4.5, 4.4, 4.3, 4.2, 4.1; preferably the RD / H ratio is at least 0.1, 0.11, 0.25, 0.26, 0.27, 0.28, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.6, 0.7, 0.8, 0.9, 1.2, 1.3, 1.4, 1.5, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, or 4.4. More preferably, the column comprises a RD / H ratio ranged from 0.1, and 4, more preferably a RD / H ratio of 1.4, even more preferably a RD / H ratio of 4.

[0254] In an embodiment that is suitable for lab scale, it is possible that the RD / H ratio of the column is 0.1, 0.11, 0.25, 0.26, 0.27, 0.28, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5. In this invention, the preferred RD / H ratio of the column suitable for lab scale is ranged from 0.10 to 0.50, or is 0.11 or 0.28 or 0.48. In an embodiment that is suitable for plant or industrial scale, it is possible that the RD / H ratio of the column is 1.3, 1.4, 1.5, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, or 4.4. In this invention, the preferred RD / H ratio of the column suitable for plant or industrial scale is ranged from 1.3 to 4.2, or is 1.4 or 4.

[0255] In some embodiments, the column is an IEC column.

[0256] In an embodiment, it is noted that adjusting the dimensions of the column, such as adjusting the diameter (D), the height (H), and / or the ratio thereof (RD / H) may contribute to an optimization of the purification of the protein. The objective of adjusting the dimensions of the column was to increase the section of the column so that protein aggregation may be avoided and thus it may improve the protein load entering to the column. However, advantageously, it appears that an increase in the column dimensions and therefore the contact surface between the protein (present in the load mixture) and the IEC resin at the top of the column would lead to a better purification via better solubilization of the protein of interest. Thus, it may further facilitate promoting protein entering to the resin, therefore avoiding protein precipitation at the column head. Moreover, it has been unexpectedly discovered that increasing the D of the column contributes to an improvement of the yield (as demonstrated in example 1). This is hypothesized to be due to the increase of interaction surface with the mixture loaded in the column due to the optimization of the column dimensions (diameter (D), the height (H), and / or the ratio thereof (RD / H)).

[0257] It is noted that the dimensions of the laboratory tests, such as RD / H, may be extrapolated to the industrial process accordingly to exhibit similar results or similar improvement of the purification process. The term “purify” or “purification” or “purifying” a protein of interest from a mixture or solution comprising said protein and one or more contaminants refers to enhancing the purity of the target protein by eliminating at least one contaminant from the mixture or solution. In a preferred embodiment for a lab scale, there is provided a process according to the invention, wherein the load density is 31 mg TP / cm2; the elution flow rate is 21 cm / h; the flow stop is 2h; and a total of elution time 168-170, in particular 168 minutes. Preferably, the diameter of the column of this embodiment is 2.6 cm and the height is 5.4 cm. Preferably, the RD / H of the column of this embodiment is 0.48. This embodiment results in about 90% yield, which is the best result in the lab scale. This may give an increase of about 20% yield compared to the reference lab scale column (“Wide” or 2.6x5.4 cm versus “Narrow” or 1.6x14.3 cm columns). This optimized process step may be upscaled or implemented at a plant or an industrial scale to improve the yield of the protein purification process of the invention.

[0258] In a preferred embodiment for a lab scale, there is provided a process according to the invention, wherein the load ratio is 6 mg TP / mL resin; the load density is 31 mg TP / cm2; the load concentration is 0.4 mg / mL; the elution flow rate is 21 cm / h; the flow stop is 2h; and a total of elution time 168-170, in particular 168 minutes. Preferably, the diameter of the column of this embodiment is 2.6 cm and the height is 5.4 cm. Preferably, the RD / H of the column of this embodiment is 0.48. This embodiment results in about 90% yield, which is the best result in the lab scale. This may give an increase of about 20% yield compared to the reference lab scale column (“Wide” or 2.6x5.4 cm versus “Narrow” or 1.6x14.3 cm columns). This optimized process step may be upscaled or implemented at a plant or an industrial scale to improve the yield of the protein purification process of the invention.

[0259] In a further embodiment, there is provided a process according to the invention, wherein the load density is ranged from 20 to 257 or from 25 to 250 or from 30 to 247 mg TP / cm2; the elution flow rate ranged from 21 to 22 cm / h and 2h flow stop, preferably in the wide column, or 48 cm / h and 2h flow stop, preferably in the narrow column; and / or a total of elution time ranged from 168 to 190 minutes.

[0260] In a further embodiment, there is provided a process according to the invention, wherein the load ratio is ranged from 4.5 to 12.5 or from 5.0 to 12.0 or from 5.5 to 11.5 mg TP / mL resin; the load density is ranged from 20 to 257 or from 25 to 250 or from 30 to 247 mg TP / cm2; the load concentration is ranged from 0.10 to 0.65 or from 0.15 to 0.60 or from 0.20 to 0.55 mg / mL; the elution rate ranged from 21 to 22 cm / h and 2h flow stop, preferably in the wide column, or 48 cm / h and 2h flow stop, preferably in the narrow column; and / or a total of elution time ranged from 168 to 190 minutes.

[0261] Preferably, the diameter of the column of this embodiment is 20 or 30 cm and the height is 7.5 or 14.3 cm. Preferably, the embodiment with elution rate ranged from 21 to 22 cm / h and 2h flow stop is for the column having 30 cm diameter or the wide column. Preferably, the embodiment with elution rate of 48 cm / h 2h flow stop is for the column having 20 cm diameter or the narrow column. Preferably, the RD / H of the column of this embodiment is 1.4 or 4. This embodiment related to a preferred plant or an industrial scale. This optimized process steps may be upscaled or implemented at a plant or an industrial scale to improve the yield of the protein purification process of the invention. In a further embodiment, there is provided a process according to the invention, wherein the load density is ranged from 20 to 247 mg TP / cm2; the elution flow rate ranged from 21 to 22 cm / h and 2h flow stop, preferably in the wide column, or 48 cm / h and 2h flow stop, preferably in the narrow column; and / or a total of elution time ranged from 168 to 190 minutes.

[0262] In a further embodiment, there is provided a process according to the invention, wherein the load ratio is ranged from 5.5 to 11.5 mg TP / mL resin; the load density is ranged from 20 to 247 mg TP / cm2; the load concentration is ranged from 0.20 to 0.55 mg / mL; the elution rate ranged from 21 to 22 cm / h and 2h flow stop, preferably in the wide column, or 48 cm / h and 2h flow stop, preferably in the narrow column; and / or a total of elution time ranged from 168 to 190 minutes.

[0263] Preferably, the diameter of the column of this embodiment is 20 or 30 cm and the height is 7.5 or 14.3 cm. Preferably, the embodiment with elution rate ranged from 21 to 22 cm / h and 2h flow stop is for the column having 30 cm diameter or the wide column. Preferably, the embodiment with elution rate of 48 cm / h 2h flow stop is for the column having 20 cm diameter or the narrow column. Preferably, the RD / H of the column of this embodiment is 1.4 or 4. This embodiment related to a preferred plant or an industrial scale. This optimized process steps may be upscaled or implemented at a plant or an industrial scale to improve the yield of the protein purification process of the invention.

[0264] In a further embodiment, there is provided a process according to the invention, wherein the load density is ranged from 20 to 65 or from 25 to 60 or from 30 to 55 mg TP / cm2; and the load concentration is ranged from 0.25 to 0.55 or from 0.30 to 0.50 or from 0.35 to 0.45 mg / mL

[0265] In a further embodiment, there is provided a process according to the invention, wherein the load ratio is ranged from 5.5 to 6.5 or from 5.7 to 6.3 or from 5.9 to 6.1. mg TP / mL resin; the load density is ranged from 20 to 65 or from 25 to 60 or from 30 to 55 mg TP / cm2; and the load concentration is ranged from 0.25 to 0.55 or from 0.30 to 0.50 or from 0.35 to 0.45 mg / mL.

[0266] Preferably, the diameter of the column of this embodiment is 20 or 30 cm and the height is 7.5 or 14.3 cm. Preferably, the embodiment with elution rate of ranged from 21 to 22 cm / h and 2h flow stop is for the column having 30 cm diameter or the wide column. Preferably, a total of elution time ranged from 168- to 190 minutes. Preferably, the embodiment with elution rate of 48 cm / h 2h flow stop is for the column having 20 cm diameter or the narrow column. Preferably, the RD / H of the column of this embodiment is 1.4 or 4. This embodiment encompasses a preferred plant or industrial scale. This optimized process steps may be the upscaled or implemented at plant or industrial scale to improve the yield of the protein purification process of the invention.

[0267] In a further embodiment, there is provided a process according to the invention, wherein the load density is ranged from 30 to 55 mg TP / cm2; and the load concentration is ranged from 0.35 to 0.45 mg / mL.

[0268] In a further embodiment, there is provided a process according to the invention, wherein the load ratio is ranged from 5.9 to 6.1. mg TP / mL resin; the load density is ranged from 30 to 55 mg TP / cm2; and the load concentration is ranged from 0.35 to 0.45 mg / mL. Preferably, the diameter of the column of this embodiment is 20 or 30 cm and the height is 7.5 or 14.3 cm. Preferably, the embodiment with elution rate of ranged from 21 -to 22 cm / h and 2h flow stop is for the column having 30 cm diameter or the wide column. Preferably, a total of elution time ranged from 168 to 190 minutes. Preferably, the embodiment with elution rate of 48 cm / h 2h flow stop is for the column having 20 cm diameter or the narrow column. Preferably, the RD / H of the column of this embodiment is 1.4 or 4. This embodiment encompasses a preferred plant or industrial scale. This optimized process steps may be the upscaled or implemented at plant or industrial scale to improve the yield of the protein purification process of the invention.

[0269] In a preferred embodiment for a plant or industrial scale, there is provided an improved process according to the invention, wherein the load density is 85-86 mg TP / cm2.

[0270] In a preferred embodiment for a plant or industrial scale, there is provided an improved process according to the invention, wherein the load ratio is 6 mg TP / mL resin; the load density is 85-86 mg TP / cm2; and the load concentration is 0.4 mg / mL.

[0271] Preferably, the elution rate is of 48 cm / h and a 2h flow stop is carried out. Preferably, the elution time is 170-180, in particular 176 minutes. Preferably, the diameter of the column of this embodiment is 20 cm and the height is 14.3 cm. Preferably, the RD / H of the column of this embodiment is 1.4. This embodiment relates to a preferred plant or industrial scale. This optimized process steps may be upscaled or implemented at a plant or an industrial scale to improve the yield of the protein purification process of the invention.

[0272] In another preferred embodiment for a plant or industrial scale, there is provided an improved process according to the invention, wherein the load density is 45 mg TP / cm2.

[0273] In another preferred embodiment for a plant or industrial scale, there is provided an improved process according to the invention, wherein the load ratio is 6 mg TP / mL resin; the load density is 45 mg TP / cm2; and the load concentration is 0.4 mg / mL.

[0274] Preferably, the diameter of the column of this embodiment is 30 cm and the height is 7.5 cm. Preferably, the elution rate is of 21-22 cm / h and a 2h flow stop is carried out. Preferably, the elution time is 185-190, in particular 187 minutes. Preferably, the RD / H of the column of this embodiment may be 4. This embodiment relates to a preferred plant or industrial scale. This optimized process steps may be upscaled or implemented at a plant or an industrial scale to improve the yield of the protein purification process of the invention.

[0275] Characterisation of the improved read out of the purification process of the invention

[0276] In one preferred embodiment of a laboratory scale and / or an industrial scale, there is provided a process wherein:

[0277] the total elution time is ranged from 168 to 202 minutes; and / or

[0278] the load density is ranged from 30-55 mg TP / cm2; and optionally

[0279] the load ratio is ranged from 5.9 to 6.1 mg TP / mL resin;

[0280] the load concentration is ranged from 0.35 to 0.45 mg / mL;

[0281] the flow rate is ranged from 21 to 48 cm / h; and / or

[0282] the flow stop is about 2 hour.

[0283] In another preferred embodiment of a laboratory scale and / or an industrial scale, the process provided may also further comprise the preferred diameter (D), the height (H), and / or the ratio thereof (RD / H) according to this invention.

[0284] In an embodiment, the process of the invention for purifying a protein, preferably represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1, wherein said process comprises a step of purifying said protein from a mixture by a chromatography column, using at least one column comprising a resin and wherein said process is with optimised elution time and / or optimised characteristics of the load mixture as earlier defined herein. In an embodiment, this process of the invention is carried out at an industrial scale and is improved compared to a control process carried out under the same conditions, including the same column but not having an optimised elution time and / or optimised characteristics of the load mixture.

[0285] Such improved process carried out at an industrial scale is expected to exhibit at least of the following improvements compared to a control process carried out at an industrial scale:

[0286] an increase of the yield of the protein of interest compared to the yield obtained using process with no optimisation of the elution time and / or of the characteristics of the mixture to be loaded to the column (up to 40% increase, see example 1) and

[0287] an increase in the amount of protein of interest produced (grams of final purified drug substance, protein Q) obtained per batch (up to 41 % increase, see example 2).

[0288] In an embodiment, the increase of the yield of the protein of interest may be of at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%.

[0289] In an embodiment, the increase of the amount of protein of interest produced may be of at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%. The inventors demonstrated in example 4 that this process could be applied with success to a protein having at least 60% identity to Protein Q. Two distinct proteins (Protein 2 having 59.8% identity with Protein Q, and Protein 3 having 87.4% identity with Protein Q) have been successfully purified in example 4. An increase in the amount of protein of interest produced (grams of final purified drug substance, protein 2) obtained per batch (up to 70% increase, see example 4), An increase in the amount of protein of interest produced (grams of final purified drug substance, protein 3) obtained per batch (up to 31% increase, see example 4),

[0290] Such improved process carried out at an industrial scale is expected to exhibit at least of the following improvements compared to a control process carried out at an industrial scale:

[0291] an improved yield (or percentage of protein of interest recovery): yield up to 90% (see example 1, figure 1),

[0292] a more reproducible process (see lower coefficient of variation in example 2 compared to coefficient de variation obtained with control process) and

[0293] a process which is easier and more flexible to handle (see example 1).

[0294] The yield of the process of the invention may be up to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of protein of interest. In an embodiment, the yield is assessed using method known to the skilled person, preferably by BCA as explained in the experimental part.

[0295] Furthermore, the advantageous effects are that this process helps to better solubilize the protein remaining in the column. Increasing contact time of the elution buffer with the target protein during the elution is advantageous for proteins with low solubility and, and proteins having low detachment and resolubilization kinetics. Moreover, reducing load ratio and load density are aiming to reduce overloading of the resin, especially at the column head. In this way, the optimized process may significantly prevent protein aggregation which results in poorer solubilization kinetics.

[0296] Protein Q

[0297] The Protein Q vaccine is authorized for commercialization in the European Union under the tradename LetiFend® (Laboratorios LETI, Spain). LetiFend® is the sole commercial vaccination based on a recombinant protein known as Protein Q (SEQ ID NO:1). It is composed of a recombinant protein (Protein Q) obtained through the genetic fusion of five antigenic determinants. The five antigenic fragments that were isolated from four distinct L. infantum proteins, namely the N- and C-terminal regions of histone H2A as well as the ribosomal proteins LiP2a, LiP2b, and LiPO. In W02000039298, which is included herein by reference in its entirety, a protein is disclosed which is highly homologous (98%) with Protein Q having SEQ ID NO:1. This highly homologous protein Q is functionally characterized in this patent application. In WO2000039298, this highly homologous protein Q is identified by SEQ ID NO:1. In this study, vaccination with Protein Q in mice and dogs have also shown its high immunogenicity, as well as their safety profile, protective capability and efficacy against the development of the disease. A single dose of Protein Q has demonstrated to be immunogenic and to protect dogs against experimental L. infantum infection in the absence of an adjuvant. In a pre-licensing phase III experiment, LetiFend® comprising Protein Q shown a 72% vaccination efficacy in avoiding CanL clinical symptoms in 549 dogs exposed to natural infection over a two-year period in two CVL-endemic locations in France and Spain. Comparing vaccinated dogs to placebo dogs, the vaccination also decreased the chance of confirmed CanL cases and the onset of clinical symptoms by 5 and 9.8 times, respectively (Iniesta V, Fernandez-Cotrina J, Solano-Gallego L, Monroy I, Gomez-Luque A, Munoz-Madrid R. Vaccination with LetiFend, a novel canine leishmaniosis vaccine, does not interfere with serological diagnostic tests. Proceedings of the X Southern European Veterinary Conference / 51 Congreso Nacional Avepa, 20-22 October 2016, Granada (Spain) Poster SEVC00678, 2016).

[0298] In an embodiment, the protein purified in the process of the invention comprises the following amino acid sequence (SEQ. ID NO. 1):

[0299] MRGSHHHHHHTDPHASSNNNNNNNNNNLGIEGRPLATPRSAKKAVRKSGSKSAKCGL IFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHD DDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTKYLAAYA LASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPA ASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSG KTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGA VSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPEESDEDD FGMGGLF

[0300] In a preferred embodiment, a protein Q is a protein target or a protein of interest. Preferably, the protein Q is represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:1. In preferred embodiment, the protein Q has at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity or similarity with SEQ ID NO:1.

[0301] It is also encompassed by the present invention that proteins of interest separated by I EX chromatography do not need His-tags, because this method relies on the intrinsic properties of the proteins of interest, rather than affinity tags. In a preferred embodiment, protein Q, represented by SEQ ID NO:1, may be produced by fermenting a host cell, and subsequently purified using the IEX chromatography column comprising characteristics and process described in this invention.

[0302] In the process comprising the IEX chromatography column, the stationary phase contains charged functional groups (preferably positive). Then, the Protein Q, with opposite charges to the stationary phase, binds to it. Bound Protein Q is eluted by gradually changing the ionic strength or pH (elution pH at about 8) of the mobile phase.

[0303] In the context of the disclosure, it is to be understood that preferably Protein Q is the protein of interest in all aspects of the invention. General part dedicated to definitions

[0304] Polypeptide / nucleic acid

[0305] A “wild type” protein / polypeptide amino acid sequence can refer to a sequence that is naturally occurring and encoded by a germline genome. A species can have one wild type sequence, or two or more wild type sequences (for example, with one canonical wild type sequence and one or more non-canonical wild type sequences). A wild type protein amino acid sequence can be a mature form of a protein that has been processed to remove N-terminal and / or C-terminal residues, for example, to remove a signal peptide. In some embodiments, a reference sequence used herein is a wild type sequence. Protein Q is a non-naturally occurring chimeric protein comprised of the antigenic determinants of four proteins of Leishmania infantum.

[0306] An amino acid sequence that is “derived from” a wild type sequence or reference sequence or another amino acid sequence disclosed herein can refer to an amino acid sequence that comprises an amino acid modification, for example an amino acid sequence that differs by one or more amino acids compared to the wild type or reference amino acid sequence, for example, containing one or more amino acid insertions, deletions, or substitutions as described herein.

[0307] In the context of the disclosure, a polypeptide comprises an amino acid sequence. In the context of the disclosure, a nucleic acid such as a nucleic acid molecule encoding a protein chimera Q, which is the product of a chimeric gene encoding the antigenic determinants of four proteins of Leishmania infantum. A nucleic acid molecule may comprise a regulatory region.

