Lysate concentration

Abstract

The current invention provides an improved manufacturing process, in particular improved downstream processing, of pharmaceutical products especially comprising proteins, useful for the prevention or treatment of leishmaniosis. The pharmaceutical composition may comprise a protein chimera Q, which is the product of a chimeric gene encoding the antigenic determinants of four proteins of Leishmania infantum.

Description

[0001] Lysate concentration - P62038693WO 1

[0002] Lysate concentration

[0003] DESCRIPTION

[0004] Field

[0005] 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, in particular canine leishmaniasis, further defined herein.

[0006] Background of the invention

[0007] Proteins are widely utilised in the pharmaceutical and medical industries as hormones, therapeutic medications, vaccinations, and / or clinical research. The biotechnology industry today employs recombinant bacteria, mammalian cells, and transgenic animals for the production of high-value therapeutic proteins. 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. For example, biopharmaceutical downstream processing is applicable in monoclonal antibody (mAb) or protein processes, as well as in the manufacture of polysaccharides and / or various vaccines. Upscaling of the manufacturing and / or downstream processing (also known as DSP) was required to meet the needs of the industries. Such upscaling could only have been made possible with optimization of these processes. The concentration of protein production and protein purification have been steadily improved over the years.

[0008] In order to make the manufacturing process economically more feasible, improvements at each step of the process are needed. As observed in recent years, historical investment in upstream cell line development, metabolite feed controls, biomass improvements and product output per biomass have collectively made large scale bioprocesses economically and engineering feasible. Upstream and downstream processes altogether comprise the production process of biopharmaceuticals. The consequence of such modernization and upstream improvement has now placed new burdens and bottlenecks on most downstream processing performances, such as significant yield loss and process inefficiencies. The upstream improvements and new challenges imposed on downstream processing have put a new focus on downstream improvements and investments. For instance, most of the biotechnology proteins are present in complex mixtures of products and this makes the task of purifying these molecules very difficult. Moreover, it is suggested that the downstream processing costs 70% of the total production cost of a particular biopharmaceutical. Hence, improving the downstream processing steps is crucial for also establishing an economically sustainable production method (Mehta, A. (2019). Downstream Processing for Biopharmaceuticals Recovery. In: Arora, D., Sharma, C., Jaglan, Lysate concentration - P62038693WO 2

[0009] S., Lichtfouse, E. (eds) Pharmaceuticals from Microbes. Environmental Chemistry for a Sustainable World, vol 26. Springer, Cham).

[0010] Despite the fact that a downstream process may function effectively at a laboratory scale, upscaling to an industrial scale might present numerous difficulties. The efficacy of a separation procedure might decrease at a greater scale, or it may become more challenging to sustain uniform concentration, temperature or pH levels. The expenses associated with downstream processing might escalate substantially with scale.

[0011] This invention in particular focuses on the process of producing, or at least downstream processing, 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 Leishmania infantum (L. infantum) is the source of visceral leishmaniasis (LV) in canines and humans. It is extensively dispersed over various Mediterranean regions. Dogs infected by L. 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.

[0012] 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).

[0013] 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 downstream processing of such protein. It is further the object of the invention to design an improved manufacturing and / or downstream processing that may be implemented in producing other proteins, especially other proteins that are highly homologous with protein Q. It is further the object of the invention to optimize manufacturing and / or downstream processing proteins in, for instance, a laboratory scale as well as an industrial scale.

[0014] Summary of the invention

[0015] In one aspect of the invention, there is provided a process for downstream processing 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, from a biomass comprising cells, wherein said downstream processing comprising the following steps:

[0016] suspending the biomass in a suspension buffer;

[0017] subsequently lysing the cells present in the suspended biomass to obtain a lysate; and Lysate concentration - P62038693WO 3

[0018] further downstream processing the protein from said lysate;

[0019] wherein the Total Protein (TP) concentration in the suspended biomass before and / or after the lysing step is ranged from 2 to 12 g / L.

[0020] Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L, or optionally the TP concentration before adjustment and / or dilution is 11 g / L. Preferably, suspending biomass to 10, 11, 12 g / LTP concentration is configured for a reference process or when a further dilution is applied post lysis.

[0021] In one embodiment of this aspect, there is provided a process wherein the TP concentration in the suspended biomass before and / or after the lysing step is ranged from 2 to 7 g / L or 2 to 6 g / L.

[0022] In one embodiment of this aspect, there is provided a process wherein the TP concentration in the suspended biomass before and / or after the lysing step is ranged from 2.5 to 3.5 g / L.

[0023] In one embodiment of this aspect, there is provided a process wherein the TP concentration is kept as the indicated value by carrying out a diluting step before and / or after the lysing step.

[0024] In one embodiment of this aspect, there is provided a process wherein the further downstream processing step comprises the following steps: a solubilizing step, followed by a clarifying step.

[0025] In one embodiment of this aspect, there is provided a process wherein the TP concentration in the biomass suspension before lysis is adjusted up to 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9. 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, 10.0, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, or up to 12.0 g / L. In one embodiment of this aspect, there is provided a process wherein the TP concentration in the lysate is adjusted up to 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / L.

[0026] In one embodiment of this aspect, there is provided a process wherein the TP concentration of the lysate is adjusted up to 7.7, 7.8, 7.9, or up to 12.0 g / L.

[0027] In one embodiment of this aspect, there is provided a process wherein the process or at least part of it is carried out at a temperature ranged from 1°C to 10°C or is 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C.

[0028] In one embodiment of this aspect, there is provided a process wherein the part of the process which is carried out at the temperature range of 1°C to 10°C is the lysis step and the subsequent downstream processing steps, in particular at least during the solubilizing step.

[0029] In one embodiment of this aspect, there is provided a process wherein the isoelectric point of the protein is from 1 to 3 units away from the pH of the lysate (lower or higher). In one embodiment of this 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 lysate. In an embodiment, the pH of the lysate is comprised between 7.5 and 10.0 or is Lysate concentration - P62038693WO 4

[0030] 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9,9 or 10.0.

[0031] In one embodiment of this aspect, there is provided a process wherein the TP concentration is adjusted up to 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / L, preferably by diluting before and / or after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 to 10.0. The lysate is kept at such temperature preferably for example, but not limited to, on ice, jacketed tanks, and / or chillers.

[0032] In one embodiment of this aspect, there is provided a process wherein the TP concentration is adjusted up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / L, preferably by diluting before and / or after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, at least during the lysis and downstream step, and optionally the lysate pH is ranged from 7.5 to 10.0. The lysate is kept at such temperature preferably for example, but not limited to, on ice, jacketed tanks, and / or chillers. In one embodiment of this aspect, there is provided a process wherein the TP concentration is adjusted up to 7.8, or 7.9, or 12.0 g / L, preferably by diluting before and / or after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 to 10.0. The lysate is kept at such temperature preferably for example, but not limited to, on ice, jacketed tanks, and / or chillers.

[0033] In one embodiment of this aspect, there is provided a process wherein the protein is represented by an amino acid sequence having at least 60% or at least 80% identity or similarity with SEQ ID NO:1, preferably having 100% identity of SEQ ID NO:1.

[0034] Detailed description of the invention

[0035] 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.

[0036] The inventors surprisingly discovered that the downstream processing of a protein of interest (also referred to as POI) such as the one having at least 80% identity or similarity with SEQ ID NO:1 as described in this invention and characterised by maintaining a relatively low Total Protein (TP) concentration (ranged from 2 to 12 g / L) in the suspended biomass before and / or after lysing the cell comprised in said biomass leads to an improvement of the amount of TP, and more surprisingly to a further improvement of the amount of the protein of interest recovered (increase of up to 44% Protein Q, increase of up to 9% of TP, with a ratio PQ / TP that is increased from 10 to 13%, see examples 1 and 2). Quite attractive results were obtained applying a dilution to the suspended biomass before lysing the cells to keep the TP concentration as 2-12 g / L Lysate concentration - P62038693WO 5

[0037] It is to point out that the quality of the protein of interest is not affected by these optimisations of the downstream process. Furthermore, the quantity of the protein of interest is increased through the optimization of the downstream process as demonstrated in the experimental part (Examples 1 and 2). Moreover, the inventors surprisingly discovered that the downstream processing of the invention could be successfully applied to a protein of interest having at least 60 % identity or similarity with SEQ ID NO:1 (Protein 2 has 59,8% identity with Protein Q and Protein 3 has 87.4% identity with Protein Q) by maintaining a relatively low Total Protein (TP) concentration (3 g / L) in the suspended biomass before lysing the cells comprised in said biomass. This process leads to an improvement of the amount of TP, and more surprisingly to a further improvement of the amount of the protein of interest recovered (increase of up to 19% for both Proteins 2 and 3 tested, increase of up to 39% of TP in the case of Protein 3 (and 10% for Protein 2),( see example 5).

[0038] It is to point out that the quality of the protein of interest is not affected by these optimisations of the downstream process. Furthermore, the quantity of the protein of interest is increased through the optimization of the downstream process as demonstrated in the experimental part (Example

[0039] 5). Downstream processing

[0040] Biopharmaceutical production is primarily categorised into two principal processes: upstream and downstream, each encompassing multiple steps. Upstream processes, as known in the art, focus on converting the substrate into targeted products, whereas downstream processes concentrate on purifying the intended product with high yield (Gronemeyer P, Ditz R, Strube J (2014) Trends in upstream and downstream process development for antibody manufacturing. Bioengineering 1 (4):188-212). In an embodiment, downstream processing of a protein may also be called purifying said protein. Downstream processing (also known as DSP) refers to the purification (and thus recovery) of a substance typically a product from natural sources, such as animal, plant, or bacterial cells. The source could also come from fermentation broth. This process is beneficial for use in the manufacture of antibiotics, hormones, antibodies, vaccines, diagnostic enzymes, industrial enzymes, and / or natural fragrance and flavor compounds. The main purpose of DSP is to remove contaminants and / or impurities.

[0041] 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.

[0042] The aim of carrying out downstream processing remains the same whether the biomolecule of interest is produced by the transformed cells intracellularly or extracellularly, although there are differences relating to the procedure of each method. In case of extracellular production of target biomolecule, the culture medium is usually concentrated, and then purification can be carried out. In case the target biomolecule is produced intracellularly, it is important to harvest by lysing the cells, followed by contaminants removal. In this invention, it is preferable to produce the protein of interest intracellularly. The advantage of producing the protein of interest intracellularly is that the cells can produce higher yields of protein because the protein is not limited by the secretion capacity of the cell, and / or the protein Lysate concentration - P62038693WO 6

[0043] may be less prone to degradation from extracellular conditions, and / or it may be more cost-effective as it may not require medium optimization used for extracellular production. It is also viable to produce the protein of interest extracellularly, for easier purification steps.

[0044] The term “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.

[0045] The biomolecules could be polymers. 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. These covalent bonds are crucial for the structure and function of biomolecules, providing stability and enabling the formation of complex molecular structures.

[0046] The term “biomass” refers to a total mass of living organisms within a specific area or ecosystem at a particular moment. It encompasses flora, fauna, microbes, and their derivatives. Biomolecules are the building blocks of biomass. Essentially, biomass comprises biomolecules, as they make up the tissues and structures of living organisms. The biomolecule comprises the protein of interest in this invention. The protein of interest is preferably a recombinant protein, even more preferably a recombinant overexpressed protein. The protein of interest is preferably protein Q represented by SEQ ID NO:1 or having at least 80% identity or similarity with SEQ ID NO:1.

[0047] In one aspect of the invention, there is provided a process for downstream processing a protein, preferably represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:1, from a biomass comprising cells, wherein said downstream processing (purification) comprising the following steps:

[0048] suspending the biomass in a suspension buffer;

[0049] subsequently lysing the cells present in the suspended biomass to obtain a lysate; and further downstream processing (or further purifying) the protein from said lysate; wherein the Total Protein (TP) concentration in the suspended biomass before and / or after the lysing step is ranged from 2 to 12 g / L.

[0050] A preferred process of this aspect for downstream processing a protein from a biomass comprising cells, is such that said downstream processing process comprising the following steps:

[0051] • suspending the biomass in a suspension buffer,

[0052] • subsequently lysing the cells present in the suspended biomass to obtain a lysate, and

[0053] • further downstream processing the protein from said lysate;

[0054] wherein the Total Protein (TP) concentration in the suspended biomass is kept at a range from 2 to 12 g / l by carrying out a diluting step before the lysing step. Lysate concentration - P62038693WO 7

[0055] As described herein, the first steps of downstream processing described in this invention comprise biomass harvesting, biomass suspension and biomass disruption to obtain a lysate. In one embodiment of this aspect, it is provided a process wherein further downstream processing steps comprise the following steps: a solubilizing step, followed by a clarifying step. A clarification step may be a centrifugation, a filtration, and / or a combination thereof. Each of these steps are further described herein in details and are applied in the experimental part. The process of the invention may comprise further steps. In an embodiment, it may comprise additional downstream processing steps.

[0056] Biomass harvesting

[0057] This step comprises a collection of the fermentation broth comprising the biomass ofthe invention. After fermentation or cell culture, the biomass (cells) is typically in suspension. One of the initial processes in downstream processing is the separation of cells from the culture supernatant. This can be accomplished by techniques such as centrifugation.

[0058] As used herein, the term “supernatant” or “culture supernatant” refers to the liquid portion of a cell culture that remains after the target cells or other particulates (biomass) have been removed, typically by centrifugation. It is generally the fluid above the pellet of cells or solid debris. Centrifugation is generally used to separate the cells comprising biomass or protein of interest from the culture (liquid) medium.

[0059] In this invention, the objective ofthe centrifugation is to separate the biomass from its surrounding broth, for example to separate the culture supernatant from the solid phase biomass.

[0060] Biomass suspension (or solubilization)

[0061] The solubilisation of the protein of interest is an important phase in downstream processing. The procedure entails dissolving or extracting the protein of interest from its source material into a solution, hence facilitating subsequent purification and processing.

[0062] In an embodiment, the biomass of the invention is suspended. The suspended biomass refers to a mixture containing cells (biomass) of interest suspended in a liquid medium, such as a suspension (and / or solubilization) buffer. By adding the buffer to the biomass, the protein of interest and / or other soluble compounds can be solubilized. The suspension may be performed before the centrifugation, after the centrifugation, or both. In an embodiment, the biomass after centrifugation is preferably resuspended, in particular as such the protein of interest is solubilized at this stage. Suspending biomass post-harvest and centrifugation is a crucial phase in downstream processing, especially when dealing with intracellular compounds. Following biomass harvesting via centrifugation, the obtained biomass ofthe invention is usually suspended in an appropriate buffer or solution. The buffer used in this invention, such as the suspension buffer used in this invention, preferably comprises monosodium Lysate concentration - P62038693WO 8

[0063] phosphate dihydrate (NaH2PO4.2H2O), urea (CH4N2O), sodium chloride (NaCI), and / or sodium hydroxide (NaOH), and optionally a non-ionic detergent (or a non-ionic surfactant).

[0064] In an embodiment, Triton, in particular Triton X-100 (Ci4H2i(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 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.

[0065] In an embodiment, it is encompassed that the suspension buffer is cold, such as at a temperature between 1-10 °C or is 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C, to preserve product integrity and prevent degradation. In a preferred embodiment, the biomass is suspended or solubilized in the cold biomass suspension buffer.

[0066] In an embodiment, the suspension (or resuspension) step is performed prior to the lysis step, for preparing the biomass or cells for lysis. The suspension step further allows for washing the cells to remove the remaining contaminants. Furthermore, the suspension step also allows for adjustment of the concentration of the biomass used in the invention for the subsequent processing steps.

[0067] As used herein, the biomass, suspended biomass, and / or the resuspended biomass used in the invention may comprise the total protein (TP). The total protein (TP) may include the protein of interest (for instance, protein of the invention or 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 mixture. The Total Protein (TP) concentration in the suspended biomass before and / or after the lysing step is ranged from 2 to 12 g / L. The TP concentration ranged from 2 to 12 g / L (or is at least 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9. 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, 10.0, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, or 12.0 g / L) is suitable for both laboratory scale and industrial scale (plant scale).

[0068] By properly suspending the harvested biomass, in particular by the suspension step of the process of this invention, the recovery of target products and the overall efficiency of the downstream processing workflow can be enhanced. Lysate concentration - P62038693WO 9

[0069] Biomass disruption (or lysis)

[0070] The term “disruption” is a broader term that encompasses various methods used to break open biomasses or cells, including but not limited to lysis. Lysis refers to the breakdown of a cell caused by damage to its plasma (outer) membrane. In this invention, the term “disruption” and “lysis” may be used interchangeably unless otherwise indicated. Generally, the primary objectives of biomass disruption are to for instance, breakdown cell walls and membranes; release of intracellular constituents therein; and / or improve the efficacy of subsequent extraction procedures.

