CHO cells with optimized host cell protein profile

Abstract

The present invention relates to CHO cells having a modified host cell protein (HCP) profile characterized by a reduced load or a reduced expression level of a hard-to-remove HCP (drHCP) and / or a rich core HCP (acHCP). The invention also relates to a method for producing at least one modified CHO cell according to the invention. The invention also relates to a method for producing at least one recombinant molecule of interest, preferably at least one recombinant protein of interest, and to the use thereof for producing at least one medicament. The invention also relates to kits that provide all suitable reagents for performing the disclosed methods.

Description

Technical Field

[0001] The present invention relates to CHO cells having a modified host cell protein (HCP) profile characterized by a reduced load or reduced expression of difficult-to-remove HCPs (drHCPs) and / or abundant core HCPs (acHCPs). The present invention also relates to a method for producing at least one modified CHO cell according to the present invention. The present invention also relates to a method for producing at least one recombinant molecule of interest, preferably at least one recombinant protein of interest, and its use for producing at least one drug. The present invention also relates to a drug kit. Background Art

[0002] The manufacture of biotherapeutics (including antibodies and other therapeutic drugs) represents an important pillar of pharmaceutical manufacturing. Generally speaking, biotherapeutics, which are also called biopharmaceuticals or biologics, are produced from cells (bacteria, yeast, plants or mammals) using recombinant DNA technology, which are called host cells or host cell lines. Chinese Hamster Ovary (CHO) cells are the workhorse for the manufacture of biopharmaceuticals. In fact, CHO cells used to manufacture biopharmaceuticals account for more than 70% of recombinant biopharmaceutical proteins, most of which are monoclonal antibodies (mAbs), and account for 35.5% of the total cumulative (1982 to 2014) biopharmaceutical product approvals (Lalonde 2017 (http: / / dx.doi.org / 10.1016 / j.jbiotec.2017.04.028), Walsh, 2014, Nat Biotechnol. 2014; 32: 992-1000). The main use of CHO as a host cell is due to its ability to correctly fold and post-translationally modify recombinant proteins and thus make them suitable for human use (Jaypal et al., 2007, Chemical Engineering Progress 103(10):40-47). In addition, CHO cells can produce human-like glycosylation patterns when producing proteins (such as antibodies and other glycosylated molecules), which advantageously avoids undesirable immune responses. In addition, CHO cells are fast-growing cells that can produce large amounts of recombinant proteins through their secretory pathway.

[0003] In a highly controlled production and sequential purification process, not only is a high yield of functional biopharmaceuticals desired, but the final purity of the administered composition is also required. Host cell proteins (HCPs) are process-related protein impurities that are endogenously produced by the host organism or host cell line during the manufacture and production of biotherapeutics. In order to recombinantly manufacture biotherapeutics in cells, laborious and cost-intensive downstream purification processes are required to remove most (>99%) HCPs from the final product. This is not only necessary to meet regulatory requirements, but the purity of therapeutic biological products is also paramount for their safe use as drug components. Specifically, HCPs can be highly immunogenic even when present at very low levels. When HCPs are co-administered to patients with biotherapeutics, HCPs in humans can trigger an immune response that causes a cytokine storm. Such cytokine storms lead to serious health problems and, if not treated, can be fatal to patients.

[0004] Typically, HCPs must be removed during downstream processing (DSP). However, the purification steps necessary to remove HCPs to achieve relevant purity levels in the final biotherapeutic product significantly increase overall production costs. Purification inherently requires cost-intensive steps performed under controlled manufacturing conditions, which also results in reduced product yields.

[0005] In addition, even if the current DSP is quite effective, residual amounts of HCP that are difficult to remove can still remain in the final and commercially sold biopharmaceutical products. Exemplary biopharmaceuticals in which such residual HCP causes problems include antibodies, such as monoclonal antibodies (mAbs), other therapeutic proteins or peptides, vaccines of any kind, and antibody-drug-conjugates (ADCs). Such residual HCPs can also trigger the immune system and cause serious adverse reactions. In addition, the HCPs in biopharmaceuticals can affect their effectiveness. For example, residual HCPs have been shown to degrade the excipients in the biopharmaceutical itself or the composition to be administered, which leads to a shortened shelf life. In addition, such degradation can inactivate biopharmaceuticals or produce immunogenic byproducts. Therefore, regulatory agencies (such as the FDA) regard HCP content as a key quality attribute and require it to be removed to an acceptable residual material level (if applicable) under relevant contamination control according to good manufacturing practice (GMP) standards, as measured by highly sensitive analytical methods.