[0308] It is to be understood that each nucleic acid molecule or polypeptide or construct as identified herein by a given Sequence Identity Number (SEQ ID NO) is not limited to this specific sequence as disclosed. Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as example) encoding a given polypeptide, one may replace it by: i. a nucleotide sequence comprising a nucleotide sequence that has at least 60% or at least 80% sequence identity with SEQ ID NO: X;

[0309] ii. a nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i);

[0310] Hi. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule of (i) or (ii) due to the degeneracy of the genetic code; or,

[0311] iv. a nucleotide sequence that encodes an amino acid sequence that has at least 60% or at least 80% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.

[0312] Throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide comprising an amino acid sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. In the context of the application, the minimum identity or similarity in relation to a protein Q or fragment thereof may mean an identity or a similarity of at least 60%. In the context of the application, the minimum identity or similarity in relation to a protein Q or fragment thereof may mean an identity or a similarity of at least 60%.

[0313] In the context of the polypeptides described herein, the term "fragment” may be replaced by the term "part”, the two terms being interchangeable. A fragment of a polypeptide lacks one or more amino acids present in the polypeptide it is derived from. A fragment of a polypeptide may correspond to at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% of the length of a polypeptide, for example as represented by an amino acid sequence with a specific SEQ ID NO, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the length of the polypeptide.

[0314] Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage (e.g. at least 60%) with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity (or a similarity where applicable) of at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the given nucleotide or amino acid sequence respectively. In a preferred embodiment, sequence identity or similarity is determined by comparing the whole length of the sequences as identified herein. In other words, sequence identity is preferably calculated based on the full length of two given sequences being compared (for example of a sequence represented by a SEQ ID NO herein and of another sequence it is being compared to). Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO means identity or similarity based on the full length of said sequence (i.e. over its whole length or as a whole).

[0315] Sequence identity

[0316] " Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The identity between two amino acid or two nucleic acid sequences is typically defined by assessing their identity within a whole length SEQ ID NO as identified herein or part thereof. Part thereof in terms of comparing the identity or similarity of two or more sequences may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0317] In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. " Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. " Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated herein by reference in its entirety.

[0318] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water (EMBLI-EBI) using default parameters share at least a certain minimal percentage of sequence identity (as described herein).

[0319] A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. When sequences have a substantially different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the EMBOSS needle and EMBOSS water default parameters are used, with a gap open penalty = 10 (nucleotide sequences) / 10 (proteins) and gap extension penalty = 0.5 (nucleotide sequences) / 0.5 (proteins). This method (EMBOSS, Needleman, Matrix: EBLOSUM62, Gap penalty: 10, Extend penalty 0.5) has been used in example 4 to assess the identity and similarity percentages of Protein 2 (SEQ ID NO:2), Protein 3 (SEQ ID NO:3) or Protein 4 (SEQ ID NO:4) to Protein Q (SEQ ID NO:1).

[0320] For nucleotide sequences the default scoring matrix used is DNAfull and for amino acid sequences the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).

[0321] In a preferred embodiment, the method used in example 4 to assess the identity and similarity to protein Q is used (EMBOSS, Needleman, Matrix: EBLOSUM62, Gap penalty: 10, Extend penalty 0.5). In this method, the alignment is carried out over the entire length (full length) of the sequence.

[0322] Sequence identity is a key metric for assessing homology between proteins. Generally, >30% identity strongly suggests evolutionary relatedness and often correlates with structural similarity. The range of 20-30% is considered the “twilight zone”, where homology cannot be confidently inferred. Below 20% identity, similarity is typically regarded as random or coincidental, with no reliable functional or structural correlation (Rost, B. (1999), DOI: 10.1093 / protein / 12.2.85. Chung, S. Y., et al (1996), Structure, DOI: 10.1016 / S0969-2126(96)00119-0 and Bartuzi, D., et al (2023) Title: Illuminating the “Twilight Zone”: Advances in Difficult Protein Modeling, Book Series: Methods in Molecular Biology, DOI: 10.1007 / 978-1 -0716-2974-1_2). It follows that the skilled person knows that identity lower than 20% may be considered as random or residual. Hence, identity below 20% may be considered to be a coincidence. Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleotide and amino acid sequences of some embodiments of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences having a certain identity with nucleic acid molecules of the disclosure. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences having a certain identity or similarity with polypeptides of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402, incorporated herein by reference. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov / .

[0323] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. " Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

[0324] Acidic Residues Asp (D) and Glu (E)

[0325] Basic Residues Lys (K), Arg (R), and His (H)

[0326] Ser (S), Thr (T), Asn (N), and

[0327] Hydrophilic Uncharged Residues

[0328] Gin (Q)

[0329] Gly (G), Ala (A), Vai (V), Leu (L),

[0330] Aliphatic Uncharged Residues

[0331] and lie (I)

[0332] Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P)

[0333] Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

[0334]

[0335] Alternative conservative amino acid residue substitution classes:

[0336] 1 A S T

[0337] 2 D E

[0338] 3 N Q

[0339] 4 R K

[0340]

[0341] 5 I L M

[0342] 6 F Y W

[0343]

[0344] Alternative physical and functional classifications of amino acid residues:

[0345] Alcohol group-containing residues S and T

[0346] Aliphatic residues I, L, V, and M

[0347] Cycloalkenyl-associated residues F, H, W, and Y

[0348] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and

[0349] Y

[0350] Negatively charged residues D and E

[0351] Polar residues C, D, E, H, K, N, Q, R, S, and T

[0352] Positively charged residues H, K, and R

[0353] Small residues A, C, D, G, N, P, S, T, and V

[0354] Very small residues A, G, and S

[0355] Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible residues Q, T, K, S, G, P, D, E, and R

[0356]

[0357] For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to lie or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyrto Trp or Phe; and, Vai to He or Leu.

[0358] Antigen

[0359] An “antigen” is a molecule or molecular structure that an antigen receptor or an antigen-binding protein can recognize (for example, bind to). An antigen can be or can comprise, for example, a peptide, a polypeptide, a carbohydrate, a chemical, a moiety, a non-peptide antigen, a phosphoantigen, a tumour-associated antigen, a neoantigen, a tumour microenvironment antigen, a microbial antigen, a viral antigen, a bacterial antigen, an autoantigen, a glycan-based antigen, a peptide-based antigen, a lipid-based antigen, or any combination thereof. In some cases, an antigen adopts a certain conformation in order to bind to an antigen receptor or antigen-binding protein, and / or to induce an immune response, e.g., adopts a conformation in response to the presence or absence of one or more metabolites. Antigen can refer to a whole target molecule, a whole complex, a or a fragment of a target molecule or complex that binds to an antigen receptor or an antigenbinding protein.

[0360] Gene or coding sequence

[0361] “Gene” or “coding sequence” or “nucleic acid” or “nucleic acid molecule” refers to a DNA or RNA region (the transcribed region) which “encodes” a particular polypeptide such as a polypeptide comprised in protein Q or fragments thereof. A coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide when placed under the control of an appropriate regulatory region, such as a promoter. A gene may optionally comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, an intron, a coding sequence and a 3’ nontranslated sequence, comprising a polyadenylation site or a signal sequence. A chimeric or recombinant gene (such as the ones described herein, or such as Protein Q) is a gene not normally found in nature, such as a gene in which for example the promoter is not associated in nature with part or all of the transcribed DNA region, or genes comprising nucleotide sequences encoding domains from multiple polypeptides. “Expression of a gene” refers to the process wherein a gene is transcribed into an RNA and / or translated into an active protein.

[0362] Codon optimization

[0363] “Codon optimization”, as used herein, refers to the processes employed to modify an existing coding sequence, or to design a coding sequence, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host cell. For example, to suit the codon preference of mammalian, insect, plant, or microbial cells, preferably human cells. Codon optimization also eliminates elements that potentially impact negatively RNA stability and / or translation (e. g. termination sequences, TATA boxes, splice sites, ribosomal entry sites, repetitive and / or GC rich sequences and RNA secondary structures or instability motifs). Codon optimization may be done according to standard methods available to skilled person.

[0364] Promoter

[0365] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes (or coding sequence), located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated depending on physiological or developmental conditions. A "tissue specific" promoter is preferentially active in specific types of differentiated cells / tissues.

[0366] Operably linked

[0367] “Operably linked” is defined herein as a configuration in which a control sequence such as a promoter sequence or regulating sequence is appropriately placed at a position relative to the nucleotide sequence of interest. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being “under the control of’ the promoter.

[0368] Nucleic acid construct

[0369] An ‘’expression construct” or "nucleic acid construct” comprises a nucleic acid molecule, such as the ones described herein, which may be expressed in a host cell. Expression constructs disclosed herein could be prepared using recombinant techniques which result in nucleotide sequences being expressed in a suitable cell, e.g., cultured cells or cells of a multicellular organism, such as described in Ausubel et al., and in Sambrook and Green (supra).

[0370] Typically, a nucleic acid molecule or construct is used in a vector. A vector may alternatively be called an expression vector. The phrase "expression vector" generally refers to a nucleotide sequence that is capable of effecting expression of a gene in a host compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. An additional factor necessary or helpful in effecting expression can also be used as described herein. An expression vector may optionally be suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or may be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines.

[0371] A nucleic acid molecule, construct, or vector, prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term “operably linked” has already been defined herein. DNA signal sequences may be included. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide. Generally, DNA sequences that are operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame. However, enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis. The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g., Sambrook and Green, supra). Additional examples have been provided earlier herein.

[0372] A transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognised by the host. The selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Green, supra). For example, an expression vector which includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment can be employed. In most cases, the replication system is only functional in the cell that is used to make the vector (e.g., bacterial cell as E. coli). Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are for example described in Sambrook and Green (supra). For example, suitable expression vectors can be expressed in, yeast, e.g. S. cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli. A cell may thus be a prokaryotic or eukaryotic host cell. A cell may be a cell that is suitable for culture in liquid or on solid media. In some cases, a host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal. Commercial kits comprising cells for vector expression are available, for example the LV-Max system from Thermo Fisher Scientific (Waltham, MA, USA).

[0373] A vector as described herein may be selected from any genetic element known in the art which can facilitate transfer of nucleic acids between cells, such as, but not limited to, plasmids, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, and the like. A vector may also be a chemical vector, such as a lipid complex or naked DNA. " Naked DNA” or "naked nucleic acid” refers to a nucleic acid molecule that is not contained in encapsulating means that facilitates delivery of a nucleic acid into the cytoplasm of a target host cell. Naked DNA may be circular or linear (linearized DNA sequence). Optionally, a naked nucleic acid can be associated with standard means used in the art for facilitating its delivery of the nucleic acid to the target host cell, for example to facilitate the transport of the nucleic acid through the cell membrane.

[0374] Engineered cells

[0375] The term "engineered cells" refers herein to cells having been engineered, e.g., by the introduction of an exogenous nucleic acid sequence as defined herein. Such a cell has been genetically modified for example by the introduction of for example one or more mutations, insertions and / or deletions in an endogenous gene and / or insertion of a nucleic acid construct in the genome. The modification may have been introduced using recombinant DNA technology. An engineered cell may refer to a cell in isolation or in culture. Engineered cells may be "transduced cells" wherein the cells have been infected with e.g. a modified virus, for example, a retrovirus may be used but other suitable viruses may also be contemplated such as lentiviruses. Non-viral methods may also be used, such as transfections. Engineered cells may thus also be "stably transfected cells" or "transiently transfected cells". Transfection refers to non-viral methods to transfer DNA (or RNA) to cells such that a gene is expressed. Transfection methods are widely known in the art, such as calcium phosphate transfection, PEG transfection, and liposomal or lipoplex transfection of nucleic acids. Such a transfection may be transient, but may also be a stable transfection wherein cells can be selected that have the gene construct integrated in their genome. In some cases, genetic engineering systems such as CRISPR or Argonaute may be utilized to design engineered cells that express a polypeptide described herein.

[0376] A variety of enzymes can catalyze insertion of foreign DNA into a host genome. Non-limiting examples of gene editing tools and techniques include CRISPR, TALEN, zinc finger nuclease (ZFN), meganuclease, Mega-TAL, and transposon-based systems.

[0377] A CRISPR system can be utilized to facilitate insertion of a polynucleotide sequence encoding a membrane protein or a component thereof into a cell genome. For example, a CRISPR system can introduce a double stranded break at a target site in a genome. There are at least five types of CRISPR systems which all incorporate RNAs and CRISPR-associated proteins (Cas). Types I, III, and IV assemble a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA. Types I and III both require pre-crRNA processing prior to assembling the processed crRNA into the multi-Cas protein complex. Types II and V CRISPR systems comprise a single Cas protein complexed with at least one guiding RNA. Genome editing tools as described above may also be used to introduce a genomic modification of the invention.

[0378] In some embodiments, an “engineered cell” has been transformed, modified or transduced to comprise a heterologous or exogenous nucleic acid molecule such as the one encoding protein Q as defined herein. In the application, the wording “engineered cell” may be replaced by “modified cell” or “transformed cell” or “transduced cell”. In an embodiment, said cell expresses a protein encoded by said nucleic acid molecule.

[0379] In a further aspect, there is provided a method or process for purifying a protein comprised in a load mixture as earlier defined herein, using a chromatography column as earlier defined herein. Depending on the type of proteins and column chromatography envisaged, the skilled person would know which formulations are the most suitable.

[0380] General information

[0381] It is not considered necessary to extend this description in order that someone skilled in the art can understand the scope of the invention and the advantages that it confers. The materials, form, size and disposition of the elements are susceptible to change, provided it does not suppose a change in the essence of the invention. The terms in which this disclosure has been written should always be considered as broad in nature and not limiting. Unless stated otherwise, all technical and scientific terms used herein have the same meaning as customarily and ordinarily understood by a person of ordinary skill in the art to which this invention belongs, and read in view of this disclosure.

[0382] Each aspect and / or embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0383] As used herein, the term “loading buffer” or “equilibrium buffer” refers to the buffer containing the salt or salts that is combined with the protein preparation for the purpose of loading it onto an IEC column. This buffer is utilised to equilibrate the column prior to loading and to wash the column subsequent to protein loading.

[0384] As used herein, the term “mobile phase” may be interpreted as “buffer”, “buffer solution”, “eluant”, “eluent”, “elution buffer”, “solvent” unless otherwise indicated. Eluent refers to the solvent or mixture of solvents used in a chromatographic separation process. It is typically a binary mixture with different boiling points, and its composition can be controlled and adjusted to optimize the separation.

[0385] As used herein, the term “eluate” may be interpreted as “purified sample”, “purified protein”, “purified biomolecule”, unless otherwise indicated. It may contain the separated components (the analytes) that the eluent retrieved from the stationary phase, is essentially the output solution from a chromatography operation.

[0386] As used herein, the term “ion exchange chromatography” is synonymous with “”IEC” or “IEX” or “IEX chromatography” unless otherwise indicated.

[0387] As used herein, the term “resin(s)” is synonymous with “resin bead(s)” or “bead(s)” unless otherwise indicated. As used herein, the term “stationary phase” may be interpreted as “resin” or “adsorbent” unless otherwise indicated.

[0388] As used herein, the term “column” is synonymous with “chromatography column” unless otherwise indicated.

[0389] As used herein, the term “analyte” refers to a specific substance or chemical constituent that is being analyzed or measured in the load mixture. In this invention, the analyte is the protein of interest being separated or purified. The analytes are typically comprised in the eluate. In a preferred embodiment of the invention, the analyte comprises a protein Q. As used herein, the term “laboratory scale” or “lab scale” refers to a scale based on the volume of the culture broth, such as fermentation broth, up to 10 L. As used herein, the term “industrial scale” or “plant scale” refers to a scale based on the volume of the culture broth, such as fermentation broth, higher than 10 L. The preferred embodiments of the lab scale is configured to be extrapolable to the industrial scale.

[0390] As used herein, the term “wide column” may refer to the column comprising a larger diameter used in the lab scale (such as 2.6 cm) and / or industrial scale (such as 30 cm). As used herein the term “narrow column” may refer to a column comprising a smaller diameter used in the lab scale (such as I.6 cm) and / or industrial scale (such as 20 cm). The preferred embodiments of the lab scale is configured to be extrapolable to the industrial scale.

[0391] In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The verb “to comprise” is synonymous with the verb “to have” unless otherwise indicated. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that an oligonucleotide or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0392] The word “about” when used in association with an integer (about 10) preferably means that the value may be the given value of 10 more or less 1 of the value: about 10 preferably means from 9 to I I. The word “about” when used in association with a numerical value (about 10.6) preferably means that the value may be the given value of 10.6 more or less 0.1 of the value 10.6: about 10.6 preferably means from 10.5 to 10.7. The word “about” when used in association with a numerical value (about 10.65) preferably means that the value may be the given value of 10.65 more or less 0.01 of the value 10.65: about 10.65 preferably means from 10.64 to 10.66. Notwithstanding, the skilled person knows that numerical values relating to measurements are subject to measurement errors which place limits on their accuracy. For this reason, the general convention in the scientific and technical literature is applied: the last decimal place of a numerical value indicates its degree of accuracy. Where no other error margins are given, the maximum margin is ascertained by applying the rounding-off convention to the last decimal place, e.g. for a measurement of 3.5 cm, the error margin is 3.45-3.54. When interpreting ranges of values in patent specifications, the skilled person proceeds on the same basis. It is understood that decimals may be inconsistent in the ranges and the error margins thereof might be safely assumed by the skilled person. Examples

[0393] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0394] Unless specified, reagents employed in the examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art. The examples illustrate various aspects of the invention and practice of the methods of the invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0395] It is understood that the examples herein are performed in IEC columns. However, a similar effect and / or improvement could be also expected in other chromatography columns as well, such as an affinity chromatography.

[0396]

[0397] 1: at the scale

[0398] Abbreviation

[0399]

[0400] Wording Definition

[0401]

[0402] Al Active ingredient

[0403] BCA Bicinchoninic acid protein

[0404] CV Column volume

[0405] CW Column wash

[0406] DSP Downstream processing

[0407] FT Flow through

[0408] IEC Ion Exchange Chromatography

[0409] NA Not available

[0410] NG New geometry

[0411] PQ / ProtQ Protein Q

[0412] SD Standard deviation

[0413] SSP Small scale process

[0414] TP Total Protein

[0415] USP Upstream processing

[0416] Introduction

[0417] This work focuses on the Ion Exchange Chromatography (IEC) that purifies Protein Q (SEQ ID NO:1) from the eluate of a previous purification step using a strong cationic quaternary amine. The aim of the study is to improve the performance and robustness of this chromatographic step by evaluating different process conditions. To achive that, several I EC trials were performed at laboratory scale modifying the following parameters:

[0418] Elution time: defined as the duration of the elution phase. This parameter can be modified by speeding / lowering the flow rate or by including a pause to increase the contact time of the elution buffer with the target protein attached to the column.

[0419] Protein load density: defined as total protein loaded onto the I EC per column section (TP g / cm2). This parameter can be modified by means of increasing / decreasing the column diameter or by means of increasing / decreasing the amount of protein at which the product is loaded onto the chromatography column.

[0420] Load ratio: Amount of total protein loaded per volume of resin (TP g / L resin). This parameter can be modified by means of increasing / decreasing the amount of TP loaded onto the IEC or by means of increasing / decreasing the column volume. Therefore, when keeping the same column, this parameter is directly connected to protein load density.