[0071] As used herein as a preferred example, the downstream processing comprises the disruption or lysis step, wherein the breaking of the microbial membranes in order to extract the cytoplasmic compartment where 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) is present.

[0072] In an embodiment, the TP concentration in the suspended biomass before and / or after the lysing step is ranged from 2 to 7 g / L or 2 to 6 g / L, preferably adjusted by dilution.

[0073] In an embodiment, the TP concentration in the suspended biomass before and / or after the lysing step is at least 2.0, 2.1, 2.1, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3,9, 4.0.

[0074] 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 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, 6.6, 6.7, 6.8, 6.9 or at least 7.0.

[0075] In an embodiment, the TP concentration in the suspended biomass before and / or after the lysing step is 2.0, 2.1, 2.1, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3,9, 4.0. 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 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, 6.6, 6.7, 6.8, 6.9 or 7.0.

[0076] The TP concentration ranged from 2 to 7 g / L or 2 to 6 g / L is suitable for both laboratory scale and industrial scale (plant scale).

[0077] In a preferred embodiment, the TP concentration in the suspended biomass before and / or after the lysing step is ranged from 2.5 to 3.5 g / L (or is 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5 g / L). The TP concentration ranged from 2.5 to 3.5 g / L is suitable for both laboratory scale and industrial scale (plant scale).

[0078] In an embodiment, TP concentration in the suspended biomass before and / or after the lysing step is 6 g / L. More preferably, the preferred TP concentration in the suspended biomass before and / or after the lysing step is 3 g / L. These preferred embodiments are suitable for both laboratory scale and industrial scale (plant scale).

[0079] In one embodiment, the TP concentration is kept as the indicated value by carrying out a diluting step before and / or after the lysing step. Optionally, the diluting step is optionally performed before the lysing step. Optionally, the diluting step is optionally performed after the lysing step. Optionally, the diluting steps are performed before and after lysing step. The diluting step is suitable for both laboratory scale Lysate concentration - P62038693WO 10

[0080] and industrial scale (plant scale). The dilution step, in particular before lysis, has been surprisingly found to favour the obtention or the recovery of more protein of interest, such as for instance, the protein represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:1 and significantly increase the clarification yields.

[0081] In one embodiment, the TP concentration in the biomass suspension preferably before lysis is adjusted up to 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9. 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,, 8.0, 9.0, 10.0, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, or up to 12.0 g / L. These mentioned TP concentrations preferably before lysis are suitable for both laboratory scale and industrial scale (plant scale).

[0082] In a further embodiment that is suitable for both laboratory scale and industrial scale (plant scale), the TP concentration preferably before lysis is adjusted up to 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / L.

[0083] In a further embodiment that is suitable for both laboratory scale and industrial scale (plant scale), the TP concentration in the lysate is adjusted up to 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / L.

[0084] In a further embodiment, preferably at the industrial scale, the TP concentration is adjusted up to 7.7, 7.8, 7.9, or up to 12.0 g / L. For instance, the TP concentration preferably before lysis is adjusted up to 7.8 g / L.

[0085] In a further embodiment, preferably at the industrial scale, the TP concentration of the lysate is adjusted up to 7.7, 7.8, 7.9, or up to 12.0 g / L. For instance, the TP concentration of the lysate is adjusted up to 7.8 g / L.

[0086] In this invention, The TP concentration may be adjusted before lysis by diluting and / or suspension volume adjustments and / or temperature adjustments. The process of the invention provides that the biomass is diluted before the lysis, which could improve the solubilisation of the protein of interest, such as for instance, the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1, thus increasing the yields of the process.

[0087] So far in the example of downstream processing of Protein Q (the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) at an industrial scale, the TP concentration is adjusted to 6 g / L (or to 5.6, 5.7, 5.8, 5.9 or 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / l), by diluting before lysis. This is an interesting working embodiment.

[0088] According to laboratory trials, we could achieve even better yields working at 3 g / L (2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / l) before and / or after lysis, preferably before lysis. This improvement can be seen in both laboratory scale and industrial scale. This improvement can be seen in comparison to other TP concentrations, such as 6 (or 5.9 or 6.0 or 6.1) g / L. In one embodiment, this yield is named a clarification yield as it is the yield obtained at the end of the clarification step. Lysate concentration - P62038693WO 11

[0089] Advantageously, diluting the TP concentration below 12 mg / mL (or down to 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1. 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.,4, 4.5, 4.6, 4.7, 4.8, 4.9, 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, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, 10.0, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5 g / L or below 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5,8, 5.7, 5.6, 5.5, 5.4, v5.3, 5.2, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5 g / L) before and / or after lysis, preferably before lysis, shows higher protein solubilization and thus, higher amount of protein of interest, such as Protein Q (protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1), in the product being recovered (or product obtained at the end of the clarification step). Besides, protein profile is more enriched in monomer in the lysate and clarified samples (i.e. sample obtained at the end of the clarification step) compared to the reference process (above 8.0 mg / mL TP concentration) during the biomass lysis and solubilization steps.

[0090] More advantageously, diluting the TP concentration down to 6 mg / mL (or down to 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.,4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, g / l) before and / or after lysis, preferably before lysis, shows higher protein solubilization and thus, higher amount of protein of interest, such as Protein Q (protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1), in the product obtained at the end of the process (i.e. product obtained at the end of the clarification step) without compromising the quality of the product thereof.

[0091] And even more advantageously, diluting the TP concentration down to 3 mg / mL (or 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 g / l) before and / or after lysis, preferably before lysis, shows highest protein solubilization and thus, highest amount of protein of interest, such as Protein Q (protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1), in the clarified product (i.e. product obtained at the end of the clarification step). Besides, protein profile is more enriched in monomer in the lysate and clarified samples (i.e. sample obtained at the end of the clarification step) compared to the reference process (above 6 mg / mL TP concentration) during the biomass lysis and solubilization steps. Attractive results were obtained using these conditions in example 5 using proteins having either 59,8 or 87,4 % sequence identity with Protein Q.

[0092] In an exemplary embodiment, when comparing lysate concentration conditions, TP as well as protein Q recoveries are even more improved in the runs performed at 3 g / L (or 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / L) than at 6 g / L (or 5.9 or 6.0, 6.1 g / L). It indicates that proteins (protein Q (the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) and host cell proteins) are better solubilized when they are more diluted. Attractive results were obtained using these conditions in example 5 using proteins having either 59,8 or 87,4% sequence identity with Protein Q.

[0093] In an embodiment and as demonstrated in example 1, global yield is improved around 16-17 % regarding TP and around 11 % regarding PQ (or the protein represented by an amino acid sequence Lysate concentration - P62038693WO 12

[0094] having at least 60 or 80% identity or similarity with SEQ ID NO:1) when diluting the lysate with double volume of buffer. In this embodiment, the preferred TP concentration is 3 g / L.

[0095] In an embodiment and as demonstrated in example 5, global yield (TP) is increased or improved around 39 % in the case of the process for purifying Protein 2 while it is increased or improved around 10% in the case of the process for purifying Protein 3 when diluting the suspended biomass before lysis and keeping the preferred TP concentration at 3 g / L. In this embodiment, the yield of the protein of interest is increased or improved of around 19% for each of Protein 2 or 3 (example 5).

[0096] In an embodiment, the yield of TP (in total grams production), preferably the yield of the clarified protein of interest (in total grams production) (in particular the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) is increased 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% or more when the lysate is diluted with the appropriate buffer. The increase is assessed by comparison with the yield obtained with an identical process, the only difference being the absence of dilution of the lysate. In an embodiment, the TP in the lysate is at a concentration of or 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / l after dilution. In an embodiment, the dilution is made below 0 °C (for example, but not limited to, on brine, jacketed tanks, and / or chillers), or ranged from 1-10 °C (for example, but not limited to, on ice, jacketed tanks, and / or chillers) or at room temperature. In an embodiment, the yield of TP (in total grams production), preferably the yield of the clarified protein of interest (in total grams production), (in particular the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) is increased 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% or more when the suspended biomass is diluted with the appropriate buffer. The increase is assessed by comparison with the yield obtained with an identical process, the only difference being the absence of dilution of the suspended biomass. In an embodiment, the TP in the suspended biomass is at a concentration of or 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / l after dilution. In an embodiment, the dilution is made below 0 °C (for example, but not limited to, on brine, jacketed tanks, and / or chillers), or ranged from 1-10 °C (for example, but not limited to, on ice, jacketed tanks, and / or chillers) or at room temperature. The dilution is preferably performed before lysis.

[0097] In an embodiment, the downstream processing described herein or at least part of it includes keeping the temperature below 0 °C (for example, but not limited to, on brine, jacketed tanks, and / or chillers), or ranged from 1-10 °C (or is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C ) (for example, but not limited to, on ice, jacketed tanks, and / or chillers) or at room temperature. In a preferred embodiment, the process or at least part of it is carried out at a temperature ranged from 1°C to 10°C (or is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C). In a further preferred embodiment, the part of the process which is carried out at the temperature range of 1°C to 10°C (or at a temperature of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C) is the lysis step and the subsequent downstream processing steps. Lysate concentration - P62038693WO 13

[0098] In an embodiment, when paying attention to temperature adjustment, it is observed that increasing temperature promotes total protein solubility. Nevertheless, the tests performed at room temperature showed lower clarification yields in PQ (in particular the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1), resulting in lower global yields for the protein of interest. A possible explanation could be that the increased protein solubilization could cause filter fouling. For that reason, applying temperatures below zero or room temperature are not preferred. The preferred range of temperature is 1 - 10 °C (or a temperature of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C), which is proven to be beneficial for the protein yields without risking protein stability. In an embodiment, the temperature may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C during at least part of the process.

[0099] In one embodiment of this aspect, there is provided a process wherein the isoelectric point of the protein is from 1 to 3 units away from the pH of the lysate (lower or higher).

[0100] 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 lysate.

[0101] 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. 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 lysate 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 comparising the UV spectra to Protein Q and specifically, the absorbance at 350 nm.

[0102] In an embodiment, the pH of the lysate is comprised from 7.5 to 10.0. In an embodiment, the pH may be 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8., 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. Preferably, the pH of the lysate is adjusted to 8.0 ± 0.2. More preferably, the pH of the lysate is adjusted before being kept for about one hour in agitation at a range of temperature 1 - 10 °C. The adjustment may be performed after lysis. Preferably, it is performed before lysate homogenization, such as half to one hour period of homogenization.

[0103] In a preferred embodiment at lab scale, there is provided a downstream processing according to the invention comprising steps wherein TP concentration is adjusted up to 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / L, preferably by diluting before and / or after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C preferably for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 and 10.0. In an embodiment, the pH may be 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8., 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. This optimized process steps may be upscaled or implemented at a plant or an industrial scale to improve the yield and stability of the protein of the invention. Lysate concentration - P62038693WO 14

[0104] In a preferred embodiment at lab scale or industrial scale, there is provided a downstream processing according to the invention comprising steps wherein TP concentration is adjusted up to 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / L by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.5 and 10.0. In an embodiment, the pH may be 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8., 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0.

[0105] In a preferred embodiment at lab scale, there is provided a downstream processing according to the invention comprising steps wherein TP concentration is adjusted up to 3.0 g / L (or up to 2.9, 3.0, 3.1 g / L) by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent / further downstream steps, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2).

[0106] In a preferred embodiment at industrial scale, there is provided a downstream processing according to the invention comprising steps wherein TP concentration is adjusted up to 3.0 g / L (or up to 2.9, 3.0, 3.1 g / L) by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2). It is encompassed by this invention that an embodiment of the preferred industrial scale may be extrapolated from the preferred laboratory scale.

[0107] In a preferred embodiment at lab scale or industrial scale, there is provided a downstream processing according to the invention comprising steps wherein TP concentration is adjusted up to 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / L by diluting after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 and 10.0. In an embodiment, the pH may be 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8., 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0108] In a preferred embodiment at lab scale, there is provided a downstream processing according to the invention comprising steps wherein TP concentration is adjusted up to 3.0 g / L (or 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / l) by diluting after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6). Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0109] In a preferred embodiment for industrial scale, there is provided a downstream processing according to the invention comprising steps wherein TP concentration is adjusted up to 3.0 g / L (or 2.5, 2.6, 2.7, Lysate concentration - P62038693WO 15

[0110] 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / l) by diluting after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2). Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0111] In another preferred embodiment at lab scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / L, preferably by diluting before and / or after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C preferably for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 and 10.0. In an embodiment, the pH may be 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8., 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. These optimized process steps may be upscaled or implemented at a plant or an industrial scale to improve the yield and stability of the protein of interest. Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0112] In another preferred embodiment at lab scale or industrial scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / L by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.5 and 10.0. In an embodiment, the pH may be 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8., 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0113] In another preferred embodiment at lab scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 6.0 g / L (or 5.8, 5.9, 6.0, 6.1, 6.2 g / L) by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2). Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0114] In another preferred embodiment at industrial scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 6.0 g / L (or 5.8, 5.9, 6.0, 6.1, 6.2 g / L) by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2). Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L. Lysate concentration - P62038693WO 16

[0115] In another preferred embodiment at lab scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 6.0 g / L (or 5.8, 5.9, 6.0, 6.1, 6.2 g / L) by diluting after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2). Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0116] In another preferred embodiment at industrial scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 6.0 g / L (or 5.8, 5.9, 6.0, 6.1, 6.2 g / L) by diluting after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2). Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0117] In another preferred embodiment at industrial scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 7.8, or 7.90g / L, or 12 g / L, preferably by diluting before and / or after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C preferably for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 and 10.0. In an embodiment, the pH may be 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8., 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0118] In another preferred embodiment at industrial scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 7.8 g / L (or up to 7.9 g / L or 12 g / L,), by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2).

[0119] In another preferred embodiment at industrial scale, there is provided a downstream processing according to the invention comprising steps wherein the TP concentration is adjusted up to 7.8g / L (or up to 7.9 g / L or 12 g / L), by diluting after lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C for example, but not limited to, on ice, jacketed tanks, and / or chillers, at least during the lysis and subsequent downstream step, and the lysate pH is ranged from 7.8 to 8.2 (or is 7.8, 7.9, 8.0, 8.1 or 8.2). Optionally, the TP concentration is adjusted, preferably by dilution. Optionally, the TP concentration before adjustment and / or dilution is 10, 11, 12 g / L.

[0120] A preferred process includes performing a dilution of the lysate to a TP concentration of 3 g / L (or of 2.8, 2.9, 3.0, 3.1, 3.2 g / L) and keeping the temperature at around 1-10 °C (or at 1°C, 2°C, 3°C, 4°C, 5°C, Lysate concentration - P62038693WO 17

[0121] 6°C, 7°C, 8°C, 9°C, 10°C). In an embodiment, the lysate is kept for example, but not limited to, on ice, jacketed tanks, and / or chillers to get this temperature.

[0122] A more preferred process includes performing a dilution of the suspended biomass (before lysis) to a TP concentration of 3 g / L (or of 2.8, 2.9, 3.0, 3.1, 3.2 g / L) and keeping the temperature at around 1-10 °C (or at 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C). In an embodiment, the suspended biomass is kept for example, but not limited to, on ice, jacketed tanks, and / or chillers to get this temperature. An even more preferred process conditions includes performing a dilution of the suspended biomass (before lysis) to a target total protein concentration of 3 mg / mL and keeping the product at around 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C (for example, but not limited to, on ice, jacketed tanks, and / or chillers) at least during the early downstream process: biomass lysis, solubilization and clarification. Diluting suspended biomass preferably before lysis, to a total protein concentration target of 3 mg / mL shows higher protein solubilization and thus, higher yields at the end of the clarification step. Keeping protein of interest more diluted together with keeping temperature between 0°C to 10 °C enhances solubility. Moreover, keeping cool temperature (between 0°C to 10 °C) and diluted protein of interest reduces protein of interest stress and / or degradation, specially during the lysis (pressure homogenization).