[0006] Recent methods are generally intended to reduce HCP at the cell culture production level, which is referred to as upstream processing (upstreamprocessing, USP), but generally focus on a target HCP expression and therefore its final HCP load will be reduced. Specifically, US10,570,397B2 and US 9,932,591 B2 are intended to reduce a widely known HCP called lipoprotein lipase (lipoprotein lipase, LPL) at USP levels by interfering RNA or knocking out at least one copy of the gene encoding endogenous LPL in the host cell. This results in a reduction in the level of LPL during upstream production. US 2016 / 251411A1 discloses a recombinant host cell, in which the coding sequence of the polynucleotide encoding phospholipase B-like 2 (PLBD2) protein is modified, thereby reducing the amount of PLBD2 (referred to as rich HCP) as an undesirable by-product during the production of recombinant biotherapeutics.

[0007] WO 2022 / 225880 A1 discloses a long list of potential proteins endogenous to CHO cells whose expression can be reduced or eliminated to specifically reduce the expression of endogenous retrovirus-like particles of mammalian cells, which represent key contaminants to be removed during downstream processing.

[0008] However, to date, there has been a lack of a systematic approach that specifically defines and evaluates the most critical HCP patterns in host cells in a first step to establish, test and provide generally applicable platform host cells, preferably modified CHO cells, with a significantly reduced profile of HCPs as undesirable co-purifying contaminants while maintaining or even improving the overall product yield and upstream and downstream processing parameters during the production of recombinant biotherapeutics in mammalian cells, preferably CHO cells.

[0009] Therefore, there is still a great need to reduce the content of HCPs in biopharmaceutical products. Therefore, one object of the present invention is to reduce the content of HCPs in biopharmaceutical products or even eliminate one or more HCPs. Another object is to reduce the content of HCPs in biopharmaceutical products or even eliminate one or more HCPs at USP levels. Summary of the Invention

[0010] The present invention addresses the above-mentioned problems and objectives by providing genetically optimized CHO cell lines characterized by a specific knockout pattern targeting an HCP or group of HCPs, resulting in an improved impurity profile while maintaining host cell stability and integrity, as measured by upstream processing performance. In particular, the present invention provides generally applicable host cells and multiplexed methods for producing the same, wherein the host cells are characterized by a genetic background that ensures a much lower HCP profile suitable for cost-effective production of biotherapeutics at desired yields.

[0011] In a first aspect, CHO cells having a modified host cell protein (HCP) profile are provided, characterized by a reduced load or reduced expression level of at least one endogenous protein, the endogenous protein being a difficult-to-remove HCP (drHCP) and / or an abundant core HCP (acHCP), wherein the CHO cells: comprise at least one modification in at least one endogenous coding sequence encoding at least one drHCP and / or acHCP selected from the group summarized in Table 1, or any homolog, ortholog or paralog thereof.

[0012] In a second aspect, a CHO cell having a modified host cell protein (HCP) profile is provided, characterized by a reduced load or reduced expression level of at least one endogenous protein, wherein the endogenous protein is a difficult-to-remove HCP (drHCP) and / or an abundant core HCP (acHCP), wherein the CHO cell: comprises two or more modifications in two or more coding sequences encoding two or more drHCPs and / or acHCPs defined / shown in Tables 2, 3, 4 or 5, or any homologs, orthologs or paralogs thereof.

[0013] Furthermore, there is provided a CHO cell according to the first or second aspect, wherein the at least one modification or the two or more modifications affect a drHCP, preferably wherein the at least one modification or the two or more modifications affect one, two or more proteins classified as both a drHCP and an acHCP, or wherein the at least one endogenous protein has an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, or any homologue, orthologue or paralogue thereof having an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, respectively. %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, preferably wherein the at least one endogenous protein has an amino acid sequence corresponding to SEQ ID NO: 2, or any homologue, orthologue or paralogue thereof having an amino acid sequence corresponding to SEQ ID NO: 3 The sequence of NO:2 has an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0014] Furthermore, in one embodiment of all aspects disclosed herein, a CHO cell according to any one of the preceding aspects or embodiments is provided, wherein the at least one modification results in reduced transcription and / or reduced functional expression of the endogenous protein, thereby reducing the total content of drHCP and / or acHCP.