[0421] Protein concentration: is the concentration of protein in mg / mL loaded onto the IEC column. This variable can be modified by diluting the protein that comes from the previous purification step with more or less buffer volume.

[0422] Applying modifications of the mentioned parameters over the reference process corroborated the relevance of these operational variables over the IEC performance. The final aim is to find the best combination of these parameters to optimize the IEC in terms of yield and robustness, allowing to transfer it to the industrial scale.

[0423] Equipment, Materials and solutions

[0424] Equipment

[0425] The following pieces of equipment were used to perform the laboratory trials:

[0426] Table 1.1. List of equipment used.

[0427] Equipment

[0428] Freezer

[0429] Refrigerator

[0430] FPLC Chromatograph

[0431] Stirring plate

[0432] Precision scale

[0433] pHmeter

[0434] Centrifpge

[0435] Ultrapure Water Purification System

[0436] Micropipette P1000

[0437] Micropipette P200

[0438]

[0439] Micropipette multichannel P300 Equipment

[0440] Micropipette P20

[0441]

[0442] Micropipette P50

[0443] Materials

[0444] The following table gathers the materials employed to perform the laboratory trials.

[0445] Table 1.2. List of materials used.

[0446] Material Model

[0447] Filter PES membrane

[0448] IEC resin Q Sepharose FF

[0449] IEC Chromatography column Long / Narrow Column: 14.3 cm bed height and 1.6 cm diameter

[0450]

[0451] IEC Chromatography column Short / Wide Column: 5.4 cm bed height and 2.6 cm diameter Solutions

[0452] The composition of the solutions used in the I EC process is described in the following tables:

[0453] Urea 8 M solution

[0454] Table 1.3. Urea 8 M solution composition.

[0455] Material Cone. (g / L)

[0456] Urea, CH4N2O 480.48

[0457]

[0458] Water, H2O c.s.p. liter

[0459] IEC load solution or pH adjustment solution (pH 10)

[0460] Buffer is prepared weighing the following reagents:

[0461] Table 1.4. pH adjustment solution (pH 10) composition.

[0462] Material Cone. (g / L)

[0463] Monosodium phosphate dihydrate,

[0464] NaH2PO4.2H2O 7.41

[0465] Urea 8M solution 370.0

[0466] Water, H2O 614

[0467] Sodium hydroxide, NaOH 4M c.s.p = pH 10

[0468]

[0469] Triton X-100, Ci4H2i(C2H4O)nOH 9.51

[0470] IEC Equilibration and Wash solution

[0471] Buffer is prepared according to the following table:

[0472] Table 1.5. IEC Equilibration and Wash solution composition.

[0473] Material Cone. (g / L)

[0474] Monosodium phosphate dihydrate,

[0475] NaH2PO4.2H2O 7.31

[0476] Urea 8M solution 469

[0477]

[0478] Sodium Chloride, NaCI 2.74 Water, H2O 512

[0479] Sodium hydroxide, NaOH 4M c.s.p = pH 9

[0480]

[0481] Triton X-100, Ci4H2i(C2H4O)nOH 9.37

[0482] IEC Elution solution

[0483] Buffer is used to elute Protein Q from IEC column, and it is prepared according to the following table:

[0484] Table 1.6. IEC elution solution composition.

[0485] Material Cone. (g / L)

[0486] Monosodium phosphate dihydrate,

[0487] NaH2PO4.2H2O 6.7

[0488] Urea 8M solution 960

[0489] Sodium Chloride, NaCI 25.1

[0490] Sodium hydroxide, NaOH 4M c.s.p = pH 8

[0491]

[0492] Triton X-100, Ci4H2i(C2H4O)nOH 9.19

[0493] Small scale process description

[0494] The IEC step has been scaled down from the current existing industrial process. To do that, the dimensions and the resin of the current industrial column were taken as a reference to design the laboratory trials.

[0495] Conditioned eluate from the previous purification step is used as starting material for the laboratory trials. It means, the product to be loaded on to the IEC has been adjusted to pH = 10, urea = 4 M and TP < 0.5 g / L. The required mg of total protein to be loaded onto the IEC for each run are calculated to start from the right product volume. This is made based on:

[0496] ■ Load density: Amount of TP to be loaded on to the column per column section. The reference according to the current example is < 127 mg / cm2.

[0497] ■ Load ratio: Amount of TP to be loaded on to the column per mL of resin. The reference according to the current example is < 8.9 mg / mL.

[0498] ■ Load concentration: TP concentration of the IEC load. The reference according to the current example is < 0.5 g / L.

[0499] The calculated necessary volume per run is tempered and homogenized in gentle agitation. If needed, pH is adjusted to 10.0 ± 0.2. Then, the product is filtered before being loaded onto the column. IEC chromatography is run in achromatography system operating at a flow rate range 15 -60 L / h and the method consists of the following stages:

[0500] Equilibration with 4 CV IEC Equilibrium and Washing Sol., pH 9.0

[0501] Product load, CV variable depending on product concentration.

[0502] Column wash with 3 CV Dilution and equilibrium buffer Elution with 3.14 CV IEC Elution buffer, pH 8.0

[0503] The elution flow rate in the current ISP is 40 L / h (127 cm / h), which results in an elution time of 21 min. Elution flow rates are adjusted in the method depending on the conditions to be tested in each run. The elution peak is gathered according to the column volumes stated in the current process: 2.34 CV are collected after running 0.8 CV of elution phase.

[0504] Analytical methods

[0505] Total protein is quantified by BCA. The method is described in Example 3.

[0506] Experimental design

[0507] This report gathers the results of several trials looking at the improvement of the IEC step with respect to the current Protein Q manufacturing process. For this purpose, the following parameters are evaluated:

[0508] • Elution time: slower elution linear velocities (48, 22 and 15 cm / h vs. the reference 127 cm / h) are studied with the purpose of increasing product contact time with the elution buffer from 21 min. up to 180 min., thus promoting protein solubilization and increasing IEC yield by eluting the remanent PQ in the column. Adding a pause during the elution: in this scenario we had to adapt the slow flow rates to the ISP capabilities, where the minimum flow rate of the chromatograph was 15 L / h, equivalent to 48 cm / h for a narrow column or 21 cm / h for a wide column. In the middle of the elution phase, the flow was stopped for two hours, allowing the elution buffer to be in contact with PQ emulating 15 cm / h flow contact time of 180 min. In this document, the best IEC load conditions are also evaluated considering the industrial process capacity:

[0509] • IEC load density (mg TP / cm2): TP loaded onto the IEC combining the different column geometries and load ratios TP g / L resin. Range tested from 31 to 247 mg TP / cm2. This parameter is modified by changing the column section and / or the amount of protein loaded onto the IEC. To assess that, three different column geometries are tested: a wider IEC column 2NG (XK26 / 20 with 2.6 cm diameter and 5.4 cm height) is compared to the reference column 1 NG (HiScale 16 / 20 with 1.6 cm diameter and 14.1 cm height) aiming to improve the contact surface and the solubility of the protein at the head of the column. A third column geometry was also tested 3NG (H=9.2 cm, D=2.6 cm) keeping the wide diameter and increasing the height, more in line with the needs of the industrial scale process. See Table 7 for column details.

[0510] • Ratio TP g / L resin: three different protein loads onto the IEC column have been tested, contemplating possible scenarios in the industrial process, depending on the previous purification step yield: low = 6.0 g TP / L resin, current = 8.9 g TP / L resin and high = 11.1 g TP / L resin. • IEC load concentration: three different IEC load concentrations have been tested, contemplating possibles scenarios in the industrial process, depending on the previous purification step yield or diltuion applied: low = 0.25 mg / mL, current = 0.40 mg / mL and high = 0.50 mg / mL.

[0511] In order to test all the variables in a combined way, different set of runs were designed combining 0-Ocolumn geometries, elution regimes and loading conditions.

[0512] The main parameters for the three evaluated column geometries are gathered in the next table, compared to the current industrial IEC column (narrow). Geometry 1NG represents the reference process at laboratory scale. It was downscaled keeping the industiral IEC height and reducing the column diameter to process the laboratory intended volumes. Geometry 2NG keeps 1NG resin volume (CV) but increases the section with the intention to reduce load density. Geometry 3NG keeps 2NG column section but increases the height from 5.4 to 9.2 cm in order to fulfill with industrial packing column limitations. Thus, 3NG increases slightly the resin volume, allowing to reduce TP load ratios.

[0513] Table 1.7. Summary of columns dimensions.

[0514] Industrial 1NG 2NG 3NG Column

[0515] BPG200 / 500 HiScale 16 / 20 XK26 / 20 HiScale 26 / 20 CV (mL) 4500 29 29 49 Height (cm) 14.3 14.3 5.4 9.2 Diameter (cm) 20 1.6 2.6 2.6

[0516]

[0517] If the reference narrow column (industrial BPG200 / 500 or laboratory 1NG) operates at a continuous flow rate of 15 cm / h instead of the reference flow rate of 127 cm / h, it means increasing the elution time from 20 min to 180 min. In order to equal the elution time, the flow rates have been adapted to the minimum possible industrial flow rate (15 L / h or 250 mL / min) adding a pause in the middle of the eluate collection of 120 min. It means running the narrow column (BPG200 / 500 or 1NG) at 48 cm / h or the wide column (2NG or 3NG) at 21 cm / h.

[0518] Two set of experiments were carried out. The first one was more focused on studying the effect of the elution time by means of varying the linear flow rates and or adding a pause in the elution phase for the three column geometries. The second set was more focused on studying the IEC loading conditions. Tables 1.8 and 1.9 summarize the IEC trials performed in the two set of experiments and the varied conditions. These runs are grouped in the results section to find out how these variables or their combination may affect the chromatography performance. Table 1.8. Summary of the first set of IEC runs conditions.

[0519] IEC Colum Geometry IEC Elution IEC Load Conditions

[0520] Run ID Geometry Diameter Height Ratio Surface Linear Flow Rate Elution Time TP Ratio TP Qty. TP Density TP Cone.

[0521] ID (cm) (cm) D / H (cm2) (cm / h) (min) (mg / mL resin) (mg) (mg / cm2) (mg / mL) 1NG04 1NG 1.6 14.3 0.11 2.0 22 123 8.9 258 128.4 0.4 1NG05 1NG 1.6 14.3 0.11 2.0 127 21 8.9 258 128.4 0.4 1NG07 1NG 1.6 14.3 0.11 2.0 15 180 8.9 258 128.4 0.4 1NG08 1NG 1.6 14.3 0.11 2.0 15 180 8.9 258 128.4 0.4 1NG09 1NG 1.6 14.3 0.11 2.0 22 123 8.9 258 128.4 0.4 1NG12 1NG 1.6 14.3 0.11 2.0 48 + 2h stop 176 8.9 258 128.4 0.4 1NG13 1NG 1.6 14.3 0.11 2.0 48 56 8.9 258 128.4 0.4 1NG14 1NG 1.6 14.3 0.11 2.0 48 + 2h stop 176 8.9 258 128.4 0.4 1NG15 1NG 1.6 14.3 0.11 2.0 48 + 2h stop 176 8.9 258 128.4 0.4 2NG03 2NG 2.6 5.4 0.48 5.3 22 46 8.9 258 48.6 0.4 2NG04 2NG 2.6 5.4 0.48 5.3 127 8 8.9 258 48.6 0.4 2NG07 2NG 2.6 5.4 0.48 5.3 21 + 2h stop 168 8.9 319 60.1 0.4 3NG01 3NG 2.6 9.2 0.28 5.3 21 + 2h stop 202 8.9 436 82.1 0.4

[0522]

[0523] Table 1.9. Summary of the second set of IEC runs conditions.

[0524] IEC Colum Geometry IEC Elution IEC Load Conditions

[0525] Run ID Geometry Diameter Height Ratio Surface Linear Flow Rate Elution Time TP Ratio TP Qty. TP Density TP Cone.

[0526] ID (cm) (cm) D / H (cm2) (cm / h) (min) (mg / mL resin) (mg) (mg / cm2) (mg / mL) IEC 01 1NG 1.6 14.3 0.11 2.0 127 21 8.9 266 132.1 0.4 IEC 02 1NG 1.6 14.3 0.11 2.0 127 21 8.9 252 125.5 0.4 IEC 05 2NG 2.6 5.4 0.48 5.3 127 8 8.9 256 48.1 0.4 IEC 03 1NG 1.6 14.3 0.11 2.0 48 + 2h stop 176 8.9 246 122.2 0.4 IEC 04 2NG 2.6 5.4 0.48 5.3 21 + 2h stop 168 8.9 256 48.1 0.4 IEC 06 1NG 1.6 14.3 0.11 2.0 48 + 2h stop 176 8.9 250 124.3 0.25 IEC 07 2NG 2.6 5.4 0.48 5.3 21 + 2h stop 168 8.9 250 47.1 0.25 IEC 08 1NG 1.6 14.3 0.11 2.0 48 + 2h stop 176 8.9 496 246.7 0.5 IEC 09 2NG 2.6 5.4 0.48 5.3 21 + 2h stop 168 8.9 493 92.9 0.5 IEC 10 1NG 1.6 14.3 0.11 2.0 48 + 2h stop 176 6.0 173 85.9 0.4 IEC 11 2NG 2.6 5.4 0.48 5.3 21 + 2h stop 168 6.0 164 30.9 0.4 IEC 12 1NG 1.6 14.3 0.11 2.0 48 + 2h stop 176 11.1 311 154.6 0.4 IEC 13 2NG 2.6 5.4 0.48 5.3 21 + 2h stop 168 11.1 319 60.0 0.4 IEC 14 3NG 2.6 9.2 0.28 5.3 21 + 2h stop 202 6.0 293 55.2 0.4 IEC 15 3NG 2.6 9.2 0.28 5.3 21 + 2h stop 202 6.0 293 55.2 0.4 IEC 16 3NG 2.6 9.2 0.28 5.3 21 + 2h stop 202 6.0 293 55.2 0.4

[0527]

[0528] Results

[0529] For all the runs, the total protein was quantified by BCA, so that protein recoveries could be compared. The IEC step yield was calculated as g TP in IEC eluate / g TP in IEC load.

[0530] Next, the results are evaluated grouped, depending on the variable that is being studied:

[0531] Elution time

[0532] The first set of experiments evaluated different elution times for the three column geometries that range from 8 min. up to 202 min. These times are achieved by varying the linear flow rates and / or adding a 2 hour pause in the middle of the elution phase. In this battery of runs, the load ratio (TP g / L resin) and load concentration (TP g / L) are fixed as in the current ISP. Table 1.10 gathers the runs conditions and yields achieved.

[0533] Table 1.10. First set of IEC runs performed to evaluate the impact of elution time in the protein recovery for three column geometries. All these runs have same TP load ratio and TP load concentration.

[0534] IEC Colum IEC Elution Load conditions Geometr Geometry Yield Run Linear Flow Rate Time TP Ratio1TP Cone.

[0535] y (cm)

[0536] ID ID Heigh

[0537] Diameter (cm / h) (min) (mg / mL) (mg / mL) (%) t

[0538] 1NG0

[0539] 1NG 1.6 14.3 22 123 8.9 0.4 74% 4

[0540] 1NG0 1NG

[0541] 1.6 14.3 127 21 8.9 0.4 61% 5

[0542] 1NG0 1NG

[0543] 1.6 14.3 15 180 8.9 0.4 72% 7

[0544] 1NG0 1NG

[0545] 1.6 14.3 15 180 8.9 0.4 79% 8

[0546] 1NG0 1NG

[0547] 1.6 14.3 22 123 8.9 0.4 73% 9

[0548] 1NG1 1NG

[0549] 1.6 14.3 48 + 2h stop 176 8.9 0.4 85% 2

[0550] 1NG1 1NG

[0551] 1.6 14.3 48 56 8.9 0.4 67% 3

[0552] 1NG1 1NG

[0553] 1.6 14.3 48 + 2h stop 176 8.9 0.4 72% 5

[0554] 1NG1 1NG

[0555] 1.6 14.3 127 21 8.9 0.4 62% 6

[0556] 2NG0 2NG

[0557] 2.6 5.4 22 46 8.9 0.4 73% 3

[0558]

[0559] 2NG0 2NG

[0560] 2.6 5.4 127 8 8.9 0.4 75% 4

[0561] 2NG0 2NG

[0562] 2.6 5.4 21 + 2h stop 168 8.9 0.4 86% 7

[0563] 3NG0 3NG

[0564] 2.6 9.2 21 + 2h stop 202 8.9 0.4 86% 1

[0565]

[0566] 1Amount of total protein loaded per mL of resin.

[0567] Table 1.11 intends to summarize the results from the first set of experiments grouping them by elution time and column geometry.

[0568] Table 1.11. TP average IEC yields using the data obtained in runs from Table 1.9 (first set of runs).

[0569] Elution Linear Flow

[0570] Column geometry Elution time (min) Mean yield n Rate

[0571] 21 127 cm / h 62% 2 1NG: HiScale 16 / 20 123 22 cm / h 74 % 2 (H=14.3 cm, D=1.6 cm) 180 15 cm / h 76% 2

[0572] 176 48 + 2h stop 79% 2 8 127 cm / h 75% 1 2NG: XK26 / 20

[0573] 46 22 cm / h 73% 1 (H=5.4 cm, D=2.6cm)

[0574] 168 21 + 2h stop 86% 1 3NG: XK26 / 20

[0575] 202 21 + 2h stop 86% 1 (H=9.2 cm, D=2.6 cm)

[0576] ISP (BP200 / 500) 21 127 cm / h* 45% 48

[0577]

[0578] *Data obtained from industrial batches manufactured in 2020 and 2023.

[0579] A clear trend is observed for both column geometries at which different elution times have been applied: the longer the elution time the better IEC recoveries. In addition, it can be noted that, on average, for the wider geometries (2NG and 3NG) yields are slightly higher than for the narrow ones (1NG) applying equivalent linear flow rates.

[0580] It is confirmed eluting at lower flow rates than the current process (127 cm / h) is clearly beneficial to increase the IEC yield: the amount of Protein Q recovered increases by 17 % in the narrow column and by 11 % in the wide column, with respect to the reference laboratory runs. This increment is even larger when comparing to the current ISP yield average (45%). Focusing on the average IEC yields applying slow flow with 2 hours stop elution vs. continuous slow flow rates, a slight increase is observed too for both column geometries.

[0581] Comparable runs results are depicted together in Figure 1, to visualize the strong direct correlation between elution time and IEC yield. The elution time shows to have a huge impact on protein recovery. Next, a second set of experiments was conducted applying the reference elution conditions 127 cm / h, which means 20 min. for 1NG and 8 min. for 2NG geometry and the best found conditions in the first set of experiments: around 168 -202 min. of elution time achieved by means of applying the slowest flowrate at industrial scale plus a 2 hour pause. The aim of these second set was to check the reference laboratory runs results and the increment provided by the increment of elution time. Table 1.12 summarized the trials conditions and results obtained.

[0582] Table 1.12. Second set of IEC runs performed to evaluate the impact of elution time in the protein recovery for three column geometries. All these runs have same protein load concentration.

[0583] IEC Geometry IEC Elution Load conditions

[0584] Run Yield Geometry Diameter Height Linear Flow Rate Time TP Ratio TP Cone.