[0123] As described herein in an exemplary embodiment, carrying out the dilution process step before the lysis of the biomass suspension has a positive impact on the amount of TP as well as protein Q (in particular the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) recovered: carrying the lysis on a TP concentration of 3 g / L (or of 2.8, 2.9, 3.0, 3.1, 3.2 g / L) instead of around a 4 times more concentrated TP leads to an improved recovery of TP and even more surprisingly, an even more improved recovery of PQ. It indicates that proteins (such as protein Q (the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) and host cell protein) are better extracted and solubilized in the buffer when they are more diluted during the lysis. The yield of TP and also of the protein of interest may be improved by 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% (preferably at least 15 %). Moreover, higher total amounts of TP and surprisingly even higher amount of protein Q (the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) may be obtained under such conditions at the end of the further downstream processing steps such as solubilizing and clarifying steps. This is surprising that the effect on the recovery of protein Q is stronger than the effect on TP. This is evidenced by a higher protein Q / TP ratio when dilution is applied before the lysis. This effect and improvement is seen at lab scale and at industrial scale.

[0124] In particular, the best tested conditions would be applying a dilution of the biomass to a TP concentration of 3 mg / mL (or of 2.8, 2.9, 3.0, 3.1, 3.2 g / L) before the lysis and control product temperature at 1-10 °C (or at 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C) during the lysis and further downstream processing steps such as solubilization (or solubilizing) steps. As seen in Example 1, but not limited to, Lysate concentration - P62038693WO 18

[0125] solubilization step is the variable optimized in Example 1, where the temperature and concentration are adjusted according to the invention.

[0126] In particular, the best tested conditions would be applying a dilution of the biomass to a TP concentration of 3 mg / mL (or of 2.8, 2.9, 3.0, 3.1, 3.2 g / L) before the lysis and control product temperature at 1-10 °C (or at 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C) during the lysis and further downstream processing steps such as solubilization (or solubilizing) steps. Example 5 further confirms that such process conditions are quite attractive also for proteins that are different from Protein Q, especially proteins having at least 60 % identity with Protein Q (or 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%).

[0127] Clarification

[0128] The primary objective of the clarification step is to eliminate cellular remnants and other solid particles from the lysate, resulting in a clearer solution that contains the proteins or biomolecules of interest. Generally, a clarification step is performed after lysis to prevent contamination or degradation of the lysate. It is also encompassed to adjust the pH after lysis and during clarification (or after lysis and before clarification) process to achieve a suitable pH, preferably ranged from 7.5 and 10.0 (or 7.5, 7.6, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0), even more preferably 7.8 to 8.2 (or is 7.8, 7.9. 8.0, 8.1, 8.2). In one embodiment,, after having lysed the cells, it is possible to apply a half to one hour homogenization step or resuspension step. In this embodiment, the temperature is preferably kept between 1 to 10 °C, to assure the protein of interest is released from inclusion bodies and is solubilized in the buffer previous to the clarification step.

[0129] In one embodiment, after lysis and solubilization, the product is clarified. In one embodiment, clarification is carried out by filtration. In one embodiment, filtration is carried out by applying at least two filtrations. In one embodiment, a first filtration is to remove bigger particles (such as cellular debris) and a second filtration is to remove smaller particles (such as particles smaller than cells). The filters may comprise the range of 0.1 to 1 pm, preferably 0.2 to 0.8 pm. It is also possible to apply a third filtration, preferably before purifying by chromatography step, such that the protein of interest is sterilized. In another example, the filtration may be carried out by performing Tangential Flow Filtration (TFF), also known as Cross-Flow Filtration. Applying a higher number of filtration steps allows to get a cleaner product, but it is expected to have a negative impact on the amount of protein recovered. In a non-limitative example, a reference process comprises a biomass at TP concentration of 11 g / L (or 10.8, 10.9, 11.0, 11.1, 11.2 g / L) which is suspended and solubilized one hour after lysis without dilution. The dilution of the TP concentration to 6 mg / mL is applied after clarification step. The clarification (may also be called filtration step) is also important for preparing the lysate for subsequent purification, such as chromatography steps, in the downstream processing of the protein of interest (in particular protein Q (the protein represented by an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:1)) according to this invention. Lysate concentration - P62038693WO 19

[0130] In a non-limitative embodiment of the optimized process according to this invention, an initial suspension of biomass is at TP concentration of 11 (or 10.8, 10.9, 11.0, 11.1, 11.2 g / L) g / L and followed by dilution after lysis to 6 (or 5.8, 5.9, 6.0, 6.1, 6.2 g / L) or 3 (or 2.8, 2.9, 3.0, 3.1, 3.2 g / L) g / L, and one hour solubilization.

[0131] In a non-limitative embodiment of the optimized process according to this invention, an initial suspension of biomass is at TP concentration of 6 (or 2.8, 2.9, 3.0, 3.1, 3.2 g / L) or 3 (or 2.8, 2.9, 3.0, 3.1, 3.2 g / L) g / L, and one hour solubilization.

[0132] Characterisation of the improved read out of the purification process of the invention

[0133] In an embodiment, the process of the invention for downstream processing a protein, preferably represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1, from a biomass comprising cells, wherein said downstream process comprising the following steps:

[0134] • suspending the biomass in a suspension buffer,

[0135] • subsequently lysing the cells present in the suspended biomass to obtain a lysate

[0136] and further downstream processing said protein by solubilising and clarifying it and wherein the concentration of the Total Protein (TP) is optimised (preferably ranged from 2 to 12 or preferably from 3 to 6 g / L) in the suspended biomass before lysing it as earlier defined herein.

[0137] 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 steps but not having an optimised TP concentration and not having a dilution step to optimise the TP concentration. Such improved process may be carried out at lab scale or at industrial scale. In both cases, it is expected to exhibit at least one of the following improvements compared to a control process (i.e. no optimisation of the TP concentration and no dilution step of the suspended biomass before lysis) carried out at an industrial scale:

[0138] an increase of the yield of TP compared to the yield of TP obtained carrying out a control process: the increase 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%, or at least 45% (up to 9% increase in example 2 or increase up to 10% or even up to 39% in example 5),

[0139] an increase of the yield of the protein of interest (such as the protein represented by an amino acid sequence having at least 60 or 80% identity or similarity with SEQ ID NO:1) compared to the yield obtained carrying out a control process: the increase 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%, Lysate concentration - P62038693WO 20

[0140] 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or at least 45% (up to 44% increase, see examples 1 and 2, up to 19% increase in example 5), for instance this increase is seen in at least after the lysis and / or at the end of the clarification step; and

[0141] an increase of the protein of interest / TP average ratio compared to the corresponding ratio obtained carrying out a control process: the increase 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%, or at least 45% (ratio went from 10 to 13% in example 2).

[0142] Protein Q

[0143] 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. 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 W02000039298, this highly homologous protein Q is identified by SEQ ID NO:1.

[0144] 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.

[0145] 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).

[0146] In an embodiment, the protein of the invention comprises the following amino acid sequence (SEQ. ID NO. 1):

[0147] MRGSHHHHHHTDPHASSNNNNNNNNNNLGIEGRPLATPRSAKKAVRKSGSKSAKCGL Lysate concentration - P62038693WO 21 IFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHD DDIGTLLKNVTLSHSGWPNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTKYLAAYA LASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPA ASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSG KTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGA VSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPEESDEDD FGMGGLF

[0148] In a preferred embodiment, a protein Q is a protein target or protein of interest. Preferably, the protein Q is represented by an amino acid sequence having at least 60% or 80% identity or similarity with SEQ ID NO:1. In a 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. 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.

[0149] Figure legends

[0150] Figure 1: this figure shows the total protein grams in the adjusted clarified product for all industrial batches, as well as the Protein Q grams of the analyzed batches. It is remarkable the upward trend in the batches according to the current invention, after the process improvement is applied. It is shown that the yield of total protein TP (g), the yield average of TP, the yield of protein Q (g), and the yield average of protein Q are higher than the previous known batches.

[0151] Figure 2: Diagram of the solubility trial.

[0152] This experiment was performed with three proteins: PQ, Protein 2 and Protein 3. Only 1 gram of each pellet was taken and suspended in biomass suspension buffer to a target biomass concentration 0.11 g / mL (this concentration is obtained from industrial scale process). The next step was to homogenize the samples at a setpoint temperature of 5°C. Due to the small volumes used, a sonicator was applied instead of a disruptor, then the samples were centrifuged (3000 g, 10 minutes) and filtered.

[0153] Figure 3. Diagram of lysis concentration trial.

[0154] Two tests were performed with each protein: one under reference conditions (biomass suspension up to 11 mg / mL TP and dilution after lysis and clarification up to 6 mg / mL TP) and another under optimal conditions (biomass suspension previous to lysis to 3 mg / mL TP). Lysate concentration - P62038693WO 22

[0155] Figure 4: Absorption spectrum in the clarified product of the three proteins studied.

[0156] Figure 5: Absorption spectrum in the clarified product for rPQ (Protein 5, SEQ ID NO:4) compared to PQ (SEQ ID NO:1).

[0157] General part dedicated to definitions

[0158] Polypeptide / nucleic acid

[0159] 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.

[0160] 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.

[0161] 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, or chimeric antigen-recognition receptor comprises a nucleic acid or nucleotide sequence which encodes such a polypeptide. A nucleic acid molecule may comprise a regulatory region.

[0162] 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:

[0163] i. a nucleotide sequence comprising a nucleotide sequence that has at least 60% or at least 80% sequence identity with SEQ ID NO: X;

[0164] ii. a nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i);

[0165] 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,

[0166] 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. Lysate concentration - P62038693WO 23

[0167] 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%.

[0168] 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.

[0169] 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).

[0170] Sequence identity

[0171] " 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%. Lysate concentration - P62038693WO 24

[0172] 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.

[0173] “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).

[0174] 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) 110 (proteins) and gap extension penalty = 0.5 (nucleotide sequences) 10.5 (proteins). This method (EMBOSS, Needleman, Matrix: EBLOSUM62, Gap penalty: 10, Extend penalty 0.5) has been used in example 5 to assess the identity and similarity percentages of Protein 2 (SEQ ID NO:2), Protein 3 (SEQ ID NO:3) to Protein Q (SEQ ID NO:1).

[0175] 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).

[0176] In a preferred embodiment, the method used in example 5 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.

[0177] 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% Lysate concentration - P62038693WO 25

[0178] 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 ofthe 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 / .

[0179] 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.

[0180] Acidic Residues Asp (D) and Glu (E)

[0181] Basic Residues Lys (K), Arg (R), and His (H)

[0182] Ser (S), Thr (T), Asn (N), and

[0183] Hydrophilic Uncharged Residues

[0184] Gin (Q)

[0185] Gly (G), Ala (A), Vai (V), Leu (L),

[0186] Aliphatic Uncharged Residues

[0187]

[0188] and He (I) Lysate concentration - P62038693WO 26

[0189] Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P)

[0190]

[0191] Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

[0192] Alternative conservative amino acid residue substitution classes:

[0193] 1 A S T

[0194] 2 D E

[0195] 3 N Q

[0196] 4 R K

[0197] 5 I L M

[0198]

[0199] 6 F Y W

[0200] Alternative physical and functional classifications of amino acid residues:

[0201] Alcohol group-containing residues S and T

[0202] Aliphatic residues I, L, V, and M

[0203] Cycloalkenyl-associated residues F, H, W, and Y

[0204] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and

[0205] Y

[0206] Negatively charged residues D and E

[0207] Polar residues C, D, E, H, K, N, Q, R, S, and T

[0208] Positively charged residues H, K, and R

[0209] Small residues A, C, D, G, N, P, S, T, and V

[0210] Very small residues A, G, and S

[0211] Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T

[0212]

[0213] Flexible residues Q, T, K, S, G, P, D, E, and R

[0214] 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- Lysate concentration - P62038693WO 27

[0215] 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 He or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thrto Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.

[0216] Antigen

[0217] 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 embodiments, an antigen is capable of inducing an immune response. In some examples, an antigen binds to an antigen receptor or antigen-binding protein, or induces an immune response, when present in a complex e.g., presented by MHC. In some cases, an antigen adopts a certain conformation in order to bind to an antigen receptor or antigenbinding 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 ora fragment of a target molecule or complex that binds to an antigen receptor or an antigenbinding protein.

[0218] Gene or coding sequence

[0219] “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) 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.

[0220] Codon optimization Lysate concentration - P62038693WO 28

[0221] “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.

[0222] Promoter

[0223] 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.

[0224] Operably linked

[0225] “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.

[0226] Nucleic acid construct

[0227] 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).

[0228] 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 Lysate concentration - P62038693WO 29

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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).

[0233] 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 Lysate concentration - P62038693WO 30

[0234] 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.

[0235] Engineered cells

[0236] 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". T ransfection 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. 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. In some embodiments, an “engineered cell” has been transformed, modified or transduced to comprise a heterologous or exogenous nucleic acid molecule. 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.

[0237] 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.

[0238] General information

[0239] It is not considered necessary to extend this description in order that someone skilled in the art can Lysate concentration - P62038693WO 31

[0240] 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.

[0241] 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.

[0242] 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.

[0243] As used herein, the term “g / L” is synonymous with “mg / mL” when referring to the TP or lysate concentration, unless otherwise indicated.

[0244] As used herein, the term “total protein” or “TP” maybe interpreted as “protein lysate” or “lysate” when referring to the TP or lysate concentration, unless otherwise indicated.

[0245] 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.

[0246] 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.

[0247] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting 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". 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 11. 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 Lysate concentration - P62038693WO 32

[0248] 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. Furthermore, in all aspects of this invention, the skilled person might understand that one decimal is sufficient for the precision of the analytical results achieved. Lysate concentration - P62038693WO 33

[0249] Examples

[0250] 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. 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.

[0251] E

[0252]

[0253] xample 1 - Optimization process at the laboratory scale

[0254] Abbreviation

[0255]

[0256] Wording Definition

[0257] DSP Downstream processing

[0258] HCP Host cell protein

[0259] ISP Industrial scale process

[0260] NA Not available

[0261] PQ / ProtQ Protein Q

[0262] SD Standard deviation

[0263] SSP Small scale process

[0264] TP Total Protein

[0265] USP Upstream processing

[0266] Introduction

[0267] This work focuses on the downstream process for the Protein Q (SEQ ID NO:1) production and purification, comprising the following steps:

[0268] • suspending the biomass in a suspension buffer.

[0269] • subsequently lysing the cells present in the suspended biomass to obtain a lysate where Protein Q is solubilized, and

[0270] • further clarifying the protein from said lysate.

[0271] This study focuses on extraction of the target protein from the biomass, which comprises from lysis to clarification steps. The studied variables are the suspension volume added and the temperature applied during the process to keep the product soluble and stable. It is also evaluated the process step where the dilution of the biomass is applied: before or after cells lysis. Lysate concentration - P62038693WO 34

[0272] More precisely, the laboratory trials consisted on testing two different working concentrations: 3 mg / mL and 6 mg / mL of TP in the solubilization step and the following filtration steps and three different temperature ranges: keeping the lysate on brine, (-8 - 0) °C (S), on ice (1 - 10) °C (H) and room temperature (approx. 25 °C) (RT) during 1h solubilization after the lysis step.

[0273] In addition, in the current process the biomass is diluted after the lysis, and these trials intended to assess if applying this dilution before the lysis would help Protein Q solubilization, thus increasing the yields of the process.

[0274] To carry out this study, the industrial current process was adapted to a small-scale process in LETI Pharma, applying different conditions in several runs. In order to compare the performance of each trial, several analyses were performed at every process step: Protein profile was analyzed by electrophoresis (WB) and protein productivity is analyzed by BCA and ELISA-Ni. The best combination of conditions for this part of the process is intended to be applied based on the obtained results.

[0275] Equipment and materials

[0276] Equipment

[0277] The following pieces of equipment were used to perform the laboratory trials:

[0278] Table 1.1. List of equipment used.

[0279] Equipment

[0280] Freezer

[0281] Refrigerator

[0282] Cell Disruptor

[0283] Stirring plate

[0284] Precision scale

[0285] pHmeter

[0286] Centrifuge

[0287] Sonicator

[0288] Ultrapure Water

[0289] Purification System

[0290] Micropipette P1000

[0291] Micropipette P200

[0292] Micropipette

[0293] multichannel P300

[0294] Micropipette P20

[0295]

[0296] Micropipette P50

[0297] Materials

[0298] The following table gathers the materials employed to perform the laboratory trials. Lysate concentration - P62038693WO 35

[0299] Table 1.2. List of materials used.

[0300] Material Model

[0301] Filter capsule with membrane

[0302] Clarification Filters

[0303]

[0304] porous diameter 0.1 - 1 μm

[0305] Solutions

[0306] The composition of the solutions employed in the process are described in the following tables:

[0307] Table 1.3. Urea 8 M solution composition

[0308] Material Quantity

[0309] Urea, CH4N2O 480.48 g / kg

[0310]

[0311] Water, H₂O q.s.p 1 kgr

[0312] Buffer is prepared weighing the following reagents.