[0015] In another embodiment, a CHO cell according to any one of the preceding aspects or embodiments is provided, wherein the CHO comprises at least one recombinant gene encoding at least one recombinant protein of interest and / or encoding at least one recombinant RNA molecule of interest, preferably, wherein the at least one recombinant protein of interest is a therapeutic molecule.

[0016] In a further embodiment, a CHO cell according to any one of the preceding aspects or embodiments is provided, wherein the at least one modification is selected from at least one insertion, at least one deletion and at least one substitution, including base editing, or any combination thereof, preferably, wherein the at least one modification is present in exon 1 of the corresponding endogenous coding sequence, and / or wherein the at least one modification in the coding sequence is a frameshift mutation or a point mutation, which point mutation produces a stop codon.

[0017] In a further embodiment, a CHO cell according to any one of the preceding aspects or embodiments is provided, wherein the at least one modification in the endogenous coding sequence allows for optimized downstream processing and / or ensures a reduced load or reduced expression level of total HCPs, (i) wherein the CHO cell comprises at least one recombinant non-endogenous gene encoding at least one recombinant protein or RNA of interest, and wherein the optimized downstream processing is characterized by a reduced load or reduced expression level of total HCPs in the recombinant protein or RNA of the product of interest, wherein the total load of HCPs is reduced by at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, preferably at least 1%, at least 1.25%, at least 1.5%, at least 1.75%, at least 2%, at least 2.25%, at least 2.5%, at least 3. or (ii) wherein said at least one modification in said endogenous coding sequence results in an improvement in upstream processing performance, wherein said improvement in upstream processing performance is characterized by an increase in viable cell concentration and / or an increase in specific productivity and / or an increase in final titer and / or an increase in process duration compared to a wild-type cell that does not carry said at least one modification in its genome. In another embodiment, a CHO cell according to any of the preceding embodiments is provided, wherein all alleles of said at least one endogenous coding sequence comprise at least one or more of said modifications.

[0018] Additionally, there is provided a CHO cell according to any one of the preceding aspects or embodiments, wherein the at least one endogenous protein has an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128,or corresponding to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 ,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90 , 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, Any of the sequences of 126, 127, and 128 has an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0019] In another embodiment, a CHO cell according to any one of the preceding aspects or embodiments is provided, wherein the CHO cell comprises at least one modification in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty or more different HCP coding sequences, wherein each HCP is preferably selected from acHCP and / or drHCP.

[0020] In another aspect, a method for producing at least one modified CHO cell according to the preceding aspect or embodiment is provided, the method comprising the steps of: (i) providing at least one CHO cell comprising at least one endogenous target nucleic acid segment; (ii) providing at least one genome or transcriptome editing agent comprising at least one RNAi agent or at least one site-directed endonuclease, the site-directed endonuclease preferably being selected from a mega-nuclease, a ZFN, a TALEN, a CRISPR nuclease, or a nickase or nuclease-inactive variant thereof, or a nucleic acid molecule encoding these, and optionally in the case of a CRISPR nuclease The invention further comprises the steps of: (i) providing at least one suitable functional guide RNA molecule, or a nucleic acid molecule encoding the same; (iii) introducing the at least one genome editing agent of step (ii) into the at least one CHO cell; (iv) obtaining at least one modified CHO cell comprising at least one modification in the at least one endogenous target nucleic acid segment according to step (i); (v) optionally: selecting another target nucleic acid segment and repeating steps (i) to (iv) at least once to obtain at least one modified CHO cell comprising at least one additional modification in another endogenous target nucleic acid segment.

[0021] In yet another aspect, a method for producing at least one recombinant molecule of interest, preferably at least one recombinant protein of interest, is provided, comprising the following steps: (a) providing at least one modified CHO cell as defined in the preceding aspect or embodiment, wherein the at least one modified CHO cell comprises at least one recombinant gene encoding at least one recombinant protein of interest, DNA or RNA; (b) culturing the at least one target cell in a culture medium such that the at least one recombinant molecule of interest is transcribed and / or translated; (c) harvesting the at least one recombinant molecule of interest; (d) purifying and / or clearing up the at least one recombinant molecule, preferably at least one recombinant protein of interest.