[0585] ID ID (cm) (cm) (cm / h) (min) (mg / mL) (mg / mL) (%) IEC 01 1NG 1.6 14.3 127 21 8.9 0.4 36% IEC 02 1NG 1.6 14.3 127 21 8.9 0.4 29% IEC 05 1NG 2.6 5.4 127 8 8.9 0.4 41% IEC 03 1NG 1.6 14.3 48 + 2h stop 176 8.9 0.4 63% IEC 04 2NG 2.6 5.4 21 + 2h stop 168 8.9 0.4 81% IEC 14 3NG 2.6 9.2 21 + 2h stop 202 6.0 0.4 85% IEC 15 3NG 2.6 9.2 21 + 2h stop 202 6.0 0.4 85% IEC 16 3NG 2.6 9.2 21 + 2h stop 202 6.0 0.4 72%

[0586]

[0587] Table 1.13 intends to summarize the results from the first set of experiments grouping them by elution time and column geometry.

[0588] Table 1.13. TP average IEC yields using the data obtained in runs from Table 1.12 (second set of runs).

[0589] Elution Linear Mean Column geometry Elution time (min) n Flow rate yield

[0590] 1NG: HiScale 16 / 20 21 127 cm / h 33% 2 (H=14.3 cm, D=1.6 cm) 176 48 + 2h stop 63% 1 2NG: XK26 / 20 8 127 cm / h 41% 1 (H=5.4 cm, D=2.6cm) 168 21 + 2h stop 81% 1 3NG: XK26 / 20

[0591] 202 21 + 2h stop 81% 3 (H=9.2 cm, D=2.6 cm)

[0592]

[0593] This new battery of runs confirms the significant increment in yields when increasing the elution time to around 168 - 202 min. The observed recovery increase is 30 % for the narrow geometry and 40 % for the wide geometry. The difference in protein recovery found between the reference lECs (127 cm / h) run in both set of experiments is notable, nevertheless, the yields improvement when increasing the elution time is confirmed, reaching recoveries higher than 80%, this value seems very consistent. Besides, the findings at this second set of experiments show a clearer tendency to increase the yield with a wider column run under comparable flow rates.

[0594] For all the trials evaluated in this section, the best condition was identified running a wider column and eluting during 168 - 202 min using slow flow rate with 2 hours stop. The stop implies employing the same elution time but consuming less elution buffer and requiring less supervision, so it is considered a logistic improvement as well.

[0595] IEC load density (mg TP / cm2)

[0596] IEC load density is dependent on the column geometry and the amount of protein loaded onto the column. For that reason, the impact of this parameter is evaluated considering the runs that are loaded with different protein amounts, and which are performed with the three column geometries, meaning two column sections: narrow (1 NG) and wide (2NG and 3NG). In this case, elution regimes which are considered comparable are applied in all the runs: slow flow rates + 2h stop. Table 1.13 gathers the conditions and results of the IEC runs conducted to assess this variable.

[0597] Table 1.14. Second set of IEC runs performed to evaluate the impact of load density (TP mg / cm2) and load ratio (TP g / L resin) in the protein recovery for three column geometries.

[0598] IEC Geometry IEC Elution Load conditions Run TP Yield Geometry Diameter Height Linear Flow Rate Time TP Ratio TP Cone.

[0599] ID density

[0600] ID (cm) (cm) (cm / h) (min) (mg / cm2) (mg / mL) (mg / mL) (%) IEC

[0601] 1NG 1.6 14.3 48 + 2h stop 176 85.9 6.0 0.4 70% 10

[0602] IEC

[0603] 1NG 1.6 14.3 48 + 2h stop 176 122,2 8.9 0.4 63% 03

[0604] IEC

[0605] 1NG 1.6 14.3 48 + 2h stop 176 154.6 11.1 0.4 49% 12

[0606] IEC

[0607] 2NG 2.6 5.4 21 + 2h stop 168 30,9 6.0 0.4 90% 11

[0608] IEC

[0609] 2NG 2.6 5.4 21 + 2h stop 168 48.1 8.9 0.4 81% 04

[0610] IEC

[0611] 2NG 2.6 5.4 21 + 2h stop 168 60.0 11.1 0.4 69% 13

[0612] IEC

[0613] 3NG 2.6 9.2 21 + 2h stop 202 55.2 6.0 0.4 85% 14

[0614] IEC

[0615] 3NG 2.6 9.2 21 + 2h stop 202 55.2 6.0 0.4 85% 15

[0616] IEC

[0617] 3NG 2.6 9.2 21 + 2h stop 202 55.2 6.0 0.4 72% 16

[0618]

[0619] It is observed that the I EC recoveries improve when decreasing the TP load density. This is translated in the best results for the wide geometries (2NG and 3NG) and when loading lower TP ratios (6.0 mg / mL resin).

[0620] Data gathered in Table 14 is represented in the graph from Figure 3 in order to visualize the strong correlation between I EC recoveries and load density.

[0621] Figure 2 further evidenced the high correlation between IEC yield and load density. The high determination coefficient (R2= 0.82) obtained in the graph demonstrates that a high density of proteins in the top of the column is detrimental for the IEC recovery. It is checked that working with load protein density < 55.2 mg / cm2achieves protein recoveries above 70%.

[0622] IEC load ratio (TP g / L resin)

[0623] Three different IEC load ratios (TP g / L resin) were tested contemplating possibles scenarios in the industrial process: low = 6.0 g / L resin, current = 8.9 g / L resin and high = 11.1 g / L resin. These runs are compared for the wide and narrow geometries, keeping the slow flow rates + 2 hour stop in the elution phase. Since this parameter depends on the amount of TP loaded onto the IEC and to the column geometry, the same IEC trials are used to study the effect of the TP ratio and the load density. Thus, Table 1.13 gathers the results that are of interest to evaluate this parameter. The difference lies on load ratio depends on the column volume (being different 3 NG, with 49 mL, from 1NG and 2NG, with 29 mL) and load density depends on column surface (being equal for 2NG and 3NG but different for 1NG).

[0624] Looking at the results from Table 1.13, it is noted the yields improve when decreasing the loading ratio, being the optimal one 6.0 TP g / L resin. Nonetheless, all runs show recoveries above 60 %, except one, which demonstrate it is feasible to operate in the whole studied range. This allows applying a flexible working range in the ISP, since the total protein to be loaded onto the IEC column may vary depending on the previous purification step yield.

[0625] IEC load TP concentration

[0626] Three different IEC load TP concentrations are tested as well: low = 0.25 mg / mL, current = 0.40 mg / mL and high = 0.50 mg / mL. These concentrations have been chosen contemplating possibles scenarios in the industrial process, since the TP concentration at which the IEC is loaded depends on the previous purification step yield.

[0627] The following table and graph show the effect of loading the IEC with the same amount of proteins but at different concentrations. Only runs with load ratios of 8.9 g / L resin and elution regimes which are considered comparable are selected: slow flow rates + 2h stop. Table 1.15. Second set of I EC runs performed to evaluate the impact of IEC load concentration. Only runs with load ratios of 8,9 g / L resin are considered. Elution flow rates that lead to similar elution time are considered comparable.

[0628] IEC Geometry IEC Elution Load conditions

[0629] Yield Run Geometry Diameter Height Linear Flow Rate Time TP

[0630] ID density TP Ratio TP Cone.

[0631] ID (cm) (cm) (cm / h) (min) (mg / cm2) (mg / mL) (mg / mL) (%) IEC 1NG 1.6 14.3 48 + 2h stop 176 124.3 8.9 0.25 39% 06

[0632] IEC 1NG 1.6 14.3 48 + 2h stop 176 122.2 8.9 0.40 63% 03

[0633] IEC 1NG 1.6 14.3 48 + 2h stop 176 246.7 8.9 0.50 56% 08

[0634] IEC 2NG 2.6 5.4 21 + 2h stop 168 47.1 8.9 0.25 62% 07

[0635] IEC 2NG 2.6 5.4 21 + 2h stop 168 48.1 8.9 0.40 81% 04

[0636] IEC 2NG 2.6 5.4 21 + 2h stop 168 92.9 8.9 0.50 69% 09

[0637]

[0638] Figure 3 shows the IEC yield percentage obtained for different protein concentration load and for each column geometry tested. Figure 3 suggests that the optimal TP load concentration is the reference one, which is the intermediate tested: 0.40 mg / mL. This parameter has been demonstrated to affect differently to both column geometries, the wide (2NG) reached higher IEC recoveries applying the same protein concentration load, for the three tested levels.

[0639] Protein profile evaluation

[0640] Protein profile was evaluated by SDS-PAGE and WB in all the tested runs with the aim to prove that the current product quality keeps unaltered with the variations applied on the IEC process.

[0641] All the analyzed samples of IEC eluate show comparable protein profiles, concluding that none of the operational parameters modified in these trials affected the product quality. CONCLUSIONS

[0642] Numerous laboratory trials were conducted in order to optimize the IEC process step by modifying several operational parameters: the elution time, by means of modifying the elution flow rates and / or adding a pause in the middle of the elution phase; the protein load density, by varying the column geometry and amount of TP loaded, the load ratio TP / L resin; and the protein load concentration. It was found that the elution time was the most relevant parameter in terms of increasing protein recoveries in this chromatographic step. It was proven that incrementing the contact time of the elution buffer with the IEC resin from 21 min., as in the current process, to around 168-202 min. produced yield gains of up to 40 %. This was achieved by means of reducing the linear flow rate during the elution phase from 127 cm / h, used in the ISP, up to 15 cm / h or using intermediate flow rates and adding a 2 hour pause in the middle of the elution phase. The pause was applied as a solution to adapt to the minimum flow rate achieved by the industrial chromatograph, but it turned out to reach as high yields as the slowest continuous flows, in addition to save buffer consumption and supervision in the industrial plant.

[0643] It is also demonstrated the importance of the IEC load conditions: the load density (mg / cm2) and the protein ratio loaded onto the column (TP g / L resin) showed a significant impact in terms of protein recovery. Applying lower protein amounts onto the IEC column showed to be beneficial, which is translated into the lowest load densities and ratios gave the best IEC yields. A TP density between 31 and 55 mg / cm2and a TP ratio of 6.0 g / L resin, seemed to be the optimal loading conditions resulting in yields ranging from 70 to 90 %.

[0644] In terms of loading TP concentration, 0.40 mg / mL seems to be optimal; this is the average concentration at which the IEC is load at the current industrial process. However, concentrations up to 0.5 mg / mL are shown to be acceptable, which allows keeping a certain concentration margin when processing the product from the previous purification step.

[0645] It is noted that within the studied column geometries, applying a wider section, maintaining or slightly increasing the volume of resin, showed an improvement in the IEC yield in all the tested conditions. The protein profiles of the IEC eluate are not affected by the tested process changes. It is, indeed, the expected result considering that the elution is isocratic, and the impurities removed in this chromatography step are separated during the product load and column wash phases, which remain unchanged.

[0646] In conclusion, the best combination of tested variables would be to run a wider column (lab scale 2.6 cm) in order to load the IEC with a TP density between 31 and 55 mg / cm2and a ratio of 6.0 g / L resin. The product load should be at a concentration of 0.4 TP mg / mL and the elution phase should last 168-202 min, which can be achieved by using slow flow rates plus 2h pause in the middle of the elution phase. The combination of these conditions during the IEC chromatography has shown to improve protein recoveries from 45 %, as the ISP average, to 86% and to achieve a more robust process. Example 2: Ionic Exchange chromatography protein Q process optimization at industrial scale LIST OF ABBREVIATIONS

[0647] Abbreviation

[0648]

[0649] Wording Definition

[0650]

[0651] CV Column volume

[0652] CW Column wash

[0653] DSP Downstream processing

[0654] FT Flow through

[0655] IEC Ion-Exchange Chromatography

[0656] ISP Industrial scale process

[0657] NA Not available

[0658] PQ / ProtQ Protein Q

[0659] SD Standard deviation

[0660] TP Total Protein

[0661] USP Upstream processing

[0662] This work focuses on the Ion Exchange Chromatography (IEC) that purifies Protein Q from the eluate of a previous purification step using a strong cationic quaternary amine. The aim of the study is to improve the performance and robustness of this chromatographic step by evaluating different process conditions. According to the development studies carried out at laboratory scale (see example 1), the studied parameters of major impact on the IEC process are elution time and protein load density.

[0663] Elution time is defined as the duration of the elution phase. This parameter can be modified by modulating the flow rate or by including a pause (stop-flow step) to increase the contact time of the elution buffer with the target protein attached to the column.

[0664] The current report demonstrates that the improvements in the chromatographic conditions lead to considerably IEC purification step yields.

[0665] Equipment, Materials and solutions

[0666] Equipment

[0667] The following pieces of equipment are used to carry out the IEC process at industrial scale:

[0668] Table 2.1. List of equipment used.

[0669] Equipment

[0670] Scale

[0671] Multimeter (pH and conductivity)

[0672] Dilution Pump

[0673]

[0674] Agitator Equipment

[0675] Filtration pump

[0676] Filtration manometer

[0677]

[0678] FPLC Chromatograph

[0679] Materials

[0680] The following table gathers the materials employed to perform the IEC process at industrial scale.

[0681] Table 2.2. List of materials used.

[0682] Material Description

[0683] Dilution tank liner 200 L tank liner

[0684] IEC wash & eluate tank liners 50 L tank liner

[0685] IEC flowthrough tank liner 100 L tank liner

[0686] IEC load filtrate collecting bag 200 L tank liner

[0687] IEC eluate collecting bag 20 L tank liner

[0688] Impeller 15 x 15 cm

[0689] IEC resin Q Sepharose FF

[0690]

[0691] IEC Chromatography column 14.3 cm bed height and 20 cm diameter

[0692] Solutions

[0693] The composition of the solutions used in the process are comprised and described in the following tables:

[0694] 8 M Urea solution

[0695] Table 2.3. 8 M urea solution composition.

[0696] Component Qty.

[0697] Urea CO(NH2)2480.48 g / kg

[0698] Water for injection (WFI) c.s.p. 1 kg

[0699]

[0700] Dilution solution “step 1”

[0701] Table 2.4. Dilution solution “step 1” composition.

[0702] Component Qty.

[0703] NaOH 30.0 g / kg

[0704] Dilution solution “step 2” c.s.p. 1 kg

[0705]

[0706] Dilution solution “step 2”

[0707] Table 2.5. Dilution solution “step 2” composition.

[0708] Component Qty.

[0709] Urea solution 370 g / kg Sodium dihydrogen phosphate dihydrate

[0710] 7.4 g / kg (NaH2PO4.2H2O)

[0711] Triton X-100 (C14H21(C2H4O)nOH) 9.5 g / kg Water for injection (WFI) 614 g / kg

[0712]

[0713] Dilution solution “step 3”

[0714] Table 2.6. Dilution solution “step 3” composition.

[0715] Component Qty.

[0716] Urea solution 469 g / kg Sodium dihydrogen phosphate dihydrate

[0717] 7.3 g / kg (NaH2PO4.2H2O)

[0718] Sodium chloride (NaCI) 2.7 g / kg Triton X-100 (C14H21(C2H4O)nOH) 9.4 g / kg Water for injection (WFI) 512 g / kg

[0719]

[0720] 1 M NaOH Solution

[0721] Table 2.7. 1 M NaOH Solution composition.

[0722] Component Qty.

[0723] Sodium hydroxide (NaOH) 40.0 g / kg Water for injection (WFI) c.s.p.1 kg

[0724]

[0725] 2 M HCL Solution

[0726] Table 2.8. 2 M HCI Solution composition.

[0727] Component Qty.

[0728] Hydrochloric acid 37 % (HCI 37 %) 197.1 g / kg Water for injection (WFI) c.s.p. 1 Kg

[0729]

[0730] IEC Equilibrium & Washing solution

[0731] Table 2.9. IEC Equilibrium & Washing solution composition.

[0732] Component Qty.

[0733] 8 M urea solution 469 g / kg

[0734] Water for injection (WFI) 512 g / kg

[0735] Sodium dihydrogen phosphate dihydrate

[0736] (NaH2PO4.2H2O) 7.3 g / kg

[0737] Sodium chloride (NaCI) 2.7 g / kg

[0738] Triton X-100 (Ci4H2i(C2H4O)nOH) 9.4 g / kg

[0739]

[0740] IEC Elution solution

[0741] Table 2.10. IEC Elution solution composition.

[0742] Component Qty.

[0743] 8 M urea solution 960 g / kg

[0744] Sodium dihydrogen phosphate dihydrate

[0745] (NaH2PO4.2H2O) 6.7 g / kg

[0746] Sodium chloride (NaCI) 25.1 g / kg

[0747] Triton X-100 (Ci4H2i(C2H4O)nOH) 9.2 g / kg

[0748]

[0749] Control Industrial Scale Process description

[0750] In this section, a control industrial-scale method, before applying the changes, is described. Before undergoing IEC, the eluate from the previous purification step needs to be conditioned:

[0751] • pH is adjusted to 10.0 ± 0.1 by adding Dilution solution “step 1”.

[0752] • Urea concentration is adjusted to 4 M and dilution is completed until dilution factor 1 / 5.5 by adding Dilution solution pH 10 “step 2”.

[0753] • If needed, Protein concentration is adjusted to 0.5 g / L by adding Dilution solution pH 10 “step 3”.

[0754] The maximum load on to the IEC column is 40 g of total protein, which corresponds to a density load of 127 TP mg / cm2or a load ratio of 8.9 TP g / L resin. The conditioned product is filtered through a 0.2 pm filter and tempered at room temperature before being injected.

[0755] The chromatography system is washed with IEC Equilibrium and Washing solution, pH 9.0. After that, the column is equilibrated with 4 CV of IEC Equilibrium and Washing solution, pH 9.0 at a flow rate of 45 L / h (143 cm / h). The product is loaded on to the column at 45 L / h (143 cm / h); then, a wash with 3 CV of IEC Equilibrium and Washing solution, pH 9.0 is performed at 45 L / h (143 cm / h). Protein Q is isocratically eluted at 40 L / h (127 cm / h) using I EC Elution solution, pH 8.0. This step is monitored by UV signal at 254 nm. The next table summarizes the details of every IEC step.

[0756] Table 2.11. Summary of IEC Chromatography characteristics and steps.

[0757] Column Column Resin binding capacity Load Load density dimensions (Di x volume (L) (mmol / mL medium) ratio (TP mg / cm2) H) cm (TP g / L

[0758] resin)

[0759] 20 cm x 14.3 cm 4.5 0.18-0.24 mmol CI7mL

[0760] medium 8.9 127

[0761]

[0762] Step Solution Flow Required Prepared pH rate volume volume

[0763] (L / h) (CV)

[0764] System IEC Equilibrium and Washing - 1 4.5 L 8.8-9.2 wash Solution, pH 9.0

[0765] Equilibration IEC Equilibrium and Washing 45 4 18 L 8.8-9.2

[0766] Solution, pH 9.0

[0767] Product load “Adjusted protein concentration 45 - Equivalent - product” volume to 40

[0768] g protein Column IEC Equilibrium and Washing 45 3 13.5 L 8.8-9.2 wash Solution, pH 9.0

[0769]

[0770] Elution IEC Elution Solution, pH 8.0 40 3.14 14.13 L 7.8-8.2

[0771] Applied process improvements

[0772] Some changes have been introduced that have led to an improvement in the performance of this purification step. Specifically, the process modification applied on the 16 industrial batches manufactured in 2024 is that the IEC elution flow rate has been lowered from 127 cm / h (40 L / h) to 48 cm / h (15 L / h). In addition, a pause of 2 hours is applied in the middle of the elution phase. With this variation, elution time increases from 21 min to almost 3 hours.