[0313] Table 1.4. Biomass suspension buffer composition.

[0314] Material Quantity

[0315] Monosodium phosphate dihydrate,

[0316] NaH2PO4.2H2O 6.67 g / kg

[0317] Urea 8M solution 957.00 g / kg

[0318] Sodium chloride, NaCl 24.97 g / kg

[0319] Triton X-100, C14H21(C2H4O)nOH 9.14 g / kg

[0320]

[0321] Sodium hydroxide, NaOH 4M c.s.p = pH8

[0322] Small scale process description

[0323] In this section the small-scale process is described. Frozen biomass coming from an industrial fermenter (approx. 22 g or 33 g) is suspended in cold biomass suspension buffer (ratio 0.11 g / mL, which corresponds approx, to a TP concentration of 11 mg / mL), and it is homogenized for 40 min. The resuspended biomass is subjected to two cycles of cell disruption at =750 bar and a setpoint temperature of 5 °C. Resuspended biomass is kept at 5 °C for 30 min to reduce foam between cycles. The lysate product is diluted in cold biomass suspension buffer to reach a concentration of 6 mg / mL total protein. If needed, pH is adjusted to 8.0 ± 0.2 and kept for 1 h in agitation with ice. After solubilization, the product is clarified by applying at least two nitrations, one to get rid of cellular debris and one to get rid of smaller particles.

[0324] The process changes that are evaluated are:

[0325] • Volume of suspension buffer applied. Doubling the suspension volume to dilute the lysate after cell disruption the TP concentration is around 3 mg / mL instead of 6 mg / mL, as in the ISP.

[0326] • Product temperature control during the solubilization phase. Three different temperature ranges are tested: keeping the lysate,on brine (-8 - 0) °C (S), on ice (1 - 10) °C (H) and at room temperature (approx. 25 °C) (RT) during 1 h solubilization after the lysis step. Lysate concentration - P62038693WO 36

[0327] • Process step where the dilution is applied. In the current process the biomass is resuspended in buffer keeping TP at approx. 11 mg / mL and a further dilution to aprox. 6 mg / mL is applied in the clarified product. These trials intend to assess applying a dilution up to 3 mg / mL before or right after the lysis.

[0328] Analytical methods

[0329] Total protein concentration is determined by BCA, see non-limitative Example 3 herein below.

[0330] The Protein Q (represented by SEQ ID NO:1) (protein target) concentration is determined by ELISA-Ni, see non-limitative Example 4 herein below.

[0331] Experimental design

[0332] The laboratory experiments were conducted sequentially: first, the runs regarding the concentration and temperature conditions and, afterwards, the runs regarding the process step where the dilution is applied. Therefore, the experimental design and results are divided into two sections considering the findings of the first experiment to set up the conditions of the next experiment.

[0333] Temperature and TP concentration

[0334] First, the next experimental set-up was run: 23.6 g of biomass were diluted in 214 mL of cold Biomass suspension buffer (standard ratio 0.11 g / mL), which corresponds to a TP concentration of approximately 11 mg / mL, similarto the industrial scale. The suspension was subjected to two cycles of disruption at 750 bar and a setpoint of 5 °C and the obtained lysate product was divided into 4x 50 mL aliquots. Two of them were diluted to a target TP = 6 mg / mL and the other two to TP = 3 mg / mL in cold Biomass suspension. They were kept for 1 hour in agitation: one sample of each concentration in a vessel with ice and the other two samples in a vessel with brine (ice + NaCI). After solubilization, all four aliquots were clarified by at least two filtration steps (as earlier explained herein).

[0335] In a second stage, a similar experiment was performed in order to assess solubilization of the lysate at both TP concentrations, 3 and 6 mg / mL, at room temperature. Samples were taken at every step. In all experiments, temperature control has been modified only during the solubilization step after the lysis. Previous steps, which are common for all experimental set-up, have been carried out keeping the product on ice, as in the industrial process.

[0336] Pre or Post Cell Lysis

[0337] A third round of experiments was performed with the best conditions found in the last experiments: dilution to a TP concentration of 3 mg / mL keeping the temperature on ice, at 1-10 °C, and it was compared with the reference process conditions. The experimental set-up was run: 33 g of biomass were diluted to TP 11 mg / mL (reference run) and 3 mg / mL, respectively, in cold Biomass suspension buffer. The suspension was subjected to two cycles of disruption at 750 bars keeping the temperature setpoint of the cell disruptor at 5 °C. The obtained lysate from the biomass suspended with less volume was diluted to TP = 3 mg / mL in cold Biomass suspension buffer and the lysate that was previously diluted at TP = 3mg / mL, was not diluted after lysis. All fractions were kept for 1 hour in agitation at room Lysate concentration - P62038693WO 37

[0338] temperature. After solubilization, all fractions were filtered at least two times (as earlier explained herein). A total of 5 runs were made with the Dilution post-lysis, 11 mg / mL biomass dilution before lysate (TRIAL 53, 54, 55, 59 & 62) and 2 runs were made Diluting the biomass pre-lysis, 3 mg / mL before lysis (TRIAL 64 & 65).

[0339] RESULTS

[0340] Since the trials were conducted in a sequential manner, the results are presented in two sections considering the findings of the first experiment to set up the conditions of the next experiment.

[0341] The results of the first two experiments are summarized in the next tables comparing all the tested conditions. This part of the study was carried out in different dates, but they were combined in order to find the best temperature and lysate concentration conditions for the process performance.

[0342] Concentrations and total amount of total proteins and, specifically, the target protein PQ are followed at each process step. With these data it is possible to calculate the process yields and ratios in order to evaluate the product quantity and quality obtained when applying each condition.

[0343] Table 1.5. Results of PQ and TP yields tracking each step of the process of runs carried out at 3mg / mL and 6mg / mL at three different temperature conditions (S: Brine, H: ice, RT: room temperature).

[0344] Total Protein by BCA [TP] Protein Q by ELISA-Ni [PQ]

[0345] TP at 3 g / L TP at 6 g / L TP at 3 g / L TP at 6 g / L Step 3S 3H 3RT 6S 6H 6RT 3S 3H 3RT 6S 6H 6RT Solubilizatio 102 122 109 112

[0346] n Yield*183% 87% % 81% 86% % % % 91% 98% 98% 95% 1st

[0347] Clarification 87% 81% 75% 76% 82% 66% 72% 65% 80% 76% 76% 99% 2nd

[0348] 88% 107 101 118 100 Clarification % % 76% 86% 93% 93% % 72% 82% % 67% Total

[0349] clarification 77% 87% 76% 58% 71% 62% 66% 77% 58% 63% 77% 67% Yield

[0350] Lysis

[0351] clarification

[0352] Global 64% 76% 77% 47% 60% 75% 72% 86% 53% 61% 75% 63%

[0353]

[0354] Yield*2

[0355] *1Solubilization yield corresponds with the protein in the adjusted lysate plus one-hour solubilization I lysate protein.

[0356] *2Global lysis-clarification yield is the accumulated yield of the whole experiment process from lysate to clarified product.

[0357] Table 1.6. PQ / PT ratios in each step of the process for runs carried out at 3mg / mL and 6mg / mL at three different temperature conditions (S: Brine, H: ice, RT: room temperature).

[0358] TP at 3 g / L TP at 6 g / L

[0359] Step 3S 3H 3RT 6S 6H 6RT Lysate 10.3% 10.3% 14.7% 10.3% 10.3% 14.7%

[0360]

[0361] Lysate concentration - P62038693WO 38

[0362] Adjusted lysate

[0363] (solubilization) 13.4% 13.2% 13.2% 12.4% 11.7% 11.4% 1st clarified product 11.0% 10.5% 14.1% 12.3% 11.0% 17.1% 2nd clarified product 11.6% 11.6% 10.0% 13.4% 12.8% 12.4%

[0364]

[0365] When comparing lysate concentration conditions, TP as well as PQ obtain better recoveries in the runs performed at 3 mg / mL than at 6 mg / mL. It indicates that proteins (PQ and HCP) are better solubilized when they are more diluted. Global yields are improved around 16-17 % regarding TP and around 11 % regarding PQ when diluting the lysate with double volume of buffer (except runs at room temperature). This means in the example that when the lysate is diluted from 6 mg / mL to 3 mg / mL the yields are not improving at RT, and when using ice and brine the diluted TP concentration of 3 mg / mL is better condition than the diluted TP concentration of 6 mg / mL.

[0366] When paying attention to temperature control, it is observed that increasing temperature promotes protein solubility. Nevertheless, the tests performed at room temperature showed lower clarification yields in PQ, resulting in lower global yields for the protein of interest. A possible explanation could be the increased protein solubilization could cause filter fouling. For that reason, applying brine (.with temperatures below zero) or room temperature are discarded. Control temperature (using ice) keeps the process in a range 1 - 10 °C, which is proven to be beneficial for the protein yields without risking protein stability.

[0367] In addition, protein profile is analyzed by SDS-PAGE and Western Blott at each step of the process and it is proven the protein profiles are comparable under all the conditions tested.

[0368] Table 1.7. Average results of Total Protein and Protein Q for runs carried out applying the biomass dilution before (n=2 trials) or after lysis (n=5 trials).

[0369] Total Protein by BCA [ TP] Protein Q by ELISA-Ni [PQ] Dilution postDilution preDilution postDilution prelysis (n=5) lysis (n=2) lysis (n=5) lysis (n=2) [TP] or [PQ]

[0370] (pg / mL) 11383 4702 1506 761 Diluted Biomass

[0371] TP or PQ 3582 4185 482 677

[0372] (mg)

[0373] [TP] or [PQ]

[0374] (pg / mL) 11233 4482 1444 761 Lysate

[0375] TP or PQ 3026 3810 391 647

[0376] (mg)

[0377] [TP] or [PQ] 4461 4796 405 771 Adjusted lysate (pg / mL)

[0378] (solubilization) TP or PQ 3566 4154 320 635

[0379] (mg)

[0380] [TP] or [PQ]

[0381] (pg / mL) 4059 4246 365 561

[0382] 1stclarified

[0383] TP or PQ 3198 3672 288 461

[0384] (mg)

[0385] 2ndpm clarified [TP] or [PQ]

[0386]

[0387] (pg / mL) 3807 4425 351 445 Lysate concentration - P62038693WO 39

[0388] Total Protein by BCA [ TP] Protein Q by ELISA-Ni [PQ] Dilution postDilution pre- Dilution postDilution pre- lysis (n=5) lysis (n=2) lysis (n=5) lysis (n=2) TP or PQ 2967 3364 275 341

[0389]

[0390] (mg)

[0391] These results show that diluting the biomass before the lysis to TP 3 mg / mL instead of 11 mg / mL, as in the industrial scale, favors the amount of TP and PQ obtained in the clarified product. Besides, this dilution seems to be more beneficial for the target protein PQ than for the TP, which includes other Host cell proteins, not related to the product of interest.

[0392] Table 1.8. PQ and TP average yields tracking each step of the process for runs carried out applying the biomass dilution before (n=2 trials) or after lysis (n=5 trials).

[0393] % YIELD BCA [TP] % YIELD ELISA-Ni [PQ]

[0394] Dilution postDilution pre- Dilution postDilution pre-lysis lysis (n=5) lysis (n=2) lysis (n=5) (n=2) Biomass lysis yield 84% 91% 81% 96% Solubilization

[0395] Yield*1118% 109% 82% 98%

[0396] 1stClarification

[0397] Yield 90% 88% 90% 73%

[0398] 2ndClarification 93% 92% 95% 74%

[0399]

[0400] Yield

[0401] *1Solubilization yield corresponds with the protein in the adjusted lysate plus one-hour solubilization I lysate protein.

[0402] It is observed the lysis performed at 3 mg / mL favors the yields for the biomass lysis and the solubilization. Interestingly, more solubilized PQ results in greater yields loss during the filtration process. Despite the clarification yields seem higher for the lysis carried out at 11 mg / mL, greater total amounts of PQ and TP are obtained at the end of clarification as well as higher PQ / PT ratios when dilution is applied before the lysis.

[0403] Table 1.9. PQ / PT ratio in each step of the process for runs carried out applying the biomass dilution before (n=2 trials) or after lysis (n=5 trials).

[0404] % PQ / PT

[0405] Dilution post-lysis (n=5) Dilution pre-lysis (n=2) Diluted Biomass 13% 16%

[0406] Lysate Biomass 13% 17%

[0407] Adjusted lysate (solubilization) 9% 16%

[0408] 1stclarified 9% 13%

[0409]

[0410] 2ndclarified 9% 10%

[0411] By means of the PQ / TP ratio it can be appreciated that the pre-lysis dilution helps to obtain more PQ amount in the initial steps, especially during solubilization, almost doubling the percentage of PQ with Lysate concentration - P62038693WO 40

[0412] respect to total proteins. The loss of PQ during the clarification steps seems to level the amount of PQ in both processes, which keeps still higher in the runs where dilution is applied before lysis.

[0413] The protein profile is checked by SDS-PAGE and Western Blott (WB) in this experiment and it is proven the bands profile keeps unaltered with respect to the reference process..

[0414] Conclusion

[0415] From the results discussed in previous section we can draw the following conclusions:

[0416] • Solubilization of the lysate product under different concentration and temperature conditions has been studied at laboratory scale. When comparing diluting the lysate to a target total protein concentration of 3 mg / mL vs. 6 mg / mL (current process average concentration) and keeping product temperature below 0 °C (on brine), 1-10 °C (on ice) or at room temperature, the following conclusions can be extracted:

[0417] • The best process conditions are performing a dilution of the lysate to a target total protein concentration of 3 mg / mL and keeping the product at around 1-10 °C (on ice) during the biomass lysis and solubilization steps.

[0418] • Diluting lysate to a total protein target of 3 mg / mL shows higher protein solubilization and thus, higher amount of Protein in the clarified product. Besides, protein profile is more enriched in monomer in the lysate and clarified samples compared to the reference process (6 mg / mL TP concentration).

[0419] • Temperatures below 0° (on brine) during the process have been shown to negatively affect the yields in both studied TP concentrations. Increasing temperature promotes protein solubilization; however, performing solubilization step at room temperature has shown lower clarification yields

[0420] • When comparing the process step to apply the biomass dilution, before or after the lysis, TP as well as PQ obtain better recoveries in the runs performing the lysis more diluted (at TP 3 mg / mL instead of the current process that takes place at around 11 mg / mL). It indicates that proteins (PQ and HCP) are better extracted and solubilized in the buffer when they are more diluted during the lysis. Yields are improved by around 15 % regarding PQ during lysis and solubilization. In spite of the clarification yields seem higher for the lysis carried out at 11 mg / mL, greater total amounts of PQ ant TP are obtained at the end of clarification as well as higher PQ / PT ratios when dilution is applied before the lysis.

[0421] To sum up, the best tested conditions would be applying a dilution of the biomass to a TP concentration of 3 mg / mL before the lysis and control product temperature at 1 -10 °C during the lysis and solubilization steps.

[0422] Example 2 - Optimization process at the industrial scale

[0423] LIST OF ABBREVIATIONS

[0424] Abbreviation

[0425]

[0426] Wording Definition

[0427]

[0428] Lysate concentration - P62038693WO 41

[0429] CV Coefficient of variation

[0430] DSP Downstream processing

[0431] ISP Industrial scale process

[0432] NA Not available

[0433] PQ / ProtQ Protein Q

[0434] q.s. Quantum satis: Add as much of the ingredient as is needed SD Standard deviation

[0435] TP Total protein

[0436] USP Upstream processing

[0437] WFI Water for injection

[0438] Introduction

[0439] The objective of this document is to give technical support to the actual patent application process for Protein Q manufacturing, more precisely for the biomass solubilization and lysis steps.

[0440] In this part of the process, the aim of the biomass disruption is to break microbial membranes in order to liberate the cytoplasmic compartment where the protein of interest, Protein Q, is expressed. After that, a filtration cascade is applied to remove cell debris and to clarify the cell lysate before further purification steps are executed.

[0441] This work focuses on increasing the biomass dilution before cell disruption to promote protein solubilization. In the manufacturing of industrial batches according to the invention, this change was performed by suspending the biomass with the solubilization buffer up to 45.0 kg instead of 34.5 kg before lysis, since it had been proven at laboratory scale that these conditions considerably improved protein Q solubilization and could increase the process yields.