[0022] In a further aspect, there is provided use of at least one modified CHO cell according to any one of the preceding aspects or embodiments for the production of at least one medicament.

[0023] In another aspect, a kit is provided comprising (a) at least one modified CHO cell according to any one of the preceding aspects or embodiments, said CHO cell or said kit comprising: (bi) at least one nucleic acid molecule suitable for expressing at least one recombinant molecule of interest, preferably at least one recombinant protein of interest; optionally means for introducing it into the genome of said CHO cell, or alternatively (b.ii) at least one nucleic acid molecule encoding at least one recombinant molecule of interest, preferably at least one recombinant protein of interest; optionally means for introducing it into the genome of said CHO cell; or alternatively (b.iii) at least one nucleic acid molecule suitable for transcribing at least one recombinant RNA molecule of interest; optionally means for introducing it into the genome of said CHO cell. means for introducing the at least one nucleic acid molecule encoding at least one recombinant RNA molecule of interest into the genome of the CHO cell; or alternatively (b.iv) at least one nucleic acid molecule encoding at least one recombinant RNA molecule of interest; optionally means for introducing the same into the genome of the CHO cell; and optionally (c) a culture medium suitable for the at least one modified CHO cell according to step (a), (i) endogenous or exogenous replication of the at least one nucleic acid molecule according to any one of steps (bi), (b.ii), (b.iii) and (b.iv); and (ii.a) expression of the at least one recombinant protein of interest according to steps (bi) and / or (b.ii), and / or transcription of the at least one recombinant RNA molecule of interest according to steps (b.iii) and / or (b.iv). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 An overview of the HCP profiles of samples measured by SWATH LC-MS and analyzed to define relevant abundant and difficult-to-remove HCPs, as further described in Example 1, is shown. "H" means harvest. Thus, "H-5" means 5 days before harvest, etc. To obtain a comprehensive and comparable dataset, several clones producing different antibody constructs were tested. ProA = Protein A column. VI = viral inactivation step. CEX = cation exchange chromatography. AEX = anion exchange chromatography.

[0025] Figure 2 A to D ( Figure 2 A to D) A schematic overview of the identification process, classification, and number of core HCPs is given. Figure 2 A: The first round of analysis categorized the 1,254 HCPs tested into core and non-core. Figure 2 B: Further characterization of the definition of core HCP clusters that are quantifiable under the chosen settings (67). Figure 2 C: Yes Figure 2B. Further characterization of the 67 quantifiable HCPs. Black = non-core; Grey = below LLOQ, 5 = 5 HCPs with membrane localization; 23 = 23 extracellular HCPs; 5 = 5 HCPs with membrane localization (total: Figure 2 67 quantifiable cores in B). Figure 2 D: shows Figure 2 B and C are percentages of the 67 quantifiable core HCPs relative to all 1,254 HCPs analyzed. See also Example 2 for further explanation.

[0026] Figure 3A to D( Figure 3A to D) show: Figure 3A The fed-batch endpoint is shown by plotting the conversion of peak VCC (=viable cell concentration) relative to the peak VCC (y-axis) determined for the clones generated against the HCP target and the corresponding CHO DG44 wild type (WT) controls (TR4, TR3, TR5, TR6 and TR7 respectively - see also Example 3 for further explanation) as detailed in the examples (see Example 3). Figure 3B : Conversion of process time. Figure 3C : Conversion of final titer on harvest day. Figure 3D : Conversion of average Qp. Once viability falls below a preset threshold, the process ends. Here, final viability, final titer [g / L] and final Qp [pg / c / d] are related. Cell specific productivity Qp is calculated using the integral of the cumulative daily viable cell concentration (IVCC [×10 5 The IVCC, process time, and Qp are calculated based on the titer (cells / d / ml). Thus, IVCC, process time, and Qp describe the course of the bioprocess and its productivity. The average Qp was calculated by averaging Qp over the culture time. (Brown-Forsythe and Welch ANOVA tests, Dunnett's test, α = 0.05 were performed.)