[0773] Experimental design

[0774] The elution flow rate of the IEC in the current process was 40 L / h, or in other words, for the industrial column BPG200 / 500, a linear flow rate of 127 cm / h.

[0775] The elution volume is 3.14 CV, that is 14.1 L for the industrial IEC, with a column volume CV = 4.5 L. This means that the elution buffer takes 21 min to pass through the column.

[0776] j-,,.. f.. ^elution (L) > 14.1 > >.

[0777] Elutiontime(min) = - 60 = ■ 60 = 21 min

[0778]

[0779] fvo1(j)

[0780] Several small-scale laboratory tests have been made to optimize the IEC conditions, showing the best yields were obtained at the slowest elution flow rates of 15 cm / h, which increased the elution time to 180 min (see more details in Example 1). Reducing the linear flow to 15 cm / h would mean working at 4.7 L / h in the current industrial column BPG200 / 500, but the chromatograph is not able to work at a flow lower than 15 L / h, which for the current IEC, is equivalent to a linear flow of 48 cm / h. The calculations are presented below.

[0781] cm fvoi (77)

[0782] flinear M h = C -ross sec,tion(,cm2) ' 1000

[0783] For the current industrial column BPG200 / 500, with a section of 314.2 cm2:

[0784] rcm\ 15

[0785] flinear BPG200 = 3^(cm2)' 1000 = 48 Cm / h

[0786]

[0787] Applying the minimum chromatograph flow rate of 15 L / h, elution times would be around 1 hour, whereas in the laboratory it was concluded that the best results are obtained by applying flow rates of 15 cm / h, which corresponds to a contact time of the protein with the elution buffer of 3h. Therefore, it is established to include a 2h stop in the middle of the eluate collection phase, to equalize elution times.

[0788] The following table shows a comparison of the different studied flow rates and, therefore, the resulting elution time in the industrial batches.

[0789] Table 2.12. Operational parameters of IEC column when applying the elution regime changes.

[0790] Industrial batches Industrial batches After Before Changes in IEC Changes in IEC Elution Elution Conditions Conditions

[0791] D (cm) 20 20

[0792] H (cm) 14.3 14.3

[0793] Cross section (cm2) 314.2 314.2 CV (L) 4.5 4.5

[0794] Vol elution (L) 14.1 14.1 Elution linear Flow (cm / h) 127 48

[0795] Elution volumetric Flow (L / h) 40 15

[0796] Elution volumetric Flow (mL / min) 665 251 Elution time (min) 21 56

[0797] Stop (min) 0 120

[0798] Total elution time (min) 21 176

[0799]

[0800] Analytical methods

[0801] Total protein is analyzed by BCA in order to evaluate IEC process performance as in example 1.

[0802] Results

[0803] Sixteen commercial batches have already been carried out on the current BPG200 / 500 column and applying a flow rate of 15 L / h (linear velocity of 48 cm / h) plus a 2h stop and an increase of TP yield in IEC has been observed.

[0804] Table 2.7 shows the average IEC yields of the 16 batches run in recent year (61%), including the IEC elution improvements, compared to those performed in previous years with the current process. It can be observed that recent year average yields are higher than the ones obtained in previous year (50%) and another previous year (42%).

[0805] Table 2.13. Comparison of average IEC Total Protein yields, grouped per years.

[0806] Min Industrial Number of Average IEC TP CV IEC TP Yield Max

[0807] yield Batches batches Yield (%) (%) yield (%)

[0808] (%) previous 23 50 21 73 37 previous 24 42 23 66 28 recent 16 61 13 72 46

[0809]

[0810] In addition, with the changes produced in this purification step, the process is more controlled allowing less variation in yields between batches, resulting in a coefficient of variation CV = 13% compared to 21% and 23% obtained in previous years, respectively.

[0811] Moreover, this improvement in IEC yields translates into an increase in grams of final purified drug substance (PQ) obtained for each batch. The modification applied represents an average increment of 41 % of drug substance per batch in 2024 with respect to the previous year’s average productivity. CONCLUSIONS

[0812] The industrial process for protein Q production has been improved by implementing some changes in the IEC process: elution flow rate has been reduced to 15 L / h (48 cm / h) and a 2 h stop is incorporated to increase the elution contact time up to 3 h.

[0813] It has been proven in 16 industrial batches that this variation has improved the quantity of Protein Q obtained and the process robustness. As shown in the results, the average IEC yield has increased from 46 % to 61 % and, thus, the average total grams of purified drug substance per batch is around 41 % superior to previous years. Furthermore, the product characteristics and profile are comparable to those obtained before the changes were made. Example 3: Total Protein quantification by BCA.

[0814] 1. INTRODUCTION

[0815] The bicinchoninic acid (BCA) protein assay is a widely used highly sensitive colorimetric method for the quantification of total protein. This assay is based on the capability of bicinchoninic acid, a sodium salt, to react with Cu+1ions generated by the reduction of Cu+2ions in alkaline media (biuret reaction). Peptide bonds and some amino acids such as cysteine, tryptophan and tyrosine, are capable to carry this reaction of reduction of Cu2+to Cu+1. This reaction causes the appearance of a purple color formed by the chelation of two BCA molecules with a Cu+1ion. The stability of the water-soluble bicinchoninic acid cuprous complex absorbs at 562 nm wavelength, and the increase in color is directly related with an increasing concentration of protein, allowing for both detection and quantification of total protein in a solution.

[0816] The reaction is shown in the following scheme:

[0817] Protein + Cu+2Cu+1

[0818]

[0819] Cu+1+ BCA purple complex BCA-Cu+1

[0820] The BCA protein assay is used for the detection and quantification of total protein in samples of the protein Q fabrication process.

[0821] 2. SOLUTIONS

[0822] Table 3.1 Composition and working pH of buffers used for BCA assay.

[0823] Solution Composition

[0824] PBS 1X BupH™ Modified Dulbecco's

[0825] Preparation according to manufacturer.

[0826] NaCI 0.9 % 0.1 M NaCI

[0827] Biomass 8 M Urea, 43 mM sodium dihydrogen resuspension buffer phosphate dihydrate, 0.4 M NaCI, 1% Triton X- 100

[0828] 8 M Urea, 50 mM sodium hydrogen phosphate

[0829] First chromatography dihydrate, 0.5 M NaCI, 280 mM imidazole, 1% step elution buffer Triton X-100

[0830] Second

[0831] chromatography step 8 M Urea, 50 mM sodium hydrogen phosphate dihydrate, 0.5 M NaCI, 1% Triton X-100

[0832]

[0833] IEC elution buffer

[0834] 3. PROCEDURE 3.1. Calibration curve and sample preparation

[0835] The calibration curve is prepared diluting standard BSA 2 mg / mL from BCA assay kit in NaCI 0.9 % between 2000 pg / mL and 50 pg / mL (Table 4)

[0836] Table 3.2. Preparation of calibration curve of BSA.

[0837] Standard Concentration (pg / mL)

[0838] A 2000

[0839] B 1000

[0840] C 600

[0841] D 500

[0842] E 400

[0843] F 300

[0844] G 200

[0845] H 100

[0846] I 50

[0847] Blank 0

[0848]

[0849] Depending on the sample to be analyzed, the assay can be carried out with its specific buffer diluted with PBS 1X or NaCI 0.9 %, considering that salts must be compensated in all the standard and sample dilutions, and absorbance at 562 nm of diluted samples need to fit between the range of the calibration curve (600- 50 ug / mL) to be quantified.

[0850] In addition, two independent positive controls are prepared to assess if the method has been properly executed: BSA 500 pg / mL and BSA 100 pg / mL

[0851] 3.2. Assay procedure

[0852] After samples, calibration curve standards, positive controls and working reagent (50: 1, Reagent A: B) are prepared, the assay in the 96 well microplate is carried out as follows: 25 pL of BSA standards, each sample dilution, positive controls and NaCI 0.9 % (blank) are loaded in triplicate before the addition of 200 pL of working reagent in each well. Then, the plate is covered with adhesive parafilm and incubated at 37 ± 1 °C with agitation for 30 ± 5 minutes. After the incubation time, the plate is tempered at room temperature for 10 ± 5 minutes and the adhesive parafilm is removed before reading the plate in a plate reader at 562 nm wavelength absorbance.

[0853] 4. RESULTS

[0854] A linear regression model (y = ax + b) is used for quantification of protein (pg protein / mL). The absorbance of samples analyzed is interpolated in the calibration curve and the dilution factor is later applied to obtain the real result in pg protein / mL. Example 4: Applying the process of the invention using Ionic Exchange Chromatography (IEC) to other proteins

[0855] LIST OF ABBREVIATIONS ABBREVIATION Wording Deli n it i on

[0856]

[0857] Al Active ingredient

[0858] BCA Bicinchoninic acid protein

[0859] CDMO Contract Development and Manufacturing Organization

[0860] CV Column volume

[0861] CW Column wash

[0862] DSP Downstream processing

[0863] FT Flow through

[0864] IEC Ion Exchange Chromatography

[0865] ISP Industrial scale process

[0866] NA Not available

[0867] ND Not detected

[0868] PQ / ProtQ Protein Q

[0869] SD Standard deviation

[0870] SDS-PAGE Sodium dodecyl-sulfate polyacrylamide gel electrophoresis SSP Small scale process

[0871] TP Total Protein

[0872] USP Upstream processing

[0873] WB Western Blot 1. Introduction

[0874] This work focuses on the method of the invention comprising the use of Ion Exchange Chromatography (IEC) step to purify a protein such as Protein Q (PQ) (SEQ ID NO:1) from the eluate of the previous purification step using a strong cationic quaternary amine. The aim of the study is to validate the use of this method to other proteins than Protein Q (SEQ ID NO:1).

[0875] To reinforce the findings in the Protein Q purification process, a series of trials using proteins with varying degrees of sequence identity to Protein Q have been designed. Three proteins have been selected because they have a similar isoelectric point to Protein Q: one of them Protein 3 presents a sequence with 87.4 % identity (87.7% similarity) to Protein Q sequence, the other one Protein 4 presents 7.4 % identity (12.9% similarity) with Protein Q. Additionally, Protein 2 with distinct isoelectric point and an intermediate identity (59.8 % identity and 65.3% similarity) level to Protein Q sequence has been tested. More details as to each of these proteins and as to how the identity and similarity to Protein Q are given in the next paragraphs. The main aim of this study is to evaluate whether the newly implemented method is universally applicable across diverse proteins, or if its effectiveness is primarily dependent on protein properties as the amino acid sequence or the isoelectric point.

[0876] 2. Proteins selected

[0877] PQ is the protein on which the process of the invention has first been successfully applied. PQ is represented by (SEQ ID N 1) PQ has a molecular weight of 50 KDa and an isoelectric point of 7.2. SEQ ID NO:1 MRGSHHHHHHTDPHASSNNNNNNNNNNLGIEGRPLATPRSAKKAVRKSGSKSAKCGL IFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHD DDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTKYLAAYA LASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPA ASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSG KTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGA VSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPEESDEDD FGMGGLF

[0878] Further proteins were selected:

[0879] Protein 2 (SEQ ID NO:2): Identity <80% with PQ (SEQ ID NO:1)

[0880] Protein 2 (SEQ ID NO:2) is a synthetic protein made up of protein fragments from six Leishmania proteins (see below). The six Leishmania proteins are: Kmp11, Pfr2, Gp63, A2, L3, L5. Protein 2 has a molecular weight of 50.32 KDa and an isoelectric point of 4.96. This protein has 59,8% identity (65,3% similarity) (EMBOSS needle alignment) when compared with PQ (SEQ ID N1) and has a histidine tail and an anti-PQ antibody recognition site.

[0881] SEQ ID NO: 2:

[0882] MGSSHHHHHHSSGLVPRGSHMNKKMHEHSEHFKQKFAELLEQQKAAQYPSKGGEEQYYIKAQLLE HLVELVADKFRIIGQTEDGGYVASVPSEEGVLAWATTCQVFSDGHPAVGVGGVDVGPLSVGPQSVGP LSVGLQAVDVSPVSGGNVAAKIALAKSLLEKEVRVDSVFQQSEACDVCSVTKGHGGFGIDEHIDLGIK YDPSTGIYGMDFYVVLGRRGERVAHRKRKCSGLKACSRPMSTKYLAAYALASLSKASPSQADVEAIC KAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDE PEEEADDDMGPSRVDPMQYLAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFD ALVAEGRTKLVGSGSAAPAGAVSTAGAAAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAA AKEEPEESDEDDFGMGGLF

[0883] Protein 3 (SEQ ID NO:3): Identity >80% with PQ (SEQ ID NO:1)

[0884] “Protein 3” (SEQ ID NO:3) is a synthetic protein derived from PQ (SEQ ID NO:1), it has a molecular weight of 40.30 KDa and an isoelectric point of 6.34 has 87.4% identity (87.7% similarity ) (EMBOSS needle alignment) when compared with PQ (SEQ ID N1) and has a histidine tail and an anti-PQ antibody recognition site.

[0885] SEQ ID NO:3:

[0886] MATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAAQSGKKR ARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTK YLAAYALASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGA AAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSGKTPSKADVQAVLKA AGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGAVSTAGAGAGAVAEAKKEEPEEEE ADDDMGPVDLQPAAAAPAAPSAAAKEEPEESDEDDFGMGGLFSSGENLYFQGLEHHHHHH

[0887] Protein 4 (SEQ ID NO:4). Identity towards the sequence of PQ is 7.4% (12.9% similarity) (EMBOSS needle alignment) (SEQ ID NO:1)

[0888] Protein 4 is myoglobine from horse (Equus caballus). It has an isoelectric point of 6.8 to 7.2 and a molecular weight of 17kDa. Protein 4 is a commercial purified protein with similar isoelectric point to the one of Protein Q but it has an unrelated sequence to the one of Protein Q.

[0889] (sp|P68082|MYG_HORSE Myoglobin OS=Equus caballus OX=9796 GN=MB PE=1 SV=2).

[0890] SEQ ID NO:4 MGLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASE DLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKH PGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

[0891] 3. Alignment of Protein 2, Protein 3 and Protein 4 with Protein Q

[0892] Alignments were carried out using EMBOSS needle alignment (Needleman): Matrix EBLOSUM62, Gap penalty: 10.0, Extend penalty: 0.5. ignment Protein 2 (SEQ ID NO:2) and Protein Q (SEQ ID NO:1): EMBOSS Needle

[0893]

[0894] #

[0895] # Aligned_seciuences: 2

[0896] # 1: PQ

[0897] # 2: PR0TEIN2

[0898] # Matrix: EBL0SUH62

[0899] f Gap_ penalty: 10.0

[0900] t Extend_penalty: 0.5

[0901] #

[0902] # Length: 490

[0903] # Identity: 293 / 490 (59.830

[0904] # Similarity: 320 / 490 (65.3%)

[0905] # Gaps: 80 / 490 (16.3JS)

[0906] # Score: 1241.0

[0907] #

[0908]

[0909] PQ 1 HtGSHHHHHifT0- - -PHAS-SNMmK«MNL6IEGRPUTP^AKKAVR 46: •■ ■ ■•£... P P S £ -’. £ M so PQ 47 KSGSK- - - - - -........76

[0910] | 4.:, | | | | -

[0911]

[0912] PiOTinc. • 44 '. ' - J. ■ - 4 J; PQ 76 - - -RIGASGAPRISEFSVKAAAQS-GKKRCRUIPRTVMIAARHDODI- -G 119

[0913] I.: - =. ■: ■ ■ I PR0TEIN2 4

[0914]

[0915] . 7 4? 4 - - 4..44

[0916]

[0917] 4 _ - 4 MB PQ 4. 4.. - 4, -, 4 - - ~ 4 - 4! 4..: 14®

[0918]

[0919] .:...:. {:. |,:...: I 4: 4 - 4 I - 4 PROTEIil 149 NVAAKIALAOLLEKEVRVOSVFQQSEACOVCSVTKGHGGFGIOEMOLG 198 PQ

[0920]

[0921] 4. 4 4 -,..................

[0922]

[0923] . 4: 4 -. 4?

[0924] .: • 4

[0925]

[0926] PRQTEIN2 199 imPSTGIYGMOFYWtSRRGERVMaKRKCSGLKACSRPHSWLMY 248 Q 173 ALASLSKASPSQAOVEAI< XAVIfIOVOQATI. AFWESVTGWVATtIAEG 222

[0927] 1 1 H 41 1 i i 1 111 1 1 4 i 1 1 i 11 1 1 11 1 1 1 H 4 H I i I 44 1 11 1 I I PROTEINS 249 ALASLSKASPSQABVEAKICAVHIOTOQATLAFVMESVTGROVATLIAEG 298 PQ 223 AAI«SAHPAASSGAAAGVT«AMDAAPAAAAAKi®EPEEEAtmDMGPSR 272

[0928] H H H H H 11 11 1 1 1 H i 1 1111 11 1 1 1 1 11 111 11 1 1 1 1111 H 11 1 PSOTEI l 299 AAtQMSAW»AASSGAAAGVTASAAGOAAPAAAAAKKDEPEEEADOOMPSR 348 PQ 273 VDPMQYLAAYALVALSGKTPSICAOVQAVLKAWSVAVOASRWAVFQEVEG 322 n 11 11 1 1 1111 111 1 1 141 1111 11 1 1 1 1 1 i 111 11 1 1 1111111 11 1 PR0TEI12 i. 4u. - - - 4 4. ' - - - M --. 4 t 4 398 PQ 323 fSFOALVAEG reLVGSGSA^AGAWTAGAGAGAVAEAKffiEPEEEEAO 372

[0929] H 11 11 1 1 1 i 1111 1 1 1 1111 101 1 11 1 1 1. 1 111 11 1 1 111 i 11 H 1 1 P10TEM2 399 FDALVAEGRTKLVfiS(»APAGAWrAGAAAGAVAEAICIffiEPEEEEAO 448 PQ 373 ODMGPVOLQPAAAAPAAPSAAAIGEEPEESDEDOFGHGGLF 412

[0930] 1 11411 1 1111 111 11 H I 1 1 H 1 1 11 11 i 11 111 111 1

[0931] PROTEIil 449 OOiGP¥BLQPa***P*APSA**KSEPEESDEOOFfiififilF 48B . Alignment Protein 3 (SEQ ID NO:3) and Protein Q (SEQ ID NO:1): EMBOSS Needle

[0932] lligBeCseQueftoes: 2

[0933] R

[0934] 21 PB0TEIM3 Matrix: EBL0SUH62 6ap_peBalty: M>® Extend_penalty: 0.5 Length: 43®

[0935] : i-. -. m

[0936] Saps: 62 / 43® (12.1SQ Scoze: 1B53.®

[0937] 16

[0938] SI tSAWEGLIHWWSGM^^ 1W PSOTEI13

[0939] Ifl4 MWTEBI3 116

[0940] BWHB 112 166

[0941] 261

[0942] 217 2i» LIOAmW«mAW:eEWG»OALVAEGfra. V®KAWAG«ST fWTHM 267 LKMam»BBBWWFlEVE6eFMLW*ES»WtBSm«mB«ST

[0943] am 4«e S80TEBS13 317

[0944] 412

[0945]

[0946] wmtnB 367 3®6 3. Alignment Protein 4 (SEQ ID NO:4) and Protein Q (SEQ ID NO:1): EMBOSS Needle Ali^ed_s«wenc«s; 2

[0947] > PBWEIJI4

[0948] ' MM Baa_penaltyi 18.0

[0949] : • • - MI;- tengtiw 434

[0950] : M -M ( 7.41) Sfcfiaxitis 56 / 434 (12. WJ

[0951] ... I M; Sc®re: li.fl

[0952]

[0953] 51 KSMC6LIFPV6R¥66BlRR6f WRI8*S6WSISEFSWa*iS6«B PBOTEnW

[0954] 1®1 CRL-- -BPBTV- HL4MM00BICTILHWTISHS6WP 134 M

[0955]

[0956] WTEIBI LASLSaS

[0957] ROW 71

[0958] M2 PSf- -WWE-*ICKMMIOW«^

[0959] 121

[0960] 229 P10TEI14 164 PWJJIIBI 166

[0961] 329

[0962] PiOIHHWI

[0963]

[0964] MOTE MB ise Sequence identity is a key metric for assessing homology between proteins. Generally, >30% identity strongly suggests evolutionary relatedness and often correlates with structural similarity. The range of 20-30% is considered the “twilight zone”, where homology cannot be confidently inferred. Below 20% identity, similarity is typically regarded as random or coincidental, with no reliable functional or structural correlation (Rost, B. (1999), DOI: 10.1093 / protein / 12.2.85. Chung, S. Y., et al (1996), Structure, DOI: 10.1016 / S0969-2126(96)00119-0 and Bartuzi, D., et al (2023) Title: Illuminating the “Twilight Zone”: Advances in Difficult Protein Modeling, Book Series: Methods in Molecular Biology, DOI: 10.1007 / 978-1-0716-2974-1_2). Therefore, Protein 4 is not related to Protein Q.