[0442] Furthermore, some trials have been carried out at laboratory scale suspending the biomass with larger amount of solubilization buffer, so that the concentration of total protein in the clarified product is 3 mg / mL instead of the current 6 mg / mL at the industrial process. Regarding the temperature, the process at industrial level is controlled at < 10°C, however after testing three different conditions (RT, brine and ice) at laboratory level, it was concluded that it is better to limit the temperature of the process between the range 1-10°C. These changes could have a positive impact on the process yields and stability, nevertheless, have not yet been implemented on an industrial scale.

[0443] Equipment and materials

[0444] Equipments

[0445] The following pieces of equipment have been used in the manufacture of the industrial batches:

[0446] Table 2.1: Equipment used in the industrial batches.

[0447] Equipment

[0448] Cell Disruptor

[0449] Mechanical agitator

[0450]

[0451] Scale Lysate concentration - P62038693WO 42

[0452] Multimeter (pH and conductivity)

[0453] Pump

[0454] Peristaltic pump

[0455]

[0456] Manometer

[0457] Materials

[0458] The following materials were used for the fabrication of the industrial batches:

[0459] Table 2.2: Materials used in the industrial batches.

[0460] Material Description

[0461] Clarification filters Filter capsule of membrane porous diameter 0.1 - 2 pm Container Tank 100 L plastic tank

[0462]

[0463] Product collecting bag 100 L Flexel tank liner

[0464] Solutions

[0465] The following solutions are prepared to manufacture the industrial batches:

[0466] Table 2.3. Urea 8 M solution composition

[0467] Material Quantity

[0468] Urea, CH4N2O 480.48 g / kg

[0469]

[0470] Water for injection, (WFI) q.s.p 1 kg

[0471] Table 2.4. Biomass Solubilization buffer composition

[0472] Material Quantity

[0473] 8 M urea solution 957 g / kg

[0474] Sodium dihydrogen phosphate dihydrate

[0475] 6.67 g / kg (NaH2PO4.2H2O)

[0476] Sodium chloride (NaCI) 24.97 g / kg

[0477] TritonX-100 (C14H21(C2H4O)nOH) 9.14 g / kg

[0478]

[0479] Table 2.5. 1 M NaOH pH adjustment solution composition

[0480] Material Quantity

[0481] Sodium hydroxide (NaOH) 40.0 g / kg

[0482]

[0483] Water for injection (WFI) c.s.p. 1 kg

[0484] Table 2.6.2 M HCI pH adjustment solution composition

[0485] Material Quantity

[0486] Hydrochloric acid 37 % (HCI 37 %) 197.1 g / kg

[0487]

[0488] Water for injection (WFI) c.s.p. 1 kg

[0489] Current industrial scale process description

[0490] In this section, the current industrial process is described. The operations in this step are as follows: 1. The biomass is transferred to a 100 L tank recovered by a liner and cooled down by ice at the base. Lysate concentration - P62038693WO 43

[0491] A determined volume of biomass solubilization buffer is added to the biomass in order to reach a total mass of 34.5 ± 0.5 kg (biomass + buffer). Then, the biomass is mechanically mixed till complete biomass re-suspension.

[0492] 2. This homogenized suspension is thereafter passed through the Cell disruptor (cooled down with glycolated water) without pressure being applied. The homogenized biomass is collected in a 100 L tank cooled down by ice. The temperature of the homogenate is < 10 °C in the current process.

[0493] 3. This homogenate is then successively passed two-times through the Cell disruptor working at a range 400 - 800 bar. After each pass the disrupted biomass is collected in a 100 L tank cooled down by ice. The temperature of the lysate is maintained at < 10 °C during each step.

[0494] 4. After cell disruption, the lysate is mechanically mixed in a tank with an impeller during 1 h and is also recirculated through the Cell disruptor without pressure being applied. The Cell disruptor is purged at the end with solubilization buffer. The temperature of the lysate is maintained at < 10 °C. The pH of the lysate is controlled and must range between 7.8-8.2. If this is not the case, the pH is adjusted with NaOH or HCI solutions under agitation.

[0495] 5. In brief, cell lysates are successively pumped through first filters and second filters The temperature of the lysate and of the clarified product is maintained at < 10 °C.

[0496] 6. During the filtration, vent valves are progressively closed and the lysate is pumped through the filters. The pump speed is controlled to have a pressure < 1 bar. When the filtration step is over, cold solubilization buffer is added to adjust the clarified product to a total mass of 50 ± 0.5 kg. The pH of the clarified product is controlled and must range between 7.8-8.2. If this is not the case, the pH is adjusted with NaOH or HCI solutions under agitation.

[0497] Process improvements

[0498] For the sixteen batches produced in the example, the biomass has been suspended prior to cell disruption with solubilization buffer in order to reach a total mass of 45 ± 0.5 kg (equivalent to aprox. 6 g / L TP) instead of 34.5 ± 0.5 kg (biomass + buffer) (equivalent to aprox. 11 g / L TP).

[0499] The increment of buffer volume promotes protein extraction from the cells and its solubilization, which has been proven to be beneficial when applying a larger dilution before lysis. This means that total protein concentration in this point is approximately 6 mg / mL, similar to the concentration after clarified product adjustment, which implies the dilution up to 50 kg after clarification is minimal. These changes have been found to favor obtaining more protein Q and increase the clarification yields. This means in this example, the modification performed at industrial scale has been diluting to aprox. 6 mg / mL before lysis, instead of working at 11 mg / mL until clarification step and diluting after clarification step to 6 mg / mL.

[0500] Furthermore, some trials have been carried out at laboratory scale suspending biomass with larger amount of solubilization buffer, so that the concentration target of total protein in the clarified product is 3 mg / mL instead of the current 6 mg / mL at the industrial process. Regarding the temperature, the process at industrial level is controlled at < 10°C, however after testing three different conditions (RT, Lysate concentration - P62038693WO 44

[0501] brine and ice) at laboratory level, it was concluded that it is better to limit the temperature of the process between the range 1-10°C. These changes could have a positive impact on the process yields and stability, nevertheless, have not yet been implemented at industrial scale.

[0502] Analytical methods

[0503] To quantify the amount of total protein and the target protein (protein Q) in the samples, analytical methods were developed based on the BCA kit and ELISA-Ni, respectively (see non-limitative Examples 3 and 4 herein below).

[0504] Results and discussion

[0505] The aim of this section is to verify the increase in yields obtained in the batches, with the change applied at industrial scale, compared with those batches manufactured in previous known batches, so the total protein and the protein Q of the lysate and the clarified adjusted product are analyzed and the yields obtained are evaluated.

[0506] Clarified product samples from numerous batches produced were analyzed by ELISA-Ni and BCA to quantify the Protein Q and the total protein, respectively (analytical methods described in Annex 1 and 2).

[0507] As can be seen in table 2.1, the average grams of total protein and protein Q increased in those batches produced according to the invention, where the dilution change was applied. The average TP grams obtained in the clarified product of the analyzed previous known batches (n = 25) are very similar (261 g), meanwhile the mean value of TP in the analyzed clarified products obtained in according to the invention, (n = 12) is 285 g, which represents an increase of 9 %. Regarding the protein Q, in previous known batches, the average value was 26.7 g compared to 38.4 g in the analyzed clarified products according to the invention, that means an improvement of 44 % in this process step. This would imply that the process change promotes an increase in the protein of interest in a greater proportion than the total proteins, which is reflected in a greater PQ / PT average ratio in the invention (13%) compared to the 10% average ratio of previous known batch.

[0508] Table 2.7. Results of PQ, TP and ratio PQ / TP in the clarified product.

[0509] Adjusted Clarified Product

[0510] Average PQ ratio

[0511] Year Batch PQ Average Average ratio TP Mean Mean TP (g) PQ / TP PQ / TP ± 2SD ± 2SD TP (g) (g) PQ (g) (%) (g) (%) (g) 8000720003 241.7 32.1 13%

[0512] 8000720006 224.4 32.8 15%

[0513] 8000720008 247.3 24.6 10%

[0514] 8000720009 261.4 34.8 13%

[0515] previous 260.7 28.7 11% 217 - 305 11 - 46

[0516] 8000720010 254.9 12.6 5%

[0517] 8000720012 281.3 23.2 8%

[0518] 8000720014 281.7 27.4 10%

[0519] 8000720016 259.3 26.4 10%

[0520]

[0521] Lysate concentration - P62038693WO 45

[0522] Adjusted Clarified Product

[0523] Average PQ ratio

[0524] Year Batc PQ Average Average ratio TP Mean

[0525] h TP (g) PQ / TP PQ / TP ± 2SD Mean ± 2SD TP (g) (g) PQ (g) (%) (g) (%) (g) 8000720018 294.5 44.2 15%

[0526] 8000723003 249.6 19.1 8%

[0527] 8000723004 265.0 30.2 11%

[0528] 8000723005 258.1 25.8 10%

[0529] 8000723006 325.0 21.0 6%

[0530] 8000723007 208.2 18.5 9%

[0531] 8000723008 221.9 21.0 9%

[0532] 8000723009 311.6 19.2 6%

[0533] 8000723011 314.9 27.9 9%

[0534] previous 261.8 25.6 10% 188 - 336 12 - 39

[0535] 8000723012 250.6 41.4 17%

[0536] 8000723013 246.7 29.2 12%

[0537] 8000723020 248.5 36.9 15%

[0538] 8000723021 287.2 27.6 10%

[0539] 8000723022 302.5 22.8 8%

[0540] 8000723023 259.9 26.0 10%

[0541] 8000723024 209.1 24.6 12%

[0542] 8000723025 229.6 19.1 8%

[0543] 8000724001 283.9 25.7 9%

[0544] 8000724002 253.3 25.6 10%

[0545] 8000724004 283.5 33.8 12%

[0546] 8000724005 238.4 36.5 15%

[0547] 8000724006 356.4 51.2 14%

[0548] 8000724007 289.7 29.2 10%

[0549] current 284.6 38.4 13% 219 - 350 12 - 64

[0550] 8000724008 254.4 23.4 9%

[0551] 8000724009 318.8 59.7 19%

[0552] 8000724010 313.1 61.8 20%

[0553] 8000724011 260.0 34.8 13%

[0554] 8000724012 275.3 35.9 13%

[0555] 8000724013 287.9 43.2 15%

[0556]

[0557] Figure 1 shows the total protein grams in the adjusted clarified product for all industrial batches, as well as the Protein Q grams of the analyzed batches. It is remarkable the upward trend in the batches according to the current invention, after the process improvement is applied.

[0558] CONCLUSIONS

[0559] From the results discussed in previous section we can draw the following conclusions:

[0560] • Twelve industrial batches have been manufactured according to the invention implementing the following process change: biomass is resuspended up to 45.0 kg (meaning approx. TP concentration of 6 g / L) with solubilization buffer instead of 34.5 kg (meaning approx. TP Lysate concentration - P62038693WO 46

[0561] concentration of 11 g / L). This increment in the biomass dilution before cell disruption has promoted protein solubilization and thus, an increase in the total protein clarified grams per batch.

[0562] • The average total protein obtained in the clarified product of the previous known batches, before implementing the change, were 261 grams, which compared to the average 285 grams obtained in 12 batches manufactured in according to the invention, implies an increase of 9 %.

[0563] • In addition, the protein of interest, Protein Q, was analyzed in the lysate and clarified product of several batches manufactured in previous known batches. The average value in the clarified was 26.7 g compared to 38.4 g in the analyzed clarified products according to the invention, that means an improvement of 44 % in this process step. This would imply that the process change promotes an increase in the protein of interest in a greater proportion than the total proteins, which is reflected in a greater PQ / PT average ratio according to the invention (13%) compared to the 10% average ratio of previous known batches.

[0564] In conclusion, it has been proven that increasing the volume of biomass dilution before lysis, rises both PQ and TP in the lysate and clarified product, although the target protein increases more in proportion, which means that the product to be loaded in the first purification step will have a greater amount of proteins, with a higher PQ content.

[0565] Example 3 - Total Protein quantification by BCA. Analytical method description.

[0566] Introduction

[0567] 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.

[0568] The reaction is shown in the following scheme:

[0569] Protein + Cu+2Cu+1

[0570] Cu+1+ BCA purple complex BCA-Cu+1

[0571] The BCA protein assay is used for the detection and quantification of total protein in samples of the protein Q fabrication process.

[0572] Materials and equipment

[0573] Table 3.1. Materials, reagents and solutions used for BCA assay and their manufacturer. Lysate concentration - P62038693WO 47

[0574] Material / Reagent / Solution

[0575] Sodium chloride (NaCI)

[0576] Sodium hydroxide (NaOH) 0.01 M

[0577] Urea > 99.5 % purity (CH4N2O)

[0578] PBS: BupH™ Modified Dulbecco's

[0579] Sodium dihydrogen phosphate

[0580] (NaH2PO4'2H2O)

[0581] Triton X-100

[0582] (C14H21(C2H4O)nOH)

[0583] Milli-Q water

[0584] 96 well microplates flat bottom

[0585] Sealing tape for 96 well plates

[0586] Pipette tips

[0587] 1.5 mL tubes (low binding)

[0588] 15 mL tubes (low binding)

[0589]

[0590] Kit Pierce BCA Protein Assay

[0591] Table 3.2. Equipment used for BCA assay and their manufacturer.

[0592] Equipment

[0593] Plate reader

[0594] Micropipettes

[0595] Millipore Milli-Q™ ultrapure water purification system

[0596] Analytical balance

[0597] Microplate shaker and incubator

[0598]

[0599] pHmeter

[0600] Solutions

[0601] Table 3.3. Composition and working pH of buffers used for BCA assay.

[0602] Solution Composition Adjusted pH BupH™ Modified Dulbecco's

[0603] PBS 1X - Preparation according to manufacturer.

[0604] NaCI 0.9 % 0.1 M NaCI - 8 M Urea, 43 mM sodium dihydrogen

[0605] Biomass phosphate dihydrate, 0.4 M NaCI, 1% Triton X- 8.0 ± 0.2 resuspension buffer

[0606] 100

[0607] 8 M Urea, 50 mM sodium hydrogen phosphate

[0608] First chromatography dihydrate, 0.5 M NaCI, 280 mM imidazole, 1% 8.0 ± 0.2 step elution buffer Triton X-100

[0609] 8 M Urea, 50 mM sodium hydrogen phosphate

[0610] IEC elution buffer 8.0 ± 0.2

[0611]

[0612] dihydrate, 0.5 M NaCI, 1% Triton X-100

[0613] Procedure

[0614] Calibration curve and sample preparation Lysate concentration - P62038693WO 48

[0615] The calibration curve is prepared diluting standard BSA2 mg / mL from BCA assay kit in NaCI 0.9 % between 2000 pg / mL and 50 pg / mL (Table 4)

[0616] Table 3.4. Preparation of calibration curve of BSA.

[0617] Standard Concentration (pg / mL)

[0618] A 2000

[0619] B 1000

[0620] C 600

[0621] D 500

[0622] E 400

[0623] F 300

[0624] G 200

[0625] H 100

[0626] I 50

[0627]

[0628] Blank 0

[0629] 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.

[0630] 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.

[0631] Assay procedure

[0632] 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.

[0633] Results

[0634] 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.

[0635] Example 4 - Protein Q quantification by ELISA-Ni. Analytical method description.

[0636] Introduction

[0637] The Enzyme-Linked Immunosorbent Assay (ELISA) is a technique that allows for detection and quantification of target molecules using an enzymatic reaction. It is based on antibody sandwich principle in which a capture molecule specific to the analyte of interest is bound to a microtiter plate to Lysate concentration - P62038693WO 49

[0638] create a solid phase. After the analyte recognizes the capture molecule, it is recognized by a different epitope by an antibody. This primary antibody is then bound to a detection antibody conjugated with HRP enzyme (horseradish peroxidase), completing the sandwich and allowing for the detection and quantification by adding a colorless substrate solution TMB / hydrogen peroxide), which changes color proportionally to the quantity of bounded analyte. In this protocol, plate wells are covered with Ni-NTA, which is specifically bound by His-tagged proteins, and making the step of blocking for unspecific binding unnecessary.

[0639] Material, equipment and reagents

[0640] Table 4.1. Materials, reagents and solutions used for ELISA and their manufacturer.