[0027] Figure 4( Figure 4A to D) show the results of the experiments detailed in Example 4 below, always measured against the 10 target HCP knockouts as detailed in that example and a wild-type (WT) control. Figure 4A : shows the peak VCC (in [×10 5 The viable cell concentration (in terms of cells* / ml) and KO score (in terms of %) were analyzed. Figure 4B : The procedure time (in days) and KO score (in %) are shown. Figure 4C : Final titer (in g / L) and KO score (in %) are shown. Figure 4D :The final Qp[pg / c / d] and KO score (in %) are shown. KO score is shown in a bar graph, according to Figure 4A Other parameters up to D are reflected by the points and the corresponding standard deviations.

[0028] Figure 5 (See Example 5) A matrix showing the multiple knockout combinations (number / no. = 4, 5, 6 or 7) of the seven targets tested in Example 5 and the number of clones identified.

[0029] Figure 6 Figure 6A D) show the results of the experiments detailed in Example 5. Figure 5 All data for 4-fold, 5-fold, 6-fold, or 7-fold KO are detailed in the summary matrix in . CHO DG44 were used as a control for all assays. Figure 6A : shows the peak VCC (in [×10 5 The viable cell concentration was calculated as [number of cells* / ml]. Figure 6B : Shows the process time (in days). Figure 6C : Final titer (in g / L) is shown. Figure 6D : Shown are the average Qp [pg / c / d].

[0030] Figure 7 shows the experimental results of the single knockout pool screening described in detail in Example 9. CHO DG44 were used as a control for all assays. Figure 7A Peak VCC (in [×10 5 The viable cell concentration was calculated as [number of cells* / ml]. Figure 7B Process times (in days) are shown. Figure 7C Final titers (in g / L) are shown. Figure 7D The average Qp [pg / c / d] is shown.

[0031] Figure 8 shows the experimental results for four exemplary test clones containing the 11x knockout (denoted as C127, C132, C139, and C141) as detailed in Example 10. DG44 was used as a control for all assays. Figure 8A Peak VCC (in [×10 5 The viable cell concentration was calculated as [number of cells* / ml]. Figure 8B Process times (in days) are shown. Figure 8C Final titers (in g / L) are shown. Figure 8D The average Qp [pg / c / d] is shown.

[0032] Sequence Description

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[0040]

[0041] definition

[0042] The terms "abundant core HCP" (acHCP), "abundant HCP" and "core HCP" are used interchangeably herein. The term refers to those HCPs that are present in high amounts in the host cell of interest (see Figure 1 and Table 4), this is in terms of the presence of the HCP in significant amounts (usually quantifiable amounts) on the day of harvest and / or throughout the culture process until harvest (and in particular at the harvest time point). It is worth noting that the definition of a core HCP essentially depends on the fact that it is present above the lower limit of quantification (Lower Limit of Quantification, LLOQ). Nevertheless, those skilled in the art are aware of the fact that the LLOQ may vary depending on the quantitative means and assay conditions used. Furthermore, with the development of more sensitive devices and techniques, the LLOQ may be even lower. Therefore, if the acHCP meets the additional criterion of being present in significant amounts throughout the culture process (and in particular at harvest), then the acHCP used herein may also refer to an acHCP below the current limit of quantification (see Figure 2 B). See also Example 1.

[0043] The term "antibody" as used herein refers to and encompasses a variety of antibody structures known to those of skill, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies composed of a single heavy chain sequence and a single light chain sequence, including multimers of such pairs), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that the antibody or fragment exhibits the desired antigen-binding activity as defined by the structure of at least one complementarity determining region (CDR) of the antibody variable region.

[0044] As used herein, "antibody fragment" or any "(functional) fragment" of an "antibody" refers to a molecule other than a full-length antibody that comprises at least a portion of an intact antibody that binds to the antigen to which the full-length antibody binds. Some examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs); and / or multispecific antibodies assembled from antibody fragments.

[0045] In the context of an "antibody" or a "fragment" thereof, the term "chimeric" refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. As is known to those skilled in the art, the "class" of an antibody refers to the type of constant domain or constant region of its cognate heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is IgG1.

[0046] As used herein, the term "difficult to remove HCP" or "drHCP" refers to an HCP that is present in quantifiable amounts in at least one sample after harvest and after at least one further purification step. In particular, the HCP is still present in Figure 1 In at least one of the samples shown in the right column of , the HCP could not be removed by at least one purification strategy used after harvesting. Further explanation is also provided in Example 1 and a list is provided in Table 5.