[0965] 4. Materials and equipment

[0966] 4.1. Equipment

[0967] The experiments were performed in Bioprocessing Area and QC laboratory in LETI Pharma, and the following equipment was used:

[0968] Table 4.1. List of equipment used.

[0969] Equipment

[0970] Freezer

[0971] Refrigerator

[0972] FPLC Chromatograph

[0973] Stirring plate

[0974] Precision scale

[0975] pHmeter

[0976] Centrifpge

[0977] Sonicator

[0978] Ultrapure Water Purification System

[0979] Micropipette P1000

[0980] Micropipette P200

[0981] Micropipette multichannel P300

[0982] Micropipette P20

[0983] Micropipette P50

[0984]

[0985] 4.2. Materials

[0986] The following table gathers the materials employed to perform the laboratory trials. Table 4.2. List of materials used.

[0987] Material Model

[0988] Filter PES membrane

[0989] IEC resin Q Sepharose FF

[0990] IEC Chromatography column Long / Narrow Column: 14.3 cm bed height and 1.6 cm diameter IEC Chromatography column Short / Wide Column: 5.4 cm bed height and 2.6 cm diameter

[0991]

[0992] 4.3. Solutions

[0993] The composition of the solutions used in the I EC process is described in the following tables:

[0994] 4.3.1. Urea 8 M solution

[0995] Solution used as the basis for the process buffers preparation.

[0996] Table 4.3. Urea 8 M solution composition.

[0997] Material Grams / L

[0998] Urea, CH4N2O 480.48

[0999] Water, H2O c.s.p. liter

[1000]

[1001] 4.3.2. IEC load solution or pH adjustment solution (pH 10)

[1002] Buffer used to dilute the protein to be loaded to the IEC. It is prepared weighing the following reagents:

[1003] Table 4.4. pH adjustment solution (pH 10) composition

[1004] Material Grams / L

[1005] Monosodium phosphate dihydrate, NaH2PO4.2H2O 7.41

[1006] Urea 8M solution 370.0

[1007] Water, H2O 614

[1008] Sodium hydroxide, NaOH 4M c.s.p = pH 10

[1009] Triton X-100, Ci4H2i(C2H4O)nOH 9.51

[1010]

[1011] 4.3.3. IEC Equilibration and Wash solution

[1012] Buffer used to equilibrate and wash the IEC column. It is prepared according to the following table:

[1013] Table 4.5. IEC Equilibration and Wash solution composition

[1014] Material Grams / L

[1015] Monosodium phosphate dihydrate, NaH2PO4.2H2O 7.31

[1016] Urea 8M solution 469

[1017] Sodium Chloride, NaCI 2.74

[1018]

[1019] Water, H2O 512

[1020] Sodium hydroxide, NaOH 4M c.s.p = pH 9

[1021] Triton X-100, Ci4H2i(C2H4O)nOH 9.37

[1022]

[1023] 4.3.4. IEC Elution solution

[1024] Buffer used to elute Protein Q from IEC column, and it is prepared according to the following table: Table 4.6. IEC elution solution composition

[1025] Material Grams / L

[1026] Monosodium phosphate dihydrate, NaH2PO4.2H2O 6.7

[1027] Urea 8M solution 960

[1028] Sodium Chloride, NaCI 25.1

[1029] Sodium hydroxide, NaOH 4M c.s.p = pH 8

[1030] Triton X-100, Ci4H2i(C2H4O)nOH 9.19

[1031]

[1032] 5. Small scale process description

[1033] The IEC step has been scaled down from the current existing industrial process. To do that, the dimensions and the resin of the current industrial column were taken as a reference to design the laboratory trials.

[1034] Conditioned eluate from the previous purification step is used as starting material for the laboratory trials. It means, the product to be loaded on to the IEC has been adjusted to pH = 10, urea = 4 M and TP < 0.5 g / L. The required mg of total protein to be loaded onto the IEC for each run are calculated to start from the right product volume. This is made based on:

[1035] • Load density: Amount of TP to be loaded on to the column per column section. The reference according to the current ISP is < 127 mg / cm2.

[1036] • Load ratio: Amount of TP to be loaded on to the column per mL of resin. The reference according to the current ISP is < 8.9 mg / mL.

[1037] • Load concentration: TP concentration of the IEC load. The reference according to the current ISP is < 0.5 g / L.

[1038] The calculated necessary volume per run is tempered and homogenized in gentle agitation. If needed, pH is adjusted to 10.0 ± 0.2. Then, the product is filtered before being loaded onto the column. IEC chromatography is run in an FPLC chromatograph and the method consists of the following stages:

[1039] • Equilibration with 4 CV IEC Equilibrium and Washing Sol., pH 9.0

[1040] • Product load, CV variable depending on product concentration.

[1041] • Column wash with 3 CV Dilution and equilibrium buffer

[1042] • Elution with 3.14 CV IEC Elution buffer, pH 8.0

[1043] • 4° CV Elution: 1 CV IEC Elution Sol., pH 8.0

[1044] • 5° CV Elution: 1 CV IEC Elution Sol., pH 8.0

[1045] • Over Night 6° CV Elution: 1 CV IEC Elution Sol., pH 8.0 The elution flow rate in the current ISP is 40 L / h (127 cm / h), which results in an elution time of 21 min. Elution flow rates are adjusted in the method depending on the conditions to be tested in each run. The elution peak is gathered according to the column volumes stated in the current process: 2.34 CV are collected after running 0.8 CV of elution phase. Additionally, as previously observed with PQ, we added three extra column volumes (4th, 5th& 6thCV) after the elution step to assess whether the protein remained bound to the resin. This allowed us to monitor residual bound protein. We have referred to this IEC step method as the “Reference” method, which serves as the standard used in the industrial scale process (ISP).

[1046] Based on this model, a series of modifications have been implemented, and we refer to the newly optimized method as the “Optimal” method. The different process parameters applied in the “Reference” and the “Optimal” trials are compared in the following table:

[1047] Table 4.7. IEC Reference and Optimal at laboratory scale

[1048] Reference Optimal

[1049] Narrow Wide

[1050] Column Geometry

[1051] H 14.3 cm, D 1.6cm H 5.4 cm, D 2.6cm

[1052] Elution linear flow rate (cm / h) 127 cm / h 15 cm / h

[1053] Elution time (min) 21 168 (2 hours stop)

[1054] Ratio TP g / L resin 8.9 6

[1055] IEC load density (mg TP / cm2) 258.1 30.9

[1056] IEC load concentration 0.4 mg / mL 0.4 mg / mL

[1057]

[1058] This new “Optimal” method incorporates all modifications that have been identified to increase the yield of PQ during the IEC step.

[1059] 6. Analythical methods

[1060] Total protein is quantified by BCA. The method is described in Example 3.

[1061] 7. Experimental design

[1062] This report presents the results of several trials directly comparing the two IEC step methods, Reference and Optimal, using a range of proteins with varying degrees of homology and isoelectric points relative to PQ.

[1063] Table 4.8. List of proteins tested in the IEC trials and their main properties

[1064] Isoelectric Molecular Name Identity to PQ% Point* Weight Protein Q 100 6.8-7.2 42kDa HH: High homology Protein 3 87,4 6.34 40kDa MH: Mid homology Protein 2 59,7 4.96 50 KDa

[1065]

[1066] LH: Low homology Protein 4 7,4 6.8-7.2 17kDa

[1067]

[1068] *Homology to PQ was calculated using Needleman / EMBOSS method.

[1069] **lsoelectric point was determined using a prediction software Expasy, this estimation could vary depending on the software used.

[1070] Each protein was subjected to both I EC methods to assess whether the “Optimal” method can be universally applied to any protein, or if its effectiveness is restricted by protein sequence, size and / or isoelectric point. The objective is to assess whether proteins with different sequence identity, but same / similar isoelectric point, behave as PQ in the method of the invention, meaning that the yield of such proteins could be improved by increasing the elution contact time and / or decreasing the protein density load.

[1071] For this study, we selected three proteins with varying degrees of sequence identity and isoelectric points relative to Protein Q (SEQ ID NO:1, isoelectric point 7.2):

[1072] • Protein 3 (SEQ ID NO:3) is a Protein Q variant with 87,4% sequence identity and a similar isoelectric point (IP) as the one of Protein Q.

[1073] • Protein 2 is a recombinant protein exhibiting 59,8% sequence identity to Protein Q but a different isoelectric point. For Protein 2, it was necessary to adjust the pH conditions during the IEC step to ensure effective binding and elution.

[1074] • Protein 4 is a commercially sourced protein with 7,4% sequence identity to Protein Q but a very similar isoelectric point, confirmed by the supplier.

[1075] Both Protein 3 and Protein 2 are recombinant proteins engineered in our laboratory, while Protein 4 serves as a control to assess the method’s applicability to unrelated proteins with similar physicochemical properties.

[1076] 8. RESULTS

[1077] For all the runs, the total protein was quantified by BCA as described in example 3, so that protein recoveries could be compared. The IEC step yield was calculated as g TP in IEC eluate / g TP in IEC load.

[1078] Next, the results are evaluated grouped, depending on the protein being studied:

[1079] 8.1. PROTEIN 3 (SEQ ID NO:3) VARIANT PROTEIN Q HIGH SEQUENCE IDENTITY

[1080] The following table summarizes the results of both the Optimal and Reference runs for Protein 3 (SEQ ID NO: 3), the protein with the highest identity, 87,4% sequence identity, and a similar isoelectric point to Protein Q (SEQ ID NO: 1). The “reference” and “optimal” methods applied are as described in the Process description of Table 4.7.

[1081] Table 4.9. Direct comparison of IEC methods (Optimal vs. Reference) for Protein 3, high homology trial. Yields and mass balance.

[1082] IEC Protein 3 01 OP IEC Protein 3 02 REF

[1083] TP / mL Resin 5.4 6.5

[1084] Elution Flow rate 15cm / h +2h (168 min) 127cm / h (21 min)

[1085]

[1086] IEC OPTIMAL REFERENCE

[1087] [TP] (pg / mL) 407 393

[1088] IEC load Indore

[1089] 445 528

[1090] liltr.ilKin

[1091] 181 208

[1092] [TP] (pg / mL) 354 361

[1093] IEC load Filtered

[1094] Vol (mL) 440 525

[1095] 0.2 pm

[1096] TP (mg) 156 189

[1097] [TP] (pg / mL) ND ND

[1098] Flow Through

[1099] TP (mg) ND ND

[1100] [TP] (pg / mL) ND ND

[1101] Column wash

[1102] TP ND ND

[1103] 4CV [TP] (pg / mL) ND 540

[1104] 5CV [TP].pg in Li ND 326

[1105] 6CV [TP] qjg m Li ND 417

[1106] Sum 4+5+6 CV ND 38

[1107] [TP] (pg / mL) 2664 2119

[1108] IEC Eluate Vol (mL) 69 69

[1109] TP (mg) 183 146

[1110] Yield Filtration 0.2 pm 87% 92%

[1111] Yield IEC 117% 77%

[1112] Yields

[1113] Global Yield filtrate +

[1114] 102% 71%

[1115] IEC FT 0% 0%

[1116] CW 0% 0%

[1117] ELUATE 102% 71%

[1118] Balance 4CV 0% 8%

[1119] 5CV 0% 5%

[1120] 6CV 0% 6%

[1121] COLUMN / LOD 0% 3%

[1122]

[1123] These results show an increase in protein recovery, up to 31 % using the optimized method with respect to the reference method, without affecting the protein profile. This outcome was somewhat expected, as both proteins share high sequence identity and similar isoelectric points. We also observed that, as with Protein Q, Protein 3 can remain bound to the column. It is reflected when the protein is still detected after passing extra elution buffer: 4th, 5thand 6thcolumn volumes (samples 4CV, 5CV and 6CV). However, by applying the same optimized method, increasing the elution buffer contact time with the protein and the column, we were able to achieve an increase in protein recovery, likely due to the sequence identity and similar physicochemical properties of both proteins. This gain aligns with the results reported for Protein Q, where we observed an increase in recovery of nearly 40% at small scale. 8.2. PROTEIN 2 (SEQ ID NO: 2) MID IDENTITY

[1124] \Ne selected this protein due to its lower sequence identity to Protein Q (59,8%). However, achieving this reduced homology resulted in a shift in its isoelectric point to 4.96, with respect to pl 6.8-7.2 of Protein Q. To accommodate this change, buffer pH conditions were adjusted to ensure proper performance during the IEC process. Product load was adjusted to pH 8 instead of 10. Equilibrium and column wash buffers were adjusted to pH 7.5, instead of 9, and the elution buffer was adjusted to pH 5.5 instead of pH 8. The “reference” and “optimal” methods applied are as described in the Process description of Table 4.7.

[1125] Table 4.10. Direct comparison of IEC methods (Optimal vs. Reference) for Protein 2, mid identity trial. Yields and mass balance.

[1126] IEC Protein 201 IEC Protein 2 02

[1127] TP / mL Resin 4.5 7

[1128] 15cm / h +2h (168

[1129] Elution flow rate 127cm / h (21 min)

[1130] min)

[1131] IEC OPTIMAL REFERENCE

[1132] [TP] (pg / mL) 435 515

[1133] IEC load In-lore

[1134] Vol.mLi 447 517

[1135] filtration

[1136] 194 266

[1137] [TP] (pg / mL) 370 449

[1138] IEC load Filtered

[1139] Vol (mL) 449 520

[1140] 0.2 pm

[1141] TP (mg) 166 234

[1142] [TP] (pg / mL) 35 48

[1143] Flow Through

[1144] 16 25

[1145] [TP].pg'mL) ND ND

[1146] Column wash

[1147] TP (mg) ND ND

[1148] 4CV [TP] (pg / mL) 149 360

[1149] 5CV [TP].pg mL) 70 269

[1150] 6CV [TP]. pg mL, 362 N / A

[1151] Sum 4+5+6 CV 17 18

[1152] [TP].'pg'mL', 2046 515

[1153] IEC Eluate Vol (mL) 69 69

[1154] TP (mg) 141 35

[1155] Yield Filtration 0.2 pm 85% 87%

[1156] Yields Yield IEC 85% 15%

[1157] Global Yield filtrate + IEC 72% 13%

[1158] FT 9% 11%

[1159] CW 0% 0%

[1160] ELUATE 85% 15%

[1161] Balance 4CV 3% 5%

[1162] 5CV 1 % 3%

[1163] 6CV 6% N / A

[1164] COLUMN / LOD 0% 66%

[1165]

[1166] The results obtained with the optimized method are outstanding, showing a 70% increase in protein recovery, without affecting the protein profile. Throughout the entire extraction and purification process, this protein demonstrated excellent behavior and stability. Similar to what was observed with Protein Q, Protein 2 remains tightly bound to the column when using the Reference method: only 15% of the total loaded protein onto the IEC is eluted. In contrast, the optimized method achieved an elution yield of 85%, highlighting its effectiveness in overcoming the strong binding and significantly improving recovery. With the reference method, it is observed that 66% of the loaded protein remains in the column or is not detected by the analytical method (COLUMN / LOD). These findings also indicate that, even when the isoelectric point is different, maintaining a certain degree of sequence identity, such as 59,7%, is sufficient for the optimized method to remain effective.

[1167] 8.3. PROTEIN 4 LOW IDENTITY

[1168] This section summarizes the data obtained for a protein with 7.4% sequence homology to Protein Q. The Protein 4, commercially sourced from Sigma (catalogue number M0630), was selected after an extensive search for proteins with a similar isoelectric point. Despite its lack of identity, its comparable physicochemical properties made it a suitable candidate for evaluating the universality of the optimized IEC method. The “reference” and “optimal” methods applied are as described in the Process description of Table 4.7.

[1169] Table 4. 11. Direct comparison of IEC methods (Optimal vs. Reference) for Protein 4, low identity trial. Yields and mass balance.

[1170] IEC Protein 401 IEC Protein 402

[1171] OP REF TP / mL Resin 4.5 7.0

[1172] 15cm / h +2h (168

[1173] Elution flow rate 127cm / h (21 min)

[1174] min)

[1175] IEC OPTIMAL REFERENCE

[1176] [TP] (pg / mL) 240 254

[1177] IEC load In-lore

[1178] 500 750

[1179] lill r.ili< HI

[1180] 120 191

[1181] [TP] (pg / mL) 263 271

[1182] IEC load Filtered

[1183] Vol (mL) 498 748

[1184] 0.2 pm

[1185] TP (mg) 131 203

[1186] [TP] (pg / mL) 227 213

[1187] Flow Through

[1188] TP (mg) 113 159

[1189] [TP] (pg / mL) 46 55

[1190] Column wash

[1191] TP (mg) 4 5

[1192] 4CV [TP] (pg / mL) ND ND

[1193] 5CV [TP] i|jg mL) ND ND

[1194] 6CV [TP] i pg mLi ND ND

[1195] Sum 4+5+6 CV ND ND

[1196] [TP] (pg / mL) 37 61

[1197] IEC Eluate

[1198] Vol (mL) 69 69

[1199]

[1200] TP (mg) 3 4

[1201] Yield Filtration 0.2 pm 110% 107%

[1202] Yields Yield IEC 2% 2%

[1203] Global Yield filtrate + IEC 2% 2%

[1204] FT 86% 79%

[1205] CW 3% 2%

[1206] ELUATE 2% 2%

[1207] Balance 4CV 0% 0%

[1208] 5CV 0% 0%

[1209] 6CV 0% 0%

[1210] COLUMN / LOD 9% 17%

[1211]

[1212] These results may suggest issues with protein solubilization in the loading buffer. The starting protein was dissolved in loading buffer to a target concentration of 400 pg / mL. Nevertheless, in both experiments the quantified TP did not reach the expected concentration, around half of the protein was solubilized and loaded onto the IEC column in both trials: 254 pg / mL for the reference run and 240 pg / mL for the optimal run. Based on the Protein 4 provider report and our initial solubilization tests, Protein 4 is 100% soluble in 0.9% NaCI; however, it was proven that solubility was not as good in Protein Q process buffers. Laboratory findings indicate that Protein 4 does not bind to the resin, as the entire protein loaded to the column was detected in the flow-through fraction (FT). Despite having a similar isoelectric point and using a loading buffer pH way above the IP, Protein 4 is not binding to the column. When using a protein with a totally different amino acid sequence, applying the optimal method does not present an improvement in recoveries, which indicates that the proposed changes may not be applicable for a protein having 7.4% identity with Protein Q.