[0641] Material / Reagent / Solution

[0642] Sodium chloride (NaCI)

[0643] Sodium hydroxide (NaOH) 0.01 M

[0644] Urea ≥ 99.5 % purity (CH4N2O)

[0645] Tween-20 10%

[0646] PBS: BupH™ Modified Dulbecco's

[0647] Sodium dihydrogen phosphate

[0648] (NaH2PO4·2H2O)

[0649] Triton X-100

[0650] (C14H21(C2H4O)nOH)

[0651] Primary Antibody 1 monoclonal mouse 2012 PPQ14-1

[0652] (Anti-protein Q)

[0653] Secondary antibody Donkey Anti-mouse IgG conjugated

[0654] with HRP

[0655] TMB Substrate Kit

[0656] Sulfuric acid 2M

[0657] BSA

[0658] Milli-Q water

[0659] PierceTM nickel coated plates

[0660] Sealing tape for 96 well plates

[0661]

[0662] Pipette tips

[0663] Table 4.2. Equipment used for ELISA and their manufacturer.

[0664] _ Equipment _

[0665] _ Plate reader _

[0666] _ Micropipettes _

[0667] Millipore Milli-Q™ ultrapure water purification system

[0668] _ Analytical balance _

[0669] _ Microplate shaker _

[0670] _ pHmeter _

[0671] _ Centrifuge _

[0672]

[0673] Sonicator

[0674] Solutions

[0675] Table 4.3. Composition and working pH of buffers used for ELISA assay.

[0676] Solution Composition Adjusted pH Biomass 8 M Urea, 50 mM sodium dihydrogen phosphate

[0677] resuspension 8

[0678] dihydrate, 0.5 M NaCI, 1% Triton X-100

[0679]

[0680] buffer Lysate concentration - P62038693WO 50

[0681] 6 M Urea, 50 mM sodium dihydrogen phosphate

[0682] Incubation solution dihydrate, 0.5 M NaCI, 0.1 % Tween-20 10%, 0.1 % 8

[0683] BSA

[0684] BupH™ Modified Dulbecco's

[0685] PBS 1X solution - Preparation according to manufacturer

[0686] Washing solution PBS 1X, 0.05 % Tween-20 - Antibody blocking - solution PBS, 0.05% Tween-20, 3% BSA

[0687] Primary antibody Primary monoclonal antibody 2012 PPQ14-1 in a - solution 1:50 000 dilution in antibody blocking solution

[0688] Secondary Secondary antibody Anti-mouse IgG, HRP in a 1: - antibody solution 50 000 dilution in antibody blocking solution

[0689] Substrate solution TMB substrate and hydrogen peroxide solutions -

[0690]

[0691] (included in the kit) are mixed in a 1:1 proportion

[0692] Procedure

[0693] Calibration curve and sample preparation

[0694] The calibration curve is prepared diluting an internally manufactured standard (active pharmaceutical ingredient from an industrial batch, e.g. Example 2) in incubation solution between 8000 ng / mL and 15.63 ng / mL (Table 4.1).

[0695] Table 4.4. Preparation of calibration curve of API batch 8000720021.

[0696] Standard ng / mL

[0697] A 8000

[0698] B 4000

[0699] C 2000

[0700] D 1000

[0701] E 500

[0702] F 250

[0703] G 125

[0704] H 62.5

[0705] I 31.25

[0706] J 15.63

[0707]

[0708] Blank 0

[0709] Depending on the sample to be analyzed, different dilutions in incubation solution are prepared considering that the absorbance at 450 nm of diluted samples need to fit between the range of calibration curve (8000- 15.63 ng / mL) to be quantified. For resuspended biomass samples, cells are first broken by sonication.

[0710] Assay procedure

[0711] After samples, calibration curve standards and solutions are prepared, the assay in the 96 well microplate is carried out as follows: 100 pL of standards and each sample are loaded either in duplicate or in triplicate and the microplate is incubated with constant agitation (450- 500 rpm) for 1 hour at room Lysate concentration - P62038693WO 51

[0712] temperature. Wells are washed three times with 200 pL of washing solution per washing before the addition of 100 pL of primary antibody 1:50000 in each well. The microplate is incubated with constant agitation (450 - 500 rpm) for 1 hour at room temperature and the washing step is repeated. Right before its addition, TMB solution and hydrogen peroxide solution are mixed in a 1: 1 proportion and 100 pL of the resulting solution are loaded. The microplate is incubated for 12 minutes at room temperature and the reaction is stopped with 100 pL of 2 M sulfuric acid before reading the plate in a plate reader at 450 nm wavelength absorbance.

[0713] Results

[0714] A 4 parameter logistic curve (4PLC) is used for quantification of protein Q (ng protQ / mL). The absorbance of samples analyzed is interpolated in the calibration curve and the dilution factor is later applied to obtain the real result un pg protQ / mL.

[0715] Example 5: Applying the biomass lysis and solubilisation process of the invention to proteins homologous and non-homologous to Protein Q

[0716] LIST OF ABBREVIATIONS ABBREVIATION

[0717]

[0718] Wording Definition

[0719]

[0720] Al Active ingredient

[0721] Anti-PQ Antibody anti Protein Q

[0722] BCA Bicinchoninic acid assay

[0723] DSP Downstream processing

[0724] ELISA-Ni Enzyme-Linked Immunosorbent assay using Nickel as ligand

[0725] HOP Host cell protein

[0726] ISP Industrial scale process

[0727] M1 LETI Resuspended Biomass

[0728] M2 LETI Lysate

[0729] M3 LETI Diluted lysate

[0730] M4 LETI Clarified

[0731] NA Not available

[0732] ND Not detected

[0733] Pl Isoelectric point

[0734] PQ / ProtQ Protein Q

[0735] q.s. Quantum satis: Add as much of the ingredient as is needed

[0736] SD Standard deviation

[0737] SP Specific protein

[0738] SDS-PAGE Sodium dodecyl-sulfate polyacrylamide gel electrophoresis

[0739] SSP Small scale process Lysate concentration - P62038693WO 52

[0740] SSQM Small scale qualification model

[0741] TP Total Protein

[0742] USP Upstream processing

[0743] WFI Water for injection

[0744] WB Western Blot

[0745] 5.1. Introduction

[0746] This document presents several laboratory trials carried out to demonstrate that the process of the invention could be successfully applied to other protein than PQ (proteins having from 59,8 to 87,4 % sequence identity with PQ, exemplified later herein as Protein 2 and Protein 3). PQ is represented by SEQ ID NO:1.

[0747] This process comprising a dilution of the total protein concentration in the biomass before lysing the cells present in said biomass leads to an increase in total protein and specific protein recoveries in the lysis and clarification steps, without altering the protein profile.

[0748] This study focuses on the extraction of the target protein from biomass and obtaining a product soluble and stable.

[0749] Protein Q production and purification process comprises the following steps: suspending the biomass in a suspension buffer, lysing the cells present in the suspended biomass to obtain a lysate where the protein is solubilized and further clarifying the protein from said lysate. More specifically, the laboratory tests consisted of testing two different working concentrations: 3 mg / mL and 11 mg / mL of total protein (TP) to suspend the biomass and applying that dilution before or after the lysis. Both conditions, reference and optimal, were tested for two proteins with different sequence identity to Protein Q. In order to compare the performance of each trial, several analyses were performed at every process step: Protein productivity is analyzed by BCA (total protein) and ELISA-Ni (specific protein), and the protein profile was analyzed by electrophoresis (SDS-PAGE and WB)

[0750] To evaluate if the protein dilution and the process step where it is applied impact on the lysis and clarification efficacy, it is needed to start from cells that express an intracellular protein. Forthat purpose, fermentations with E.coli expressing an endogenous protein with similar isoelectric point (pl) and molecular weight to Protein Q are desired.

[0751] 5.2. Proteins selected

[0752] PQ is the protein on which the process of the invention has first been succesfully 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 DDIGTLLKNVTLSHSGWPNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTKYLAAYA LASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPA ASSGAAAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSG KTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGA VSTAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPEESDEDD FGMGGLF

[0753] Further proteins were selected:

[0754] Protein 2 (SEQ ID NO:2): Identity <80% with PQ (SEQ ID NO:1) Lysate concentration - P62038693WO 53

[0755] 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 needleman alignment) when compared with PQ (SEQ ID N1) and has a histidine tail and an anti-PQ antibody recognition site.

[0756] SEQ ID NO: 2:

[0757] MGSSHHHHHHSSGLVPRGSHMNKKMHEHSEHFKQKFAELLEQQKAAQYPSKGGEEQYYIKAQLLE HLVELVADKFRIIGQTEDGGYVASVPSEEGVLAWATTCQVFSDGHPAVGVGGVDVGPLSVGPQSVGP LSVGLQAVDVSPVSGGNVAAKIALAKSLLEKEVRVDSVFQQSEACDVCSVTKGHGGFGIDEHIDLGIK YDPSTGIYGMDFYVVLGRRGERVAHRKRKCSGLKACSRPMSTKYLAAYALASLSKASPSQADVEAIC KAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKKDE PEEEADDDMGPSRVDPMQYLAAYALVALSGKTPSKADVQAVLKAAGVAVDASRVDAVFQEVEGKSFD ALVAEGRTKLVGSGSAAPAGAVSTAGAAAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAA AKEEPEESDEDDFGMGGLF

[0758] Protein 3 (SEQ ID NO:3): Identity >80% with PQ (SEQ ID NO:1)

[0759] “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 needleman alignment) when compared with PQ (SEQ ID NO:1) and has a histidine tail and an anti-PQ antibody recognition site.

[0760] SEQ ID NO:3:

[0761] MATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGASGAPRISEFSVKAAAQSGKKR ARLNPRTVMLAARHDDDIGTLLKNVTLSHSGVVPNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTK YLAAYALASLSKASPSQADVEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGA AAGVTASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSGKTPSKADVQAVLKA AGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGAVSTAGAGAGAVAEAKKEEPEEEE ADDDMGPVDLQPAAAAPAAPSAAAKEEPEESDEDDFGMGGLFSSGENLYFQGLEHHHHHH

[0762] 5.3. Alignment of Protein 2 and Protein 3 with Protein Q

[0763] Alignments were carried out using EMBOSS needle alignment (Needleman): Matrix EBLOSUM62, Gap penalty: 10.0, Extend penalty: 0.5.

[0764] 5.3.1. Alignment Protein 2 (SEQ ID NO:2) and Protein Q (SEQ ID NO:1): EMBOSS Needle Lysate concentration - P62038693WO

[0765] 54

[0766] # Aligned^secjuences: 2

[0767] # 1: PQ

[0768]

[0769] # 2: PR0TEIN2

[0770] # Matrix: EBL0SWI162

[0771] # Gap_penalty: 18.0

[0772] # Extend_penalty: 0,5

[0773] *

[0774] # Length: 490

[0775] # Identity: 293 / 490 (59.8JS)

[0776] # SiBilarity: 320 / 490 (65.3«)

[0777] # Gaps: 80 / 490 (16.3M)

[0778] # Score: 1241.9

[0779] ♦

[0780] #

[0781]

[0782] Lysate concentration - P62038693WO 55

[0783] PQ i * -...4 4.4 46

[0784] :. I.. |, I. u....;..:... PROTEIW2 1 MGSSHHHHHHSSGLVPR(^MNKKMHEHSEHFKQI0:AE1LEQQKAAQ¥PS 5© PQ 47 KSGSK- - • - • • ' 4: ‘ 4. 4 - - - - - - - - - - - 75 - 4 14 ■; ■. 4. ■ i - lee PQ 76 - I lit I I..... I M •: •, I: - 4 14. n..., • ■ ■ 4,. ■., we Pf i. \ ■■ 4: w | I: I: I:. | • I i • • I Picreiit 4 14 4 4. 4 4. 4. 44 1 4 • •.• ■'C-..- 41: < 1. '• 198 Pt 15® -KATPSAPEfg- - - - - - - -. - - - - -. - - - - - - - - - - 172

[0785] .. PROTE1112 199 IKY0PST6IWB0FY¥¥LG116ERWAMilCRKCS6LK*CSRPMSTiCYL*A¥ 248 PQ 1 4 -. 4 4 -. 4. 4 4 1.. “ 4 ■ ■■ ■. V.. 44: - 222 m H m t m 1 m H i i H 1 m 111 J m 1 m 1 1 1 i 1 1 1 m m PiOTEIiZ 249 ALASLSKASPSQADVEAICKAVHIOVDQATLAFWESVTGROVATt-IAEG 298 PQ.. - ■ ” 4 4. 4 4 4... '-A::. 4 - 4 ’ 4. 4 • 272

[0786] H m m m m 1111 m 11 m 1 m i m 1111111111 H 1 H 1 " 4744 299 AAKMSAMPAASSGAAAGVTASAAGDAAPAAAAAKi®EPEEEAM«»»PSR 348 PQ 4. 4;. 4.. 4 ’ • 4 4 • ~ ■ 1 4 ■ 4 4 7 • 322 m mi mm i i m m i i m m i m imm m m i m - 4 1 1 ’ -4 44 4 4.4 - ” - 4 1 - - 4. i 44 4 4 4 398 PQ 323 l«FOAI. VAEGRTICLVGSGSAAPAGAVSTAGAGAGAVAEAKiffiEPEEEEW 372

[0787] 1 1 f 1 1 1 1 1 I I I I I H H I H 1 1 1 1 1 1 1 I I 1 1 • I I I I I I 1 1 i 1 1 1 H I I I I

[0788]

[0789] • “ - •. 4 4 4: ’ 4;: 4 4- - - * 4. 4 ■ -. - 44 448 PQ 373 00»«P¥0l.fPAAAAPAAPSAMlCEEPeES0E00FGW6LF 412

[0790] 1 [ m 1 1 m 1 1 111 H 1 i t i f ti l m I m 1 m mr' 4. 449 ODi6P¥OLOM APA*PSAAA®EPEESOEOOFW66LF 4B8

[0791] .2. Alignment Protein 3 (SEQ ID NO:3) and Protein Q (SEQ ID NO:1): EMBOSS Needle Lysate concentration - P62038693WO

[0792] 56

[0793] #

[0794] # 2 # 1: P| it 2: PWEI13 # Matrix: EBL0SWI62 f 6at_penalty: 1®.® f. •. <.::.. ♦ ♦ # Identity: 376 / 43© (87. «) # Saps: # Sew®: 1853.0 S

[0795] tf

[0796] 1 PROTEJ13

[0797] »e tei 150 67 116 151 PWTEIIB 117 Mre*rere5SRfWrWLM¥MML5XWSOAOVEaiCKWiIO¥Ot 166 PiOTEIW 167

[0798] 251 217 A*m«OEKEE*OOOiemWSWlM^MVMS6CTPS«V?a* LKAASMWIWRVOA^OEVEGeFOAmEGRWLVeseSMfWAVST wrens 2«7 LW6VWmWDA¥FQE¥E6®F0AU»f6WKLV6Sfi»AMfiWST 316

[0799] 361 PBOTEIW 317 MA6«»BW®EPEEEE»00»SP¥0lQPAMAWWWMMEEPEE 401 S0600F6M6GLF <2

[0800]

[0801] 367 396 Lysate concentration - P62038693WO 57

[0802] 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).

[0803] 5.4. Equipment and materials

[0804] 5.4.1. Equipment

[0805] The following pieces of equipment were used to perform the laboratory trials:

[0806] Table 5.1. List of equipment used.

[0807] Equipment

[0808] Freezer

[0809] Refrigerator

[0810] Cell Disruptor

[0811] Stirring plate

[0812] Precision scale

[0813] pHmeter

[0814] Centrifuge

[0815] Sonicator

[0816] Ultrapure Water Purification System

[0817] Micropipette P1000

[0818] Micropipette P200

[0819] Micropipette multichannel P300

[0820] Micropipette P20

[0821]

[0822] Micropipette P50

[0823] 5.4.2. Materials

[0824] The following table gathers the materials employed to perform the laboratory trials.

[0825] Table 5. 2. List of materials used.

[0826] Material Model

[0827] Clarification filters Filter capsule with membrane porous diameter 0.1 - 1 μm

[0828]

[0829] 5.4.3. Buffers

[0830] The composition of the solutions employed in the process are described in the following tables:

[0831] 5.4.3.1. Urea 8 M solution

[0832] Table 5.3. Urea 8 M solution composition

[0833] Material Grams / L Urea, CH4N2O 480.48

[0834]

[0835] Water, H2O q.s.p liter Lysate concentration - P62038693WO 58

[0836] 5.4.3.2. Biomass suspension buffer

[0837] Buffer is prepared weighing the following reagents.

[0838] Table 5. 4. Biomass suspension buffer composition.