[0047] The term "downstream processing" or "DSP" as used herein (e.g. in the context of cell culture) refers to all process steps required to obtain a recombinantly produced molecule in purified form from a host cell suspension, wherein cell harvesting as the end of the respective bioprocess marks the starting point of downstream processing in contrast to upstream processing.

[0048] In this context, "harvesting" means the end point of host cell culture for obtaining the desired recombinant protein (including recombinant peptide). Harvesting can also be performed continuously throughout the culture process (e.g., in perfusion culture).

[0049] As used herein, the term "target nucleic acid segment" refers to a nucleotide sequence, typically a DNA sequence, that can be modified using a site-directed endonuclease. Typically, the target nucleic acid segment is part of a genome.

[0050] Thus, the term "upstream processing" or "USP" as used herein (eg, in the context of cell culture) refers to all culture steps that occur prior to cell harvest.

[0051] As used herein, the term "upstream processing performance" refers to an evaluation of the upstream processing of a given cell culture based on one or more parameters, wherein the parameters can be selected from peak viable cell concentration (VCC), integral viable cell concentration (IVCC), cell specific productivity, final titer, and total bioprocess time. Generally speaking, a high value determined for any of the above parameters indicates good upstream processing performance. Other parameters for evaluating the upstream processing performance of a given cell culture can also be evaluated.

[0052] The term "expression" as used herein refers to the process of transcription (e.g., for functional gRNA) and translation (to produce polypeptides) in a host cell. The level or extent of expression of a product gene to a homologous RNA in a host cell can be determined based on the amount of the corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced by the cell. For example, mRNA transcribed from the product gene is desirably quantified by northern hybridization (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). The polypeptide or protein encoded by the product gene can be quantified by assaying the biological activity of the protein or by using an assay independent of such activity (e.g., western blotting using an antibody capable of reacting with the protein) (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989). As used herein, "functionally reduced expression" further refers to a reduction and / or elimination of the expression of one or more endogenous products relative to the expression level of the endogenous product in an unmodified cell. Reduction of expression can include a reduction and / or complete elimination of a functional endogenous product (at the RNA and / or protein level).

[0053] As used herein, the term "host cell protein" (HCP) refers to endogenous proteins produced at varying levels by a host cell line (which may be an engineered host cell line). In the case where Chinese hamster ovary (CHO) cells are used as the host cell line, host cell proteins (HCPs) are those proteins produced by and endogenous to the CHO cells.

[0054] The terms "orthologs," "homologs," or "paralogs" used herein are consistent with the common understanding of these terms in the field of taxonomy. Those skilled in the art will appreciate that if two DNA sequences (or RNA or protein sequences) match to a great extent, it is believed that these sequences evolved from a common ancestral sequence by duplication or mutation (e.g., insertion, replacement, or deletion). If two homologous sequences are produced by gene duplication, they are referred to as paralogs, and if two homologous sequences differentiate due to sequence variations, they are referred to as orthologs. The term "homolog" is used as an umbrella term encompassing both paralogs and orthologs. The high degree of sequence homology in the interspecific comparison of two sequences (DNA, RNA, or amino acids) of different species is interpreted as a relationship between the two species, because it is believed that these sequences were produced from a common ancestral sequence by the genetic mutations that occurred during biological evolution. A phylogenetic family tree can be derived from the degree of molecular relationship. Based on a given sequence (database accession number or the SEQ ID NO presented herein), those skilled in the art can therefore easily use the sequence as a reference sequence and will be able to evaluate whether a sequence with a certain degree of identity to the given sequence (e.g., a sequence of the same family, order or species, particularly if the genome of the species is completely sequenced) can be classified as a "ortholog" or "paralog", and therefore as a "homolog" of the corresponding (known) query sequence for comparison therewith. "Orthologs" or "paralogs" as used herein and therefore as "homologs" generally have not only a sequence comparable to the reference sequence, but also a comparable biological function. Nevertheless, a homolog may be absolutely essential to the cell (e.g., complete knockout may be lethal to the cell), while its homolog is also related to the function of the cell, but knockout can be tolerated, for example, because a homolog has several genomic copies, but other related sequences do not.