[1213] 9. CONCLUSIONS

[1214] The optimized IEC method significantly improves protein recovery, particularly for proteins with moderate to high sequence identity to Protein Q. Protein 3 and Protein 2, both sharing > 59,8 %identity with Protein Q, showed substantial increases in yield, up to 70 % in the case of Protein 2, demonstrating the method’s ability to overcome strong column binding, issue related with Protein Q.

[1215] The results obtained with Protein 2 indicate that, even when the isoelectric point differs significantly (pl 4.96), the optimized method remains effective as long as a certain degree of sequence identity is maintained. In contrast, Protein 4, despite having a similar isoelectric point but no sequence identity, did not bind to the column. The outcomes for both proteins reinforce that the method’s effectiveness is primarily driven by sequence identity rather than physicochemical properties alone.

[1216] These findings confirm that the optimized method is highly effective for Protein Q-related proteins having at least 60% identity with Protein Q and support its specificity, as reflected in the improvements reported in our patent application.

[1217] Example 5: Increasing elution time range in ion exchange chromatography

[1218] 1. INTRODUCTION This study provides supplementary data and analysis focused on extending the elution time range in the ion exchange chromatography (I EC) process. Building upon previous trials, the objective is to evaluate the impact of prolonged elution on protein recovery and process flexibility. This assessment has been performed at laboratory scale with 4 different proteins: Protein 1 = Protein Q (SEQ ID NO:1), Protein 2 (SEQ ID NO:2) = 59.8 % identity to Protein Q sequence, Protein 3 (SEQ ID NO:3) = 87.4 % identity to Protein Q sequence and Protein 4 (SEQ ID NO:4) = 7.4 % identity to Protein Q sequence. Special attention was given to the behaviour of the PQ protein, which consistently remained partially bound to the IEC resin under both reference and optimized conditions. By incorporating extended elution periods, including up to 24 hours, we demonstrate that additional protein can be successfully recovered without compromising its structural integrity or stability. These findings support the feasibility of implementing broader elution time windows, offering enhanced adaptability in downstream processing workflows.

[1219] 2. SMALL SCALE PROCESS DESCRIPTION

[1220] The IEC step has been scaled down from the current existing industrial process. To do that, the dimensions and the resin of the current industrial column were taken as a reference to design the laboratory trials.

[1221] Conditioned eluate from the previous purification step is used as starting material for the laboratory trials. It means, the product to be loaded on to the IEC has been adjusted to pH = 10, urea = 4 M and TP < 0.5 g / L. The required mg of total protein to be loaded onto the IEC for each run are calculated to start from the right product volume. This is made based on:

[1222] • Load density: Amount of TP to be loaded on to the column per column section. The reference according to the current ISP is < 127 mg / cm2.

[1223] • Load ratio: Amount of TP to be loaded on to the column per mL of resin. The reference according to the current ISP is < 8.9 mg / mL.

[1224] • Load concentration: TP concentration of the IEC load. The reference according to the current ISP is < 0.5 g / L.

[1225] The calculated necessary load volume per run is tempered and homogenized in gentle agitation. If needed, pH is adjusted to 10.0 ± 0.2. Then, the product is filtered before being loaded onto the column. IEC chromatography is run in an FPLC chromatograph, and the method consists of the following stages:

[1226] • Equilibration with 4 CV IEC Equilibrium and Washing Sol., pH 9.0

[1227] • Product load, CV variable depending on product concentration.

[1228] • Column wash with 3 CV Dilution and equilibrium buffer

[1229] • Elution with 3.14 CV IEC Elution buffer, pH 8.0

[1230] 4th CV Elution: 1 CV IEC Elution Sol., pH 8.0

[1231] 5th CV Elution: 1 CV IEC Elution Sol., pH 8.0

[1232] • Overnight 6th CV Elution: 1 CV IEC Elution Sol., pH 8.0 The elution flow rate in the current ISP is 40 L / h (127 cm / h), which results in an elution time of 21 min. Elution flow rates are adjusted in the method depending on the conditions to be tested in each run. The elution peak is gathered according to the column volumes stated in the current process: 2.34 CV are collected after running 0.8 CV of elution phase. Additionally, three extra column volumes (4th, 5th & 6th CV) are added after the elution step to assess whether the protein remained bound to the resin. This allowed us to monitor residual bound protein. We have referred to this IEC step method as the “Reference” method, which serves as the standard used in the ISP.

[1233] Based on this model, a series of modifications have been implemented, and we refer to the newly optimized method as the “Optimal” method. The different process parameters applied in the “Reference” and the “Optimal” trials are compared in the following table:

[1234] Table 5. 1. IEC Reference and Optimal at laboratory scale

[1235] Reference Optimal

[1236] Narrow Wide

[1237] Column Geometry

[1238] H 14.3 cm, D 1.6cm H 5.4 cm, D 2.6cm

[1239] Elution linear flow rate (cm / h) 127 cm / h 15 cm / h

[1240] Standard elution time (min) 21 168 (2 hours stop)

[1241] Total elution time (h) 24 24

[1242] Ratio TP g / L resin 8.9 6

[1243] IEC density load (mg TP / cm2) 258.1 30.9

[1244] IEC load concentration 0.4 mg / mL 0.4 mg / mL

[1245]

[1246] This new “Optimal” method incorporates all modifications that have been identified to increase the yield of PQ during the IEC step.

[1247] Besides the elution time used to collect the standard IEC eluate, in all runs, reference and optimal, the extra 4th, 5th and 6th column volumes have been applied and analyzed separately in order to assess the extra protein that can be eluted from the chromatographic column. 4th and 5th CV are two extra column volumes eluted at the same flow rate as a continuation of the standard elution step, but collected aside so that they can be analysed individually. 6th CV is a third extra column volume collected aside after leaving the resin approximately 24 hours in contact with the elution buffer (without applying any flow). That would comprise the “Total elution time”.

[1248] 3. ANALYTHICAL METHODS

[1249] Total protein is quantified by BCA. The method is described in Example 3.

[1250] 4. EXPERIMENTAL DESIGN

[1251] This report presents the results of multiple trials directly comparing five ion exchange chromatography (IEC) step methods, with particular emphasis on the performance of the 4, 5 and 6 column volumes, the extended elution beyond the standard column volume and time. The analysis focuses on protein recovery, elution efficiency, and protein integrity, highlighting how variations in elution time can impact total yield without compromising protein quality.

[1252] 4.1. Protein Q experiments

[1253] Next table summarizes the runs performed in the laboratory. IEC 01 and 02 are the reference conditions (narrow column, which means greater load density, and fast flow, which means short elution time, 21 min). IEC 03 and IEC 05 present intermediate conditions: IEC 03, with narrow column and long elution time and IEC 05, with wide column and short elution time. IEC 04 represents the optimal conditions (wide column, which means lower load density, and slow flow, which means long elution time, 176 min)

[1254] Table 5. 2. List of IEC trials listing different conditions.

[1255] IEC Geometry IEC Elution Load conditions Linear Flow Yield Run Geometry Diameter Height Time TP Ratio TP Cone.

[1256] Rate

[1257] ID ID (cm) (cm) (cm / h) (min) (mg / mL) (mg / mL) (%) IEC 01

[1258] 1NG 1.6 14.3 127 21 8.9 0.4 36% (reference)

[1259] IEC 02

[1260] 1NG 1.6 14.3 127 21 8.9 0.4 29% (reference)

[1261] IEC 03

[1262] 1NG 1.6 14.3 48 + 2h stop 176 8.9 0.4 63% (combined)

[1263] IEC 04

[1264] 2NG 2.6 5.4 21 + 2h stop 168 8.9 0.4 81% (optimal)

[1265] IEC 05

[1266] 2NG 2.6 5.4 127 8 8.9 0.4 41% (combined)

[1267]

[1268] 4.2. Other protein experiments

[1269] We have also analysed this phenomenon in proteins with different identity to Protein Q to further demonstrate whether this protein retention in the column is directly linked with the identity percent with PQ or other physicochemical properties (see table 4.8. in Example 4).

[1270] Each protein was subjected to both IEC methods to assess whether the “Optimal” method can be applied to any protein, or if its effectiveness is restricted by protein sequence, size or isoelectric point. The objective is to prove if proteins with different identity, but same isoelectric point, behave as the PQ and improve the yield by increasing the elution contact time and / or decreasing the protein load density. For this study, we selected three proteins with varying degrees of sequence identity and isoelectric points relative to PQ:

[1271] • Protein 3 (SEQ ID NO:3) is a PQ variant with 87,4% sequence identity and a similar isoelectric point (IP) to PQ (SEQ ID NO:1). • Protein 2 (SEQ ID NO:2) is a recombinant protein designed in our laboratory, exhibiting 59.8% identity to PQ (SEQ ID NO:1) but a different isoelectric point. For Protein 2, it was necessary to adjust the pH conditions during the IEC step to ensure effective binding and elution.

[1272] • Protein 4 (SEQ ID NO:4) is a commercially sourced protein with low (7.4%) sequence homology to PQ (SEQ ID NO:1) but a very similar isoelectric point, confirmed by the supplier.

[1273] Table 5.3. List of proteins tested in the IEC trials and their main properties

[1274] Isoelectric Molecular Name Homology to PQ%* Point** Weight Protein 1 PQ 100 6.8-7.2 42kDa Protein 3 PQ

[1275] HH: High homology variant 87.4 6.34 40kDa MH: Mid homology Protein 2 59.7 4.96 50 KDa LH: Low homology Protein 4 7.4 6.8-7.2 17kDa

[1276]

[1277] *Homology to PQ was calculated using Needleman / EMBOSS method.

[1278] **lsoelectric point was determined using a prediction software Expasy, this estimation could vary depending on the software used.

[1279] Both Protein 2 and Protein 3 are recombinant proteins engineered in our laboratory, while Protein 4 serves as a control to assess the method’s applicability to unrelated proteins with similar physicochemical properties. For reference, sequence homology interpretation follows these thresholds: >30% suggests similarity, 20-30% is uncertain, and <20% is considered random.

[1280] 5. RESULTS

[1281] In this section, the results obtained from the different IEC runs carried out at laboratory scale are presented, first for Protein Q experiments, second for the selected proteins with more or less homology to Protein Q.

[1282] 5.1. Protein Q experiments

[1283] The chromatograms of the 5 IEC carried out with Protein Q are depicted in the next figures 4 and 5 to show how the protein is distributed along the different column volumes eluted (absorbance signal is proportional to total protein).

[1284] In the chromatograms it is clearly seen that applying slowlier elution flow rate and a 2h pause, increases the eluted protein (first and second peaks). Besides, it is seen that after applying a 24h pause, more protein is eluted, meaning higher recovery can be achieved. That is specially pronounced in Experiment 03, when a slow elution flow rate is applied and a narrow column is used, implying high load density.

[1285] Table 5.4. Results from IEC trials (1 to 5). Protein concentrations in all effluent fractions were determined via BCA assay (as described in example 3). Data includes mass balance analysis. IEC trial Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Estimated Load TP

[1286] 0.400 0.400 0.400 0.400 0.400 (mg / mL)

[1287] Load BCA value (mg / mL) 0.438 0.458 0.444 0.440 0.437 Load Protein mass (mg) 265.6 252.3 245.6 255.5 255.5 Load ratio (g / L resin) 8.7 8.9 8.9 8.9 8.9 IEC Flowthrough BCA (< LOQ) < (< LOQ) < (< LOQ) < (< LOQ) < (< LOQ) < 0.25

[1288] (mg / mL) 0.25 0.25 0.25 0.25

[1289] (< LOQ) IEC Wash BCA (mg / mL) (< LOQ) <0.25 (< LOQ) <0.25 (< LOQ) <0.25 (< LOQ) <0.25

[1290] <0.25 IEC Flowthrough Protein

[1291] < 168.5 < 160.2 <156.0 < 162.2 < 162.2 (mg)'

[1292] IEC Wash Protein (mg)‘ < 21.6 < 21.6 <21.6 < 21.5 < 21.5 Eluate BCA (mg / mL) 1.373 1.051 2.465 3.010 1.539 Eluate 4°CV BCA (mg / mL) 0.713 0.501 0.478 (< LOQ) <0.25 0.874 Eluate 5°CV BCA (mg / mL) 0.613 0.428 0.361 (< LOQ) <0.25 0.755 Eluate 6°CV BCA (mg / mL) 1.283 1.309 0.692 (< LOQ) <0.25 1.526 IEC Eluate Protein mass

[1293] 95.6 73.1 171.6 205.9 105.3 (mg)

[1294] 4°CV Eluate Protein mass

[1295] 16.5 11.6 11.1 < 5.7 19.9 (mg)

[1296] 5°CV Eluate Protein mass

[1297] 15.8 11.0 9.3 < 6.8 20.5 (mg)

[1298] 6°CV Eluate Protein mass

[1299] 39.4 40.2 21.2 < 7.7 47.0 (mg)

[1300] IEC Eluate Protein

[1301] 36.0% 29.0% 69.9% 80.6% 41.2% Recovery (%)

[1302] 4 CV Protein Recovery

[1303] 6.2% 4.6% 4.5% 2.2% 7.8% (%)

[1304] 5 CV Protein Recovery

[1305] 6.0% 4.4% 3.8% 2.7% 8.0% (%)

[1306] 6 CV Protein Recovery

[1307] 14.8% 15.9% 8.7% 3.0% 18.4% (%)

[1308] Total Protein Recovery

[1309] 63% 54% 87% 89% 75% (%)

[1310] Recovery increment by

[1311] applying 4,5,6 CV after 27% 25% 17% 8% 34% 24h pause (%)

[1312]

[1313] " Only detected protein values were considered; estimations below the limit of detection (LoD) were excluded.

[1314] The results of Figure 6 demonstrate that efficient protein elution can be achieved even after 24 hours of column incubation showing recovery increments up to 34 %. This extended elution period does not compromise the integrity or profile of the target protein. Importantly, allowing for delayed elution contributes to a higher overall protein yield, offering a practical advantage in downstream processing without introducing variability or degradation. All experiments were conducted at room temperature. Protein stability was assessed by SDS-PAGE and Western blot analysis, both of which demonstrated consistent and robust stability profiles.

[1315] Even with the optimized method, total protein recovery remains incomplete, as a fraction of the protein consistently remains bound to the column. Under optimal conditions in Experiment 04, protein yield increased from 81 % up to 89 % when collecting 4, 5 and 6 CV, indicating potential for further optimization. Improvements could be achieved by extending the elution time beyond 24 hours for all the experiments or by modifying the column geometry. Previous results have demonstrated that buffer contact time with the column and the PQ is the most critical variable influencing yield. Additionally, protein load density, directly related to geometry, is another key factor. Increasing the column surface area reduces PQ crowding, thereby enhancing recovery. Both variables are essential considerations for improving overall yield.

[1316] It has been demonstrated that the amount of protein recovered in the main elution volume correlates with the protein extracted in the subsequent 4th, 5th, and 6th column volumes (CV). The 4th and 5th CVs are essentially a continuation of the main elution, while the 6th CV yielded the highest recovery. This supports the effectiveness of the 24-hour elution protocol and highlights its efficiency in maximizing protein yield.

[1317] 5.2. Other protein experiments (figure 7)

[1318] Table 5.5 gathers the protein yields obtained for all the IEC runs executed at the laboratory with the selected proteins more or less related to Protein Q.

[1319] Table 5.5 Results from IEC trials with proteins with different homologies to Protein Q. Recoveries in all effluent fractions were determined via BCA assay (described in example 3).

[1320] Protein 3 OP Protein 3 REF Protein 2 OP Protein 2 REF Protein 4 OP Protein 4 REF IEC Eluate Protein

[1321] 102% 71% 85% 15% 2% 2% Recovery (% )

[1322] 4°CV Protein

[1323] 0% 8% 3% 4% 0% 0% Recovery (% )

[1324] 5°CV Protein

[1325] 0% 5% 1% 3% 0% 0% Recovery (% )

[1326] 6°CV Protein

[1327] 0% 6% 6% 23%* 0% 0% Recovery (% )

[1328] Total Protein

[1329] 102% 90% 95% 45% 2% 2% Recovery (% )

[1330] Recovery increment

[1331] by applying 4.5.6 CV 0% 19% 10% 30% 0% 0% after 24h pause (%)

[1332]

[1333] *Result analyzed separately due to high BCA signal. Concentration was extrapolated using Prism from OD values; this estimation is less precise but considered reliable. For Protein 4 (figure 7), for both reference and optimal conditions, only 2% of the protein is recovered in the eluate and no extra protein is yielded by including the 4th, 5th nor 6th CV.

[1334] In case of Protein 3 (figure 7), the optimal IEC already shows that all the protein is recovered, but the yield obtained under the referent conditions increases by 19% when including the protein gathered in the 4th, 5th and 6th CV.

[1335] In case of Protein 2 (Figure 7), the yield increases +30% under the referent conditions and +10% under the optimal conditions, achieving a total recovery of 95%. These trials have confirmed that the extended 24-hour elution strategy is also effective for proteins with at least 60% sequence homology to PQ. In addition, the protein profile of the main IEC eluate, the 4th, 5th and 6th CV have been analyzed by SDS-PAGE and Western Blott proving that both proteins 2 and 3 keep stable after 24h at room temperature in contact with the elution buffer (6th CV fraction). These results suggest that the approach may be applicable beyond the specific PQ construct, potentially benefiting purification processes for related proteins with similar structural or binding characteristics.

[1336] 6. CONCLUSIONS

[1337] Several IEC experiments have been compared at laboratory scale in order to prove whether prolonging the elution time up to 24h could increase protein recovery meanwhile keeping product quality. The standard eluate collection is from 0.8 to 3.14 column volumes (CV); however, the elution phase continues, and an extra 4th and 5th CV are collected and analyzed aside. The elution flow is stopped and the IEC column is left immersed in elution buffer for 24h. Then, a final 6th CV is collected. By adding the protein quantified in the 4th, 5th and 6th CV to the standard eluate, it has been checked that IEC yield can increase up to 34% when applying the reference conditions or up to 19 % when applying the optimal conditions in Protein Q trials.

[1338] The comparative analysis of both IEC methods, reference and optimal, reveals that both still have margin to improve protein recovery due to a portion of the PQ protein remains bound to the column despite procedural improvements. Notably, elution after 24 hours has proven to be a viable and effective strategy, enabling the recovery of additional protein without compromising its structural integrity or stability.