[0839] Material Grams / L Monosodium phosphate dihydrate, NaH2PO4.2H2O 6.67

[0840] Urea 8M solution 957.00 Sodium chloride, NaCl 24.97 Triton X-100, Ci4H2i(C2H4O)nOH 9.14

[0841]

[0842] Sodium hydroxide, NaOH 4M c.s.p = pH8 5.5. Analytical methods

[0843] Total protein is quantified by BCA. The method is described in Example 3.

[0844] The protein target Protein Q is quantified by ELISA-Ni. The method is described in Example 4.

[0845] 5.6. Experimental design

[0846] The laboratory experiments were carried out sequentially: first, the series relating to the solubility and stability of the proteins, and then the series relating to the stage of the process in which two different dilutions are applied to the different proteins. Therefore, the experimental design and results are divided into two sections, taking into account the results of the first experiment to establish the conditions for the next one.

[0847] 5.6.1. Solubility and stability test

[0848] An initial solubility and stability test was carried out by dissolving pellet of the selected protein in the Protein Q process buffer. Frozen biomass coming from a 30 L fermenter was used as starting material for each trial. The total protein and specific protein obtained from each fermentation were quantified so that the amount of starting biomass was standardized.

[0849] The experimental setup is shown in figure 2.

[0850] This experiment was performed with three proteins: PQ, Protein 2 and Protein 3. Only 1 gram of each pellet was taken and suspended in biomass suspension buffer to a target biomass concentration 0.11 g / mL (this concentration is obtained from industrial scale process). The next step was to homogenize the samples at a setpoint temperature of 5°C. Due to the small volumes used, a sonicator was applied instead of a disruptor, then the samples were centrifuged (3000 g, 10 minutes) and filtered.

[0851] Total protein was quantified by BCA, specific protein was measured by ELISA-Ni and protein profile was checked by SDS-PAGE from the different steps. In this way, the amount of total and specific protein per gram of biomass was defined for each clone and the protein stability was checked under the defined conditions.

[0852] 5.6.2. Experimental design of lysis with different TP concentration

[0853] Two tests were performed with each protein: one under reference conditions (biomass suspension up to 11 mg / mL TP and dilution after lysis and clarification up to 6 mg / mL TP) and another under optimal conditions (biomass suspension previous to lysis to 3 mg / mL TP). The experimental setup is shown in Figure 3. Lysate concentration - P62038693WO 59

[0854] In order to carry out the runs starting from the same total protein, the BCA findings from the previous experiment were used. Therefore, the biomass weighted for each protein was adapted aiming to start from 1.3 g of total protein.

[0855] For the reference run, approximately 12 g of biomass for Protein 2 or 30 g of biomass for Protein 3 were suspended in 445 mL of cold suspension buffer, corresponding to a TP concentration of approximately 11 mg / mL and homogenized for = 40 minutes. As explained, the equivalencies of pellet and PT were determined in the previous trial.

[0856] The suspended biomass was subjected to two cycles of cell disruption at 750 bar and a set temperature of 5 °C. Then it was maintained for 1 hour with ice agitation for solubilization, if necessary, the pH was adjusted to 8.0 ± 0.2. After that, the product was clarified from the cell debris and small particles by two consecutive filtrations. Finally, the filtrate was diluted in biomass suspension buffer to a target concentration of 6 mg / mL total protein.

[0857] For the optimal run, approximately 12 g of Protein 2 biomass or 30 g of Protein 3 biomass were suspended in 118 mL of cold suspension buffer, corresponding to a TP concentration of approximately 3 mg / mL and homogenized for = 40 minutes.

[0858] The resuspended biomass was subjected to two cycles of cell disruption at 750 bar and a setpoint temperature of 5 °C. Then, it was maintained in agitation for 1 hour for solubilization, if necessary, the pH was adjusted to 8.0 ± 0.2. After that, the product was clarified by two filtrations, one to get rid of cellular debris and one to get rid of smaller particles.

[0859] Below there is a summary table 5.5. of the volumes and weights of the tests described. Lysate concentration - P62038693WO 60

[0860] Table 5.5.. Summary table of the volumes and weights of each trial.

[0861] Protein 2 Protein 3 pQ* reference protein Run of Run Run of Run Run of Run Reference Optimal Reference Optimal Reference Optimal Pellet.grams 12 12 30 30 16 16

[0862]

[0863] Volume, mL 118 445 118 445 118 445

[0864] 5.7. Results

[0865] Since the trials were conducted in a sequential manner, the results are presented in two sections considering the findings of the first experiment to set up the conditions of the next experiment.

[0866] 5.7.1. Results of Solubility and stability test

[0867] We performed an absorbance spectrum of the three proteins in the clarified sample (M4) and observed the absorbance is around 0.14-0.15 AU at 350 nm in all proteins. See Figure 4. Turbidity is similar to Protein Q, which indicates that all proteins are soluble in the biomass suspension buffer.

[0868] We performed the analysis of the samples by BCA and ELISA-Ni. The results are shown in the following tables.

[0869] Table 5.7.1. Results of total protein by BCA.

[0870] Total Protein by BCA ProtQ Protein 2 Protein 3 TP Yield TP Yield TP Yield STEP SAMPLE (mg / mL) (%) (mg / mL) (%) (mg / mL) (%) LYSIS M2: Lysate 8.3 13.2 5.2

[0871] 7.8 94% 11.4 87% 6.7 128% CENTRIFUGATION M3: Supernatant

[0872] M3P: Pellet 1.5 18% 1.6 12% 1.8 35% FILTRATION M4: Filtered 6.1 78% 11.6 102% 5.2 77%

[0873]

[0874] Yield (%) Total 73% 88% 99%

[0875] In the lysate samples (M2), even when starting from 1 g of initial biomass for all the proteins, the TP concentration is different for each of them. Protein Q presenting an intermediate TP (8.3 mg / mL), Protein 2 (mid-homology) expressing higher concentration (13.2 mg / mL) and Protein 3 (high-homology) lower TP (5.2 mg / mL). It can be observed that, after lysis and centrifugation, most of the protein keeps in the supernatant, which proves good solubility for the three proteins in the process buffer. Besides, the global TP recovery is within 73 (ProtQ) and 99 % (Protein 3).

[0876] Table 5.7.2. Results of specific protein by ELISA-Ni.

[0877] Specific Protein by ELISA -Ni ProtQ Protein 2 Protein 3 STEP SAMPLE SP Yield Yield SP Yield (mg / mL) SP (mg / mL)

[0878] (%) (%) (mg / mL) (%) LYSIS M2: Lysate 0.75 3.71 0.58

[0879] M3: 0.73 97% 3.96 107% 0.59 102% CENTRI SupernatantFUGATION

[0880] < LOD NA 0.41 11% < LOD NA M3P: Pellet

[0881] 0.85 116% 2.58 65% 0.60 100% FILTRATION M4: Filtered

[0882] Yield (%) 113% 70% 102% Total

[0883]

[0884] We also observe that the specific protein is expressed entirely in the supernatant and there is hardly any protein left in the pellet for the three tested proteins. In addition, it is noted that Protein 2 (mid- Lysate concentration - P62038693WO 61

[0885] homology) shows much higher specific protein concentration per gram of pellet than ProtQ or Protein 3 in the lysate, supernatant and filtered product, indicating that Protein 2 is expressed more and keeps stable. To see the specific protein per total protein proportion, we calculated the ratio, which can be seen in the following table.

[0886] Table 5.7.3.. Ratio specific protein / Total Protein

[0887] Yield SP / TP (%) ProtQ Protein 2 Protein 3 STEP SAMPLE SP / TP Evolution SP / TP Evolution SP / TP Evolution (%) (%) (%) (%) (%) (%) LYSIS M2: Lysate 9.0% 28.1% 11.1% CENTRIFUGATION M3: Supernatant 9.4% 0.4% 34.6% 6.5% 8.8% -2.3%

[0888] M3P: Pellet LOD NA 25.4% NA LOD NA FILTRATION M4: Filtered 13.9% 4.5% 22.2% -12.5% 11.5% 2.6%

[0889] Yield (%) Total 4.9% -5.9% 0.4%

[0890]

[0891] It is remarkable that Protein 2 contains much higher proportion of specific protein per TP from the lysate than Protein Q or Protein 3, which shows that Protein 2 is purer and therefore contains less host cell proteins from the E.coli.

[0892] Once the total amount of protein that can be extracted per gram of biomass of each fermenter was calculated for each protein, we can estimate the total protein to be used in each lysis trial regarding Protein Q process. Protein 2 presents 110.1 mg TP / g pellet and Protein 3 has 43.4 mg TP / g pellet. The protein profile was also analyzed by SDS-PAGE and Western blot (results not shown), and similar and stable profiles were observed in all the samples of the three proteins analyzed.

[0893] 5.7.2. Results of lysis with different TP concentration

[0894] The results of these experiments are summarized in the next tables comparing the reference process to the optimal process for every tested protein.

[0895] Concentrations and total amount of specific and total proteins are followed at each step. With these data it is possible to calculate the process yields and ratios in order to evaluate the product quantity and quality obtained when applying each condition. Lysate concentration - P62038693WO 62

[0896] Table 5.7.4. Results of Total protein (BCA) or specific protein (ELISA-Ni) for runs carried out at reference (11 mg / mL) and optimal (3 mg / mL) conditions in two different proteins (Protein 2 and Protein 3).

[0897] TP (BCA) SP (ELISA-Ni)

[0898] Protein 2 Protein 3 Protein 2 Protein 3 SAMPLES Reference Optimal Reference Optimal Reference Optimal Reference Optimal [TP] or [SP]

[0899] (ug / mL) 11051 3494 14043 4306 5572 2144 782 545 Resuspended

[0900] biomass M1 Vol (mL) 118 445 121 432 118 445 121 432 PT (mg) 1304 1555 1699 1860 657 954 95 236 [TP] or [SP]

[0901] (ug / mL) 11327 3513 13465 5020 6706 2037 827 545 Lysate 1st

[0902] cycle M2a Vol (mL) 132 430 120 408 132 430 120 408 PT (mg) 1495 1511 1616 2048 885 876 99 222 [TP] or [SP]

[0903] (ug / mL) 10428 3529 13240 4926 6136 3389 959 427 Lysate 2nd

[0904] cycle M2b Vol (mL) 132 430 120 408 132 430 120 408 PT (mg) 1376 1517 1589 2010 810 1457 115 174 [TP] or [SP]

[0905] (ug / mL) 8929 3266 8125 4260 5967 3591 521 352 1st clarified

[0906] M3 Vol (mL) 124 433 120 408 124 433 120 408 PT (mg) 1107 1414 975 1738 740 1555 62 144 [TP] or [SP]

[0907] (ug / mL) 8269 3145 7813 4199 5582 2712 360 357 2nd Clarified

[0908] M4 Vol (mL) 122 432 110 398 122 432 110 398 PT (mg) 1009 1359 859 1671 681 1171 40 142 [TP] or [SP]

[0909] (ug / mL) 4736 NA 4217 NA 3810 NA 193 NA Clarified

[0910] adjusted M4b Vol (mL) 220 NA 198 NA 220 NA 198 NA PT (mg) 1040 NA 835 NA 837 NA 38 NA

[0911]

[0912] The table shows a summary of the total and specific protein results for the two proteins tested for both the reference run and the optimal run. The first thing that is observed is that more total protein is obtained from the biomass suspension when applying a bigger dilution (optimal run). In addition, total mg of protein increases after 1stand 2ndlysis, indicating better protein extraction in the optimal run for both proteins, and this is maintained during the clarification, resulting in higher productivity starting from the same amount of TP.

[0913] With regard to the specific protein, the findings from the previous experiment are corroborated: Protein 2 is expressed much more than Protein Q or Protein 3, but this is inherent to the selected protein. Next, the yield at each step is calculated comparing the reference run to the optimal run for every protein. The results are also compared with the data obtained for protein Q industrial batches (see example 2). The laboratory experiments are summarized in the following tables. Lysate concentration - P62038693WO 63

[0914] Table 5.7.5. TP yields tracking each step of the process for Protein 2 (59,8% homology) and Protein 3 (87,4% homology) runs.

[0915] % YIELD TP by BCA Protein 2 Protein 3 Samples Reference Optimal Reference Optimal Biomass lysis yield 106% 98% 94% 108% 1st Clarification Yield 80% 93% 61% 86% 2nd Clarification Yield 91% 96% 88% 96% Lysis - Clarification Global Yield

[0916]

[0917] 77% 87% 51% 90% The mid-homology Protein 2 presents 87 % TP global yield in the optimal trial, while 77% in the reference trial. This represents a total protein recovery increase of 10 % when applying the changes, similar to the average 9 % increment in yield found in the industrial batches of Protein Q. In the case of Protein 3, 90 % TP global yield is achieved in the optimal trial, while only 51% in the reference trial. In this case, the reference run has more room for improvement, increasing by 39% the TP recovery.

[0918] Table 5.7.6. SP yields tracking each step of the process for Protein 2 (59,8% homology) and Protein 3 (87,4% homology) runs.

[0919] % YIELD SP by ELISA-Ni Protein 2 Protein 3 Samples Reference Optimal Reference Optimal Biomass lysis yield 123% 153% 122% 74% 1st Clarification Yield 91% 107% 54% 82% 2nd Clarification Yield 92% 75% 63% 99% Lysis - Clarification Global Yield 104% 123% 42% 60%

[0920]

[0921] Protein 2 presents 123 % global yield for specific protein in the optimal trial, while 104 % in the reference trial. The two cycles of lysis permit extracting more protein, which explains obtaining yields >100%. In addition, the findings show that Protein 2 is a highly soluble and stable protein under the process buffer and conditions, including the referent ones. Nevertheless, Protein 3 presents 60 % global yield in the optimal trial, while only 42 % in the reference trial. The greater protein loss is given by the clarification steps, which demonstrate that applying a greater dilution of the protein previous to the lysis boost protein extraction from the cells, solubilization and thus filtration.

[0922] In any case, both proteins show an increase of specific protein global recovery of 19 % when applying the optimal conditions.

[0923] Finally, the percentage of specific protein over the total protein is calculated as the SP / PT ratio for the experiments executed at the lab.

[0924] Table 5.7.7. SP / PT ratio in each step of the process for Protein 2 (59,8% homology) and Protein 3 (87.4% homology) runs.

[0925] % YIELD SP by ELISA-Ni Protein 2 Protein 3 Samples Reference Optimal Reference Optimal Suspended Biomass 50% 61% 6% 13% Lysate 59% 96% 7% 9% 1st Clarified 67% 110% 6% 8% 2nd Clarified

[0926]

[0927] 68% 86% 5% 9% Lysate concentration - P62038693WO 64

[0928] The ratios of specific protein over total protein in each process step permits checking protein purification. Protein 2 shows much higher specific protein content per total protein than Protein 3. Besides, it increases from 50 - 61 % in the suspended biomass up to 68 - 86 % in the clarified product. In Protein 3 it keeps around 5-7% in the reference trial while 8 - 13 % in the optimal trial, then the proportion of Protein 3 over total protein keeps constant over the extraction steps.

[0929] We compared the protein profiles by SDS-PAGE and Western blot (data not shown), and the profiles are similar in the reference and the optimal runs for both proteins showing that the protein quality is not affected by the process changes.

[0930] 5.8. Conclusions

[0931] Two proteins with certain similarity to Protein Q have been selected in order to run laboratory experiments to test if the changes applied in the lysis step are successful also for other Proteins. Protein 3 is a variant from Protein Q which presents a high homology to ProtQ sequence (87,4%). Protein 2 is a fusion protein which presents a medium homology to ProtQ sequence (59,8%). In both cases, applying the process changes (increasing the biomass dilution previous to the lysis) resulted in an increase in total protein and specific protein recoveries in the lysis and clarification steps. In addition, protein profile was checked, and it was similar for both proteins after being processed applying the reference process or the optimal one.

[0932] Protein 2 turned out to have a higher expression and presented exceptional stability and solubility under the Protein Q process buffers and conditions. The lysis experiments gave an increment of 10 % in TP yield and +19 % in specific protein yield when applying the optimal conditions.

[0933] For Protein 3 the specific protein expression was more similar to Protein Q. The trials resulted in an increment of 39 % in TP yield and +19 % in specific protein yield when applying the optimal conditions. To sum up, the claimed process changes are proven to be successful not only to Protein Q, but also to proteins with 59,7% homology to ProtQ sequence or higher. By diluting the biomass previous to the lysis to a target TP concentration of 3 g / L the effectiveness of the subsequent lysis and clarification steps is significantly improved without affecting product quality.