[0055] As used herein, the term "peptibody" refers to a portion or all of an antibody fused to a peptide. Thus, peptibodies are generally characterized by a combination of the activity of the corresponding peptide with the longer duration of activity of the antibody.

[0056] The terms "polypeptide" and "protein" are used interchangeably herein and refer to the continuous stretch of amino acid sequence that defines the primary structure of a polypeptide. A properly folded protein may have structure (e.g., matrix protein) or functional activity (e.g., enzymatic activity / functional activity), or it may have specific binding or recognition properties (e.g., antibodies), or a combination thereof (binding + nuclease activity, e.g., CRISPR nuclease).

[0057] " recombinant protein of interest " used herein can be any protein, peptide or its fragment that can be produced when exogenous is added to host cell, usually as recombinant (i.e. exogenous) DNA or RNA, for example on plasmid, as viral vector, or as transgenic, for example as introduced by genome editing etc. In addition, recombinant protein can also be a protein encoded by endogenous genes modified via genome editing technology (including base editing, lead editing and InDel induced by genome editing etc.). Recombinant protein of interest can be therapeutic protein, peptide or its fragment, or any other protein, peptide or its fragment. Recombinant protein can be an antibody as defined above or its variant or functional fragment, or any other preventive or therapeutic protein. Any other protein (such as fluorescent protein, such as EGFP) is also understood to be recombinant protein, as long as the protein or its fragment is inserted, mutated and / or artificially produced in the target host cell.

[0058] The terms "RNA interference" or "RNAi" or "RNA silencing" or "gene silencing" used interchangeably herein refer to a gene downregulation (or knockdown) mechanism that has been shown to exist in all eukaryotic organisms. This mechanism was first recognized in plants and is called "post-transcriptional gene silencing" or "PTGS". In RNAi, small RNAs function to guide specific effector proteins to target nucleotide sequences through complementary base pairing, resulting in degradation of the target. A "gene silencing construct" or "RNAi agent" typically comprises a so-called "sense" sequence and an "antisense" sequence. The sense sequence and antisense sequence are complementary sequences that exist in opposite directions in the nucleic acid sequence. If the nucleic acid construct comprises a sense sequence and a corresponding antisense sequence, the two complementary sequences form an RNA duplex when transcribed, which produces an "RNA hairpin". In the RNA hairpin, the sense sequence and the corresponding antisense sequence together form a double strand and are separated by an "intervening intron loop sequence" that forms a loop of the hairpin structure. "RNAi agents" as used herein can also comprise more than one sense and antisense pair and form several loops.

[0059] As used herein, "therapeutic molecule" or "biotherapeutic molecule or biotherapeutic agent" refers to a recombinant protein of interest as defined above, or any other recombinant nucleic acid molecule that has a prophylactic or therapeutic effect when used to treat a subject in need thereof.

[0060] Whenever the present disclosure relates to percent identity of nucleic acids or amino acid sequences to each other, these values ​​define those values ​​obtained by using the EMBOSS Water Pairwise Sequence Alignment (nucleotide) program (www.ebi.ac.uk / Tools / psa / emboss_water / ) for nucleic acids or the EMBOSS Water Pairwise Sequence Alignment (protein) program (www.ebi.ac.uk / Tools / psa / emboss_water / ) for amino acid sequences. Comparison or sequence comparison as used herein refers to the comparison over the full length of two sequences compared to each other. The tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignment use a modified Smith-Waterman algorithm (see www.ebi.ac.uk / Tools / psa / and Smith, TF & Waterman, MS "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147(1):195-197). When performing the alignment, the default parameters defined by EMBL-EBI were used. Those parameters were (i) for amino acid sequences: matrix = BLOSUM62, gap opening penalty = 10 and gap extension penalty = 0.5, or (ii) for nucleic acid sequences: matrix = DNAfull, gap opening penalty = 10 and gap extension penalty = 0.5. The skilled person will be aware of the fact that a sequence encoding a protein may be "codon-optimized" if, for example, the corresponding sequence is to be used in another organism than the original organism from which the molecule was derived. DETAILED DESCRIPTION

[0061] Thus, the present invention provides CHO cell lines in which single or multiple HCPs are knocked out in a targeted manner, resulting in a more favorable HCP profile without significantly compromising the upstream performance (USP) of the cells, primarily including titer, growth, overall viability, etc. The HCP composition of such modified cells is improved in a manner that allows for simplified, more economical DSP and / or improves final product quality because: HCPs that are abundant in the cell culture harvest are knocked out in a synergistic manner and / or HCPs that are difficult to remove during DSP are knocked out and / or HCPs that may negatively impact product quality are knocked out.