[1339] Additional experiments with different proteins to PQ confirm that this extended elution strategy is also effective for proteins with at least 60% sequence homology to PQ. For Protein 4, with no homology to PQ, no yield improvement is experienced. In case of Protein 3 (88% homology), the optimal IEC already shows that all the protein is recovered, but the yield obtained under the referent conditions increases by 19% when including the protein gathered in the 4th, 5th and 6th CV. In case of Protein 2 (60% homology), the yield increases +30% under the referent conditions and +10% under the optimal conditions, achieving a total recovery of 95%. In addition, the protein profile of the main IEC eluate, the 4th, 5th and 6th CV have been analyzed by SDS-PAGE and Western Blott proving that proteins keep stable after 24h at room temperature in contact with the elution buffer (6th CV fraction). These results suggest that the approach may be applicable beyond the specific PQ construct, potentially benefiting purification processes for related proteins with similar structural or binding characteristics.

[1340] These results highlight opportunities for further optimization, either by extending the elution time beyond 24 hours or by decreasing the load density by modifying column geometry (wide columns offer better results than narrow columns), strategies that have already shown promising results.

[1341] Moreover, increased contact time of the I EC resin with the elution buffer has shown to improve recovery yields for these homologous proteins and PQ, again without affecting their integrity. These findings support the feasibility of implementing broader elution time windows, offering enhanced process flexibility from minutes to hours -or even days- without compromising protein quality.

[1342] Example 6 Ionic Exchange Chromatography (IEC) Optimization Applied to a randomized Protein, non-homologous to Protein Q segue nee

[1343] 1. INTRODUCTION

[1344] This work focuses on the Ion Exchange Chromatography (IEC) step that purifies Protein Q (PQ) from the eluate of the previous purification step using a strong cationic quaternary amine.

[1345] This addendum provides supplementary data and analysis aimed at expanding the range of proteins evaluated in example 4. Specifically, the main aim is to evaluate whether the newly implemented method is universally applicable across diverse proteins, or if its effectiveness is primarily dependent on protein properties as the amino acid sequence or the isoelectric point. Both reference and optimized conditions were tested on various proteins with varying sequence homology to Protein Q, and in this document, we focus on a protein featuring a randomized PQ (rPQ) sequence with very low homology to PQ (lower than 20%) while preserving the same isoelectric point and molecular size. The more effective way to achieve this it was to randomize the PQ sequence.

[1346] 2. Small scale process description

[1347] The IEC step has been scaled down from the current existing industrial process. To achieve this, the dimensions and the resin of the current industrial column were taken as a reference to design the laboratory trials.

[1348] Conditioned eluate from the previous purification step is used as starting material for the laboratory trials. It means, the product to be loaded on to the IEC has been adjusted to pH = 10, urea = 4 M and TP < 0.5 g / L. The required mg of total protein to be loaded onto the IEC for each run are calculated to start from the right product volume. This is made based on:

[1349] • Load density: Amount of TP to be loaded on to the column per column section. The reference according to the current ISP is < 127 mg / cm2.

[1350] • Load ratio: Amount of TP to be loaded on to the column per mL of resin. The reference according to the current ISP is < 8.9 mg / mL.

[1351] • Load concentration: TP concentration of the IEC load. The reference according to the current ISP is < 0.5 g / L. The calculated necessary volume per run is tempered and homogenized in gentle agitation. If needed, pH is adjusted to 10.0 ± 0.2. Then, the product is filtered before being loaded onto the column. IEC chromatography is run in an FPLC chromatograph, and the method consists of the following stages:

[1352] • Equilibration with 4 CV IEC Equilibrium and Washing Sol., pH 9.0

[1353] • Product load, CV variable depending on product concentration.

[1354] • Column wash with 3 CV Dilution and equilibrium buffer

[1355] • Elution with 3.14 CV IEC Elution buffer, pH 8.0

[1356] • 4thCV Elution: 1 CV IEC Elution Sol., pH 8.0

[1357] • 5thCV Elution: 1 CV IEC Elution Sol., pH 8.0

[1358] • Over Night 6thCV Elution: 1 CV IEC Elution Sol., pH 8.0

[1359] The elution flow rate in the current industrial scale process (ISP) is 40 L / h (127 cm / h), which results in an elution time of 21 min. Elution flow rates are adjusted in the method depending on the conditions to be tested in each run. The elution peak is gathered according to the column volumes stated in the current process: 2.34 CV are collected after running 0.8 CV of elution phase. Additionally, as previously observed with PQ, we added three extra column volumes (4th, 5th& 6thCV) after the elution step to assess whether the protein remained bound to the resin. This allowed us to monitor residual bound protein. We have referred to this IEC step method as the “Reference” method, which serves as the standard used in the ISP.

[1360] Based on this model, a series of modifications have been implemented, and we refer to the newly optimized method as the “Optimal” method. The different process parameters applied in the “Reference” and the “Optimal” trials are compared in the following table:

[1361] Table 6.1. Comparison between IEC methods “Reference” and “Optimal” at laboratory scale Reference Optimal

[1362] Narrow Wide

[1363] Column Geometry

[1364] H 14.3 cm, D 1.6cm H 5.4 cm, D 2.6cm

[1365] Elution linear flow rate (cm / h) 127 cm / h 15 cm / h

[1366] Elution time (min) 21 168 (2 hours stop)

[1367] Ratio TP g / L resin 8.9 6

[1368] IEC density load (mg TP / cm2) 258.1 30.9

[1369] IEC load concentration 0.4 mg / mL 0.4 mg / mL

[1370]

[1371] This new “Optimal” method incorporates all modifications that have been identified to increase the yield of PQ during the IEC step.

[1372] 3. ANALYTHICAL METHODS Total protein is quantified by BCA. The method is described in example 3.

[1373] 4. EXPERIMENTAL DESIGN

[1374] We designed a randomized sequence derived from the PQ amino acid template and conducted extensive in silico simulations to assess its structural stability and expression efficiency in E. coli. The construct maintains the original isoelectric point while reducing sequence homology to Protein Q to the lower score possible, to increase its singularity. Additionally, a polyhistidine tag (His-tag) and the monoclonal antibody (mAb) binding site were incorporated to facilitate purification and detection of the randomized protein (rPQ).

[1375] rPQ sequence (SEQ ID NO:5):

[1376] MADPVVGVLIARTDGDTTEGDVISLGASAGFVKDAESALKPIKARNPGKASAGPVSANLGSGLDSFDA LVAEGRTKSARAADRVAKYVSLDAGSPHGLADANAEMVAVYEKEVRSEAPFVCTSELEDGLKLRNM KKNMADVPKLFSPVISSDDVGDLIAAAFMERNSSPAPESVAGSKSQVNPGANAPVQEDVAGENAGL SEAAYEATMGEGSVTADNSNCPMLAGNAQTLASAPKARWSASLYSDAKAGAASEVGLPDAFTAAA AQDGGAAMAASAAEAGKISIKSREEGQPAMSARKSIDTAFTITQPPPALTKAVACGAPAPKSQGSYR AEAGKMEGAGHAMSHAKRRGHAMAAVDKLARRKVSGFSVAKRKQNSGGAMGKGKPRKAEPAAKL APVCKDDADEHHHHHH

[1377] Table 6.2. List of proteins tested in these trials and their main properties

[1378] Isoelectric Molecular Name Homology to PQ%* Point** Weight Protein 1 PQ 100 6.8-7.2 42kDa LH: Low homology Protein 5 (rPQ) 18.8 6.8-7.2 42kDa

[1379]

[1380] " Homology to PQ was calculated using Needleman / EMBOSS method.

[1381] "" Isoelectric point was determined using a prediction software Expasy, this estimation could vary depending on the software used.

[1382] 4.1 EMBOOS / Needle alignment PQ (SEQ ID NO:1) and rPQ (SEQ ID NO:5): identity %

[1383] # Aligned- sequences: 2

[1384] # 1: PQ

[1385] # 2: rPQ

[1386] # Matrix: EBLOSLR62

[1387] # Gap-penalty: 10.0

[1388] # Extend_penalty: 0.5

[1389] # Length: 528

[1390] # Identity:

[1391] # Similarity:

[1392] # Gaps: 231 / 528 (43.8JS)

[1393] # Score: 106. 5

[1394]

[1395] -

[1396] -

[1397] -

[1398] -

[1399] -

[1400] - -

[1401] - - -

[1402] -

[1403] >

[1404]

[1405] -

[1406] For reference, sequence identity is commonly interpreted using the following thresholds: values greater than 30% generally indicate significant similarity and potential structural or functional conservation; identities between 20-30% fall into an ambiguous zone where homology cannot be confidently inferred; and identities below 20% are typically considered random, suggesting no meaningful evolutionary relationship. The rPQ construct exhibits sequence identity below these thresholds, placing it in the category of low or residual homology relative to Protein Q. This new variant was tested in several trials to evaluate its behaviour under modified process conditions and to determine whether sequence homology or physicochemical properties play a critical role in the application of the optimized process.

[1407] 5. RESULTS

[1408] For all the runs, the total protein was quantified by BCA, so that protein recoveries could be compared in every step. The IEC step yield was calculated as g TP in IEC eluate / g TP in IEC load.

[1409] Table 6.3. Direct comparison of IEC methods (Optimal vs. Reference) for rPQ, low homology trial. Yields and mass balance. IEC IEC

[1410] TP / mL Resin 3.9 5.5

[1411] Elution Flow rate 15cm / h + 2h stop 127cm / h

[1412] IEC OPTIMAL REFERENCE

[1413] [TP] (pg / mL) 258 283

[1414] IEC load before filtration Vol imLi 425 600

[1415] TP <nir|) 110 170

[1416] [TP] (pg / mL) 263 268

[1417] IEC load Filtered 0.2 pm Vol (mL) 425 600

[1418] TP (mg) 112 161

[1419] [TP] (pg / mL) 239 225

[1420] Flow Through

[1421] TP <nir|) 102 135

[1422] [TP] (pg / mL) 50 68

[1423] Column wash

[1424] TP (mg) 6 11

[1425] 4CV [TP] (pg / mL) cLOD < LOD

[1426] [TP] tpg mb cLOD < LOD

[1427] [TP] i pg mL) cLOD cLOD

[1428] Sum 4+5+6 CV TP (mq) < LOD CLOD

[1429] [TP] (pg / mL) cLOD cLOD

[1430] IEC eluate Vol (mL) 68.7 68.7

[1431] TP (mg) CLOD CLOD

[1432] Yield Filtration 0.2 pm 102% 95%

[1433] Yields Yield IEC 0% 0%

[1434] Global Yield filtrate +

[1435] 0% 0%

[1436] IEC FT 91% 84%

[1437] CW 5% 7%

[1438] ELUATE 0% 0%

[1439] Balance 4CV 0% 0%

[1440] 5CV 0% 0%

[1441] 6CV 0% 0% COLUMN / LOD 4% 9%

[1442]

[1443] The results indicate that the IEC conditions used in the Protein Q process are not suitable for rPQ. Furthermore, the optimal method does not provide any significant improvement over the reference method, as protein recovery is 0% in both trials. It appears that the protein does not adsorb onto the resin, since most of the solubilized protein is lost during column loading (flowthrough, FT) rather than being recovered in the eluate.

[1444] It is worth noting that the product was calculated to be loaded onto the IEC at an approximate concentration of TP = 400 pg / mL; however, as shown in the results, this was not achieved, with the actual concentration being < 300 pg / mL, suggesting some instability under the applied conditions (pH 10, 4 M urea). Nevertheless, this deviation does not explain the zero-yield observed in the eluate. These results are consistent with previous I EC trials using other proteins (example 4) the proposed changes in the I EC chromatographic step are valid for proteins with homology > 60% relative to the Protein Q sequence. For proteins with low homology (< 20%), such as myoglobin (Protein 4) and rPQ, this process would not be effective, despite having similar isoelectric points; or even the same molecular weight and amino acid composition, although in a different order, as in the case of rPQ. This highlights that the proposed changes to the IEC process are neither obvious nor universally applicable to all proteins.

[1445] 6. CONCLUSIONS

[1446] After confirming that the proposed modifications to the ion-exchange chromatography (IEC) process for Protein Q also improve yield in other proteins with > 60% sequence homology to PQ, the study was extended to include a non-homologous protein.

[1447] The test protein was designed by randomizing the amino acid sequence of PQ while retaining the histidine tag for purification purposes and the anti-PQ antibody binding site for ELISA and Western blot analysis. Additionally, it preserved the same isoelectric point and molecular weight as Protein Q, and its stability and solubility in the target buffer were evaluated.

[1448] This new variant, termed rPQ (randomized Protein Q), was expressed and processed using both the reference IEC method for Protein Q (loading density: 258 mg TP / cm2; elution time: 21 min) and the optimized method (loading density: 31 mg TP / cm2; elution time: 168 min, including a 2-hour flow stop).

[1449] Unexpectedly, analysis of rPQ, despite sharing the same isoelectric point and amino acid composition, albeit in a different order, and exhibiting very low sequence homology to PQ, revealed that it did not bind to the IEC resin.

[1450] The minimal amount of protein recovered in the eluate from both methods indicates that the optimized process offers no improvement over the reference method. A similar effect was observed with other low-homology proteins, such as Protein 4 (myoglobin), which also failed to bind to the column despite having the same isoelectric point. These findings suggest that the method is not universally applicable; its success appears to require a certain degree of sequence similarity to PQ and solubility / stability in process buffers containing urea and high pH. Sequence homology emerges as a decisive factor for effective column binding and recovery, indicating that structural or sequence-related features play a more critical role than general physicochemical properties.

[1451] Based on these results, it can be inferred that for proteins with low sequence homology to ProtQ (19%, considered residual), the IEC process modifications do not lead to any increase in protein yield. Although rPQ shares physicochemical characteristics with ProtQ — such as isoelectric point, molecular weight, and amino acid composition — the optimized method Description of the figures

[1452] Figure 1. Correlation between IEC yield and elution time for the narrow column (1NG) and wide column (2NG).

[1453] Figure 2. Correlation between IEC yield and load density.

[1454] Figure 3. IEC yield % distributed into the different protein load concentration for each column geometry tested.

[1455] Figure 4. IEC Experiments 1, 2 and 3 chromatogram overlay.

[1456] Figure 5. IEC Experiments 4 and 5 chromatogram overlay.

[1457] Figure 6, PQ IEC Experiments 1 to 5 Protein Recovery comparison.

[1458] Figure 7. Proteins 2, 3, and 4 IEC Experiments Protein Recovery comparison.

Claims

CLAIMS1. A process for purifying a protein comprising a step of purifying said protein from a mixture by a chromatography column, using at least one column comprising a resin,wherein the total elution time is ranged from 80 minutes to 24 hours, preferably from 160 to 210 minutes, even more preferably from 168 to 202 minutes.

2. A process according to claim 1, wherein the mixture to be loaded to the column, also called the load mixture comprising said protein is characterized by its load density, which is ranged from 20 to 247mg TP / cm2.and optionallythe load ratio of total protein (TP) to the resin, ranged from 5.0 to 12 mg TP / mL resin; and / or the load concentration, ranged from 0.15 to 0.60 mg / mL.

3. A process according to claim 1 or 2, wherein the isoelectric point of the protein is from 1 to 3 units lower than the pH of the load mixture.

4. A process according to any one of the preceding claims, wherein the protein is soluble and / or stable in the presence of a chaotropic agent like urea,5. A process according to any one of claims 1 to 4, wherein the elution linear flow rate of the column is ranged from 10 to 127 cm / h, preferably from 15 to 127 cm / h, more preferably said linear flow rate is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 125, 126, 127 cm / h.

6. A process according to any one of claims 1 to 5, wherein a flow stop of at least 0.5, 1, 1.5, 2, 2.5, up to 24 hour is carried out when eluting the column, preferably said flow stop is 2 hour.

7. A process according to claim 6, wherein the flow stop is carried out during the elution phase, preferably in the middle of the elution phase.

8. A process according to any one of claims 2 to 7, wherein the load ratio is higher than 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, and / or lower than 12, 11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11.0, 10.9, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, 10.0, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6 mg TP / mL resin; preferably the load ratio is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, or 6.1 mg TP / mL resin.

9. A process according to any one of claims 2 to 8, wherein the load density is higher than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 mg TP / cm2, and / or lower than 247, 160, 155,150, 145, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95 TP / cm2; preferably the load density is ranged from 20-150, 30-140, 35-135, 40-130, 45-125, 50-120, 55-115, 60-110, 65-110, 70-105 mg TP / cm2, preferably ranged from 25-65, 25 -60, 28-58, 20-55, 20-50, 25-40, 28-35, 29-32, 31-55, 40-65, 45-60, 50-56 mg TP / cm2, more preferably is 31 and / or 55 mg TP / cm2.

10. A process according to any one of claims 2 to 9, wherein the load concentration is higher than 0.2, 0.21, 0.22, 0.23, 0.24 and / or lower than 0.55, 0.54, 0.53, 0.52, 0.51 mg / mL; preferably the load concentration is 0.38, 0.39, 0.40, or 0.41 mg / mL.

11. A process according to any one of claims 2 to 10, wherein:-the total elution time is ranged from 160 to 210 minutes, even more preferably from 168 to 202 minutes; and / or- the load density is ranged from 20-247 mg TP / cm2;And optionally:the load ratio is ranged from 5.5 to 11.5 mg TP / mL resin;the load concentration is ranged from 0.20 to 0.55 mg / mL;the flow rate is ranged from 10 to 127 cm / h; and / orthe flow stop is ranged from 1.5 to 2.5 hour.

12. A process according to any one of claims 2-10, wherein:-the total elution time is ranged from 160 to 210 minutes, even more preferably from 168 to 202 minutes; and / or- the load density is ranged from 30-55mg TP / cm2;And optionallythe load ratio is ranged from 5.9 to 6.1 mg TP / mL resin;the load concentration is ranged from 0.35 to 0.45 mg / mL;the flow rate is ranged from 10 to 127 cm / h; and / orthe flow stop is ranged from 1.5 to 2.5 hour.

13. A process according to any one of the claims 2-10, wherein:-the total elution time is ranged from 160 to 210 minutes, even more preferably from 168 to 202 minutes; and / or- the load density is ranged from 30-247 mg TP / cm2;And optionallythe load ratio is ranged from 5.5 to 11.5 mg TP / mL resin;the load concentration is ranged from 0.20 to 0.55 mg / mL;the flow rate is ranged from 10 to 127 cm / h; and / orthe flow stop is ranged from 1.5 to 2.5 hour.

14. A process according to any one of the claims 2-10, wherein:-the total elution time is ranged from 160 to 210 minutes, even more preferably from 168 to 202 minutes; and / or- the load density is ranged from 30-55 mg TP / cm2;And optionallythe load ratio is ranged from 5.9 to 6.1 mg TP / mL resin;the load concentration is ranged from 0.35 to 0.45 mg / mL;the flow rate is ranged from 10 to 127 cm / h; and / orthe flow stop is ranged from 1.5 to 2.5 hour.

15. A process according to any one of the preceding claims, wherein the column is an IEC (Ion Exchange Chromatography) column.

16. A process according to claim 15, wherein the IEC column is made of a polymer, glass, stainless steel, inert plastic, or metal; and / or the IEC column comprises the resin having quaternary amine (Q) functional group.

17. A process according to any one of the preceding claims, wherein the protein is represented by an amino acid sequence having at least 60% identity or similarity with SEQ ID NO:1, preferably having 80, more preferably 100% identity of SEQ ID NO:1.

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