[0934] Example 6: Biomass Lysis and Solubilization Optimization Applied to Non-Homologous randomized Protein Q sequence

[0935] 1. INTRODUCTION

[0936] This example provides supplementary data and analyses aimed at expanding the range of proteins evaluated in example 5. Specifically, the laboratory tests involved assessing two working concentrations, 3 mg / mL and 11 mg / mL of total protein (TP), used to suspend the biomass, with the dilution applied either before or after the lysis step. Both reference and optimized conditions were tested on various proteins (example 5) 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 (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. Lysate concentration - P62038693WO 65

[0937] 2. EXPERIMENTAL DESIGN

[0938] A randomized sequence was designed derived from the PQ amino acid template and extensive in silico simulations were conducted to assess its structural stability and expression efficiency in E. coli. The construct maintains the original isoelectric point while reducing sequence homology to increase uniqueness. Additionally, a His-tag and the monoclonal antibody (mAb) binding site were incorporated to facilitate purification and detection of the randomized PQ (rPQ) protein.

[0939] rPQ sequence (SEQ ID NO:4):

[0940] MADPVVGVLIARTDGDTTEGDVISLGASAGFVKDAESALKPIKARNPGKASAGPVSANLGSGLDSFDA LVAEGRTKSARAADRVAKYVSLDAGSPHGLADANAEMVAVYEKEVRSEAPFVCTSELEDGLKLRNMK KNMADVPKLFSPVISSDDVGDLIAAAFMERNSSPAPESVAGSKSQVNPGANAPVQEDVAGENAGLSE AAYEATMGEGSVTADNSNCPMLAGNAQTLASAPKARVVSASLYSDAKAGAASEVGLPDAFTAAAAQ DGGAAMAASAAEAGKISIKSREEGQPAMSARKSIDTAFTITQPPPALTKAVACGAPAPKSQGSYRAEA GKMEGAGHAMSHAKRRGHAMAAVDKLARRKVSGFSVAKRKQNSGGAMGKGKPRKAEPAAKLAPV CKDDADEHHHHHH

[0941] Table 6.1. Main properties of the protein tested in these trials in comparison to ProtQ.

[0942] Isoelectric Molecular Name Homology to PQ%* Point** Weight Protein 1 PQ 100 6.8-7.2 42kDa Protein 5

[0943]

[0944] LH: Low homology (rPQ) 18.8 6.8-7.2 42kDa " Homology to PQ was calculated using Needleman / EMBOSS method.

[0945] "" Isoelectric point was determined using a prediction software Expasy, this estimation could vary depending on the software used.

[0946] 2.1. EMBOSS / Needle alignment PQ and rPQ identity %

[0947] > Lysate concentration - P62038693WO 66

[0948] - - -

[0949] - >

[0950] >

[0951] >

[0952] >

[0953] >

[0954] - >

[0955] > -.

[0956] .

[0957] 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 well below these thresholds, placing it in the category of low or residual homology relative to Protein Q. This novel variant was tested in solubility and lysis trials to evaluate its behaviour under modified process conditions and to determine whether sequence homology or physicochemical properties play a more critical role in the suitability of the optimized method.

[0958] 2.2. Solubility and stability test

[0959] An initial solubility and stability test was carried out by dissolving pellet of the selected protein in the Protein Q process buffer. Frozen biomass coming from a 30 L fermenter was used as starting material for the experiments. The total protein obtained from the fermentation was quantified so that the amount of starting biomass was standardized.

[0960] The experimental setup is the same of the experiments performed with the proteins tested in example 5.

[0961] Only 1 gram of pellet was taken and suspended in biomass suspension buffer to a target biomass concentration 0.11 g / mL (this concentration is obtained from Protein Q industrial scale process). The next step was to homogenize the sample at a setpoint temperature of 5°C. Due to the small volumes used, a sonicatorwas applied instead of a disruptor, then the lysate was centrifuged (3000 g, 10 minutes) and filtered. Total protein was quantified by BCA (see example 3). Lysate concentration - P62038693WO 67

[0962] 2.3. Experimental design of lysis with diferent TP concentration

[0963] Two tests were performed with the designed protein: one under reference conditions (biomass suspension up to 11 mg / mL TP and dilution after lysis, and clarification up to 6 mg / mL TP) and another under optimal conditions (biomass suspension previous to lysis to 3 mg / mL TP). The experimental setup is the same of the trials performed with the proteins tested in example 5.

[0964] In orderto carry out the runs starting from a controlled amount oftotal protein, the BCA findings from the previous experiment were used. Therefore, the biomass weighted for RPQ protein was adapted aiming to start from 2.6 g oftotal protein, to keep proportionality with the previous tested proteins. For the reference run, approximately 23 g of biomass were suspended in 236 mL of cold suspension buffer, corresponding to a TP concentration of approximately 11 mg / mL and homogenized for = 40 minutes. As explained, the equivalencies of pellet and PT were determined in the previous trial. The suspended biomass was subjected to two cycles of cell disruption at 750 bar and a set temperature of 5 °C. Then it was maintained for 1 hour with ice agitation for solubilization, if necessary, the pH was adjusted to 8.0 ± 0.2. After that, the product was clarified from the cell debris and small particles by two consecutive nitrations. Finally, the filtrate was diluted in biomass suspension buffer to a target concentration of 6 mg / mL total protein.

[0965] For the optimal run, approximately 23 g of rPQ biomass were suspended in 867 mL of cold suspension buffer, corresponding to a TP concentration of approximately 3 mg / mL and homogenized for = 40 minutes.

[0966] The resuspended biomass was subjected to two cycles of cell disruption at 750 bar and a setpoint temperature of 5 °C. Then, it was maintained in agitation for 1 hour for solubilization, if necessary, the pH was adjusted to 8.0 ± 0.2. After that, the product was clarified by two filtrations, one to get rid of cellular debris and one to get rid of smaller particles.

[0967] 3. ANALYTICAL METHODS

[0968] Total protein is quantified by BCA. The method is described in example 3. The protein target Protein Q is quantified by ELISA-Ni. The method is described in example 4.

[0969] 4. RESULTS

[0970] An UV spectrum of the clarified product (M4) was performed and it was observed that, although rPQ gave higher absorbance signal, the spectrum of rPQ was similar to Protein Q, which indicates that rPQ is soluble in the biomass suspension buffer of the process (figure 5).

[0971] We performed the analysis of the samples by BCA and ELISA-Ni. The results are shown in the following tables.

[0972] Table 6.2. Results of total protein by BCA.

[0973] Total Protein by BCA ProtQ rPQ

[0974] STEP SAMPLE TP Yield TP Yield (mg / mL) (%) (mg / mL) (%) LYSIS M2: Lysate 8.3 12.1

[0975] M3: S 7.8 94% 11.2 93% CENTRIFUGATION upernatant

[0976] M3P: Pellet 1.5 18% 1.5 12% FILTRATION M4: Filtered 6.1 78% 11.3 101%

[0977]

[0978] Yield (%) Total 73% 93%

[0979] Total protein data indicate that rPQ biomass contains a slightly higher protein concentration than ProtQ and that rPQ protein retains good solubility in the buffer, as demonstrated by the protein recovery in the supernatant and filtrate fractions. Overall yields are satisfactory, with rPQ showing Lysate concentration - P62038693WO 68

[0980] higher recovery compared to ProtQ (93% vs. 73%). These results suggest that the process is effective for rPQ and that the protein maintains favourable solubility and stability throughout the evaluated stages.

[0981] 4.1. Results of lysis with different TP concentration

[0982] The results of these experiments are summarized in the next tables comparing the reference process to the optimal process.

[0983] Concentrations and total amount of specific and total proteins are followed at each step. With these data it is possible to calculate the process yields and ratios in order to evaluate the product quantity and quality obtained when applying each condition.

[0984] Table 6.3. Results of Total protein (BCA) or specific protein (ELISA-Ni) for runs carried out at reference (11 mg / mL) and optimal (3 mg / mL) conditions with rPQ.

[0985] TP (BCA) SP (ELISA-Ni)

[0986] SAMPLES Reference Optimal Reference Optimal

[0987] [TP] or [SP]

[0988] 7389 3342 1179 451

[0989] Resuspended (ug / mL)

[0990] biomass M1 Vol (mL) 240 860 240 860

[0991] PT (mg) 1773 2874 283 388

[0992] [TP] or [SP]

[0993] 9622 3135 1654 289

[0994] Lysate 1st (ug / mL)

[0995] cycle M2a Vol (mL) 240 860 240 860

[0996] PT (mg) 2309 2696 397 249

[0997] [TP] or [SP]

[0998] 9152 3189 1392 341

[0999] Lysate 2nd (ug / mL)

[1000] cycle M2b Vol (mL) 250 830 250 830

[1001] PT (mg) 2288 2647 348 283

[1002] [TP] or [SP]

[1003] 6788 2938 1088 313

[1004] 1st clarified (ug / mL)

[1005] M3 Vol (mL) 250 840 250 840

[1006] PT (mg) 1697 2468 272 263

[1007] [TP] or [SP]

[1008] 6731 2829 1046 290

[1009] 2nd Clarified (ug / mL)

[1010] M4 Vol (mL) 250 840 250 840

[1011] PT (mg) 1683 2376 262 243

[1012] [TP] or [SP]

[1013] 3583 - 656 - (ug / mL)

[1014] Clarified

[1015] adjusted M4b Vol (mL) 450 - 450 - PT (mg) 1612 - 295 -

[1016]

[1017] The table shows a summary of the total and specific protein results for the protein tested for both the reference run and the optimal run.

[1018] The first observation is that applying a greater dilution (optimal test) results in a higher amount of specific and total protein from the biomass suspension. Applying the optimal conditions leads to a higher yield of total protein at all stages; however, specific protein is lower in all lysates and clarified products compared to the reference process.

[1019] The yield at each step is calculated comparing the reference run to the optimal run. The laboratory experiments are summarized in the following tables. Lysate concentration - P62038693WO 69

[1020] Table 6.4. TP yields tracking each step of the process for rPQ (19% homology).

[1021] % YIELD TP by BCA rPQ

[1022] Samples Reference Optimal

[1023] Biomass lysis yield 129% 92%

[1024] 1st Clarification Yield 74% 93%

[1025] 2nd Clarification Yield 99% 96%

[1026] Lysis - Clarification Global Yield

[1027]

[1028] 95% 83%

[1029] The table shows the total protein (TP) yield at each stage of the process for rPQ (19% homology). The data indicate that the lysis yield is lower under the optimal conditions compared to the reference condition. The fact that the lysis yield in the reference assay is greater than 100% indicates more efficient protein extraction from the cells. And, although the subsequent clarification stages show a higher recovery than the reference process, overall, the total protein yield obtained using the optimal method is lower for rPQ.

[1030] Table 6.5. SP yields tracking each step of the process for rPQ (19% homology).

[1031] % YIELD SP by ELISA-Ni rPQ

[1032] Samples Reference Optimal

[1033] Biomass lysis yield 123% 73%

[1034] 1st Clarification Yield 78% 93%

[1035] 2nd Clarification Yield 96% 92%

[1036] Lysis - Clarification Global Yield

[1037]

[1038] 93% 63%

[1039] The results show that biomass lysis yield is significantly lower under optimal conditions compared to the reference run (73% vs. 123%), indicating reduced efficiency in the extraction of specific protein under optimal conditions. However, the clarification steps favor the optimal process, with higher yields observed after the first clarification (93% vs. 78%). Despite this, the overall global yield from lysis through clarification is markedly lower for the optimal run (63% vs. 93%), suggesting that the initial loss during lysis cannot be compensated by improved clarification performance.

[1040] Table 6.6. SP / PT ratio in each step of the process for rPQ (19% homology).

[1041] % YIELD SP by ELISA-Ni rPQ

[1042] Samples Reference Optimal Suspended Biomass 16% 13%

[1043] Lysate 15% 11%

[1044] 1st Clarified 16% 11%

[1045] 2nd Clarified

[1046]

[1047] 16% 10%

[1048] The ratio of specific protein to total protein at each step of the process allows verification of the purification of the rPQ protein.

[1049] The results indicate that the ratio of specific protein to total protein remains fairly stable across the different stages of the reference process (around 15-16%), while in the optimal process the ratio is consistently lower (10-13%). This suggests that, although the optimal method improves clarification recovery, it does not enhance the relative purity of the target protein compared to the reference process. Lysate concentration - P62038693WO 70

[1050] 5. CONCLUSIONS

[1051] After verifying that the proposed changes in the Protein Q lysis process also provide an improvement in performance in other proteins with > 60% homology with the PQ sequence, it was decided to extend the study by applying the reference and optimized process to a non-homologous protein.

[1052] The tested protein was designed by randomizing the amino acid sequence of PQ, while maintaining the histidine tag, to allow for purification, and the anti-PQ antibody binding site, to enable analysis by ELISA and Western Blot. Furthermore, it maintained the same isoelectric point and molecular weight as Protein Q, and its stability and solubility in the buffer of interest were evaluated.

[1053] This new variant of PQ, called rPQ (randomized Protein Q), was expressed and processed under the lysis and clarification reference method of Protein Q (biomass suspension at a TP = 11 mg / mL and post-lysis dilution to TP = 6 mg / mL) and also applying the optimized method (biomass suspension at TP = 3 mg / mL before lysis).

[1054] The results of laboratory-scale trials with rPQ showed that applying the optimal method, although it slightly increased protein recovery in the clarification steps, did not improve the overall yield of specific protein (SP) or total protein (TP) compared to the reference process. Furthermore, the SP / TP ratio was lower at all stages, especially after clarification, indicating that applying a higher pre-lysis dilution does not improve the purification of the protein of interest rPQ, compared to the reference process.

[1055] These results suggest that, for proteins with low homology to ProtQ’s sequence (19% homology, considered residual), the changes applied to the lysis process do not result in any increase in protein yield. Although the tested protein rPQ has similar physicochemical characteristics to ProtQ, such as the isoelectric point, molecular weight or the amino acids that make up the sequence - although in a different order -, the optimized method of ProtQ does not represent an improvement as it did with the tested proteins with a greater homology (> 60%). That would imply that the proposed changes in Protein Q process are not obvious nor standard for all proteins.

Claims

Lysate concentration - P62038693WO 71Claims1. A process for downstream processing a protein from a biomass comprising cells, wherein said downstream processing process comprising the following steps:• suspending the biomass in a suspension buffer,• subsequently lysing the cells present in the suspended biomass to obtain a lysate, and• further downstream processing the protein from said lysate;wherein the Total Protein (TP) concentration in the suspended biomass is kept at a range from 2 to 12 g / l by carrying out a diluting step before the lysing step.

2. A process according to claim 1, wherein the TP concentration in the suspended biomass before and / or after the lysing step is ranged from 2 to 7 g / L or 2 to 6 g / L.

3. A process according to claim 1 or 2, wherein the TP concentration in the suspended biomass before and / or after the lysing step is ranged from 2.5 to 3.5 g / L.

4. A process according to any one of claims 1 to 3, wherein the further downstream processing step comprises the following steps:- a solubilizing step,- followed by a clarifying step.

5. A process according to any one of the preceding claims, wherein the TP concentration in the biomass suspension before lysis is adjusted up to 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.

9. 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, 10.0, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, or 12.0 g / L.

6. A process according to any one of the preceding claims, wherein the TP concentration in the lysate is adjusted up to 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / L.

7. A process according to any one of claims 1 to 5, wherein the TP concentration of the lysate is adjusted up to 7.7, 7.8, 7.9, or up to 12.0 g / L.Lysate concentration - P62038693WO 728. A process according to any one of the preceding claims, wherein the process or at least part of it is carried out at a temperature ranged from 1°C to 10°C or is 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C.

9. A process according to claim 8, wherein the part of the process which is carried out at the temperature range of 1°C to 10°C is the lysis step and the subsequent downstream processing steps.

10. A process according to any one of the preceding claims, wherein the isoelectric point of the protein is from 1 to 3 units away from the pH of the lysate, preferably 1 to 3 units lower than the pH of the lysate11. A process according to any one of the preceding claims, wherein the TP concentration is adjusted up to 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / L, preferably by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 to 10.0.

12. A process according to any one of claims 1 to 10, wherein the TP concentration is adjusted up to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 g / L, preferably by diluting before lysis, the lysate is kept at up to 1 °C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 to 10.0.

13. A process according to any one of claims 1 to 10, wherein the TP concentration is adjusted up to 7.8 or 7.9 g / L, preferably by diluting before lysis, the lysate is kept at up to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, at least during the lysis and subsequent downstream step, and optionally the lysate pH is ranged from 7.5 to 10.0.

14. The process according to any 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 100% identity of SEQ ID NO:1.

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