[0062] Thus, the present inventors were able to address the above objectives by first characterizing the specific HCP contamination profiles in comparable samples, by then defining particularly problematic HCP targets and target combinations as contaminants, and by then defining prioritized knockdown and knockout strategies to provide modified CHO cells and / or modified CHO cell lines generally suitable for the production of a variety of biotherapeutics, said modified CHO cells and / or modified CHO cell lines exhibiting significantly reduced HCP profiles while exhibiting cellular integrity and, in turn, favorable upstream and downstream processing performance that is not hampered by the modification. To this end, a strategy is provided that allows for the reliable and targeted elimination of certain abundant and difficult-to-remove HCPs in the final biopharmaceutical without affecting the integrity of the host cells during upstream processing.

[0063] In a first aspect, the CHO cells have a modified host cell protein (HCP) profile characterized by a reduced load or reduced expression level of at least one endogenous protein, said endogenous protein being a difficult-to-remove HCP (drHCP) and / or an abundant core HCP (acHCP), wherein the CHO cells: comprise at least one modification in at least one endogenous coding sequence encoding at least one drHCP and / or acHCP selected from the group summarized in Table 1, or any homolog, ortholog or paralog thereof. Where two Uniprot IDs are provided in the tables herein to indicate homologues, orthologues or paralogues etc., the first one mentioned at the top in the corresponding Uniprot ID field is the one referred to by the corresponding SEQ ID NO: in the subsequent column, preferably wherein at least one modification is a modification of THBS-1 corresponding to SEQ ID NO: or any homologue, orthologue or paralogue thereof, optionally the homologue, orthologue or paralogue having an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence corresponding to SEQ ID NO: 1.

[0064] The terms "load reduction" and "reduction in expression level" are used interchangeably and are therefore synonymous herein, and are used to describe that the modified CHO cells disclosed herein comprise at least one modification that results in a reduction in the quantifiable amount of at least one acHCP and / or drHCP at the protein level. This reduction can preferably be achieved by at least one knockout (loss of function) mutation that completely eliminates the expression of the target protein. As known to those skilled in the art, reduced expression can also be achieved by knocking out or knocking down a copy of a gene encoding the target protein whose expression will be reduced. In addition, reduced expression can also be achieved by knocking down a portion of the gene transcription that results in the encoding protein whose expression will be reduced, for example, by reducing the transcription rate through a mutation in a gene regulatory region, or, for example, by silencing by RNAi methods, and thus reducing transcription and thereby reducing the expression of the target protein whose expression will be reduced (when used in the context of protein translation, expression is synonymously used for translation). Since the present invention specifically contemplates the targeting and modification of multiple HCPs, in certain embodiments, a combination of knockout and knockdown for reducing the load or expression of at least one, and particularly more than one, target can be used. Thus, one HCP can be the target for knockout modification, while another HCP can be the target for knockdown modification.

[0065] As can be seen from Table 1 below and other tables herein, specific HCP protein sequences used herein are generally referred to by Uniprot ID, corresponding SEQ ID NO, and their names and abbreviations. Several spellings of a given abbreviation may be used interchangeably herein, including uppercase or lowercase letter representations.

[0066] Table 1

[0067]

[0068]

[0069]

[0070]

[0071] In Table 1 above, and further in Tables 2 and 3 below, for some proteins, more than one Uniprot ID is given, wherein in each case the first Uniprot ID is generally the preferred one, which should be taken into account in the event that one or more proteins listed in any of Tables 1, 2 and 3 need to be distinguished from any other protein based on their respective Uniprot IDs. Nevertheless, the skilled person will be aware of the fact that orthologues, homologues and paralogues of a given sequence exist. For example, if the present disclosure refers to a lipoprotein lipase ("LPL") sequence derived from Chinese hamster, any known homologs, orthologs, or paralogs thereof within a given genus (e.g., SEQ ID NOs: 14 and 127) and any known homologs, orthologs, or paralogs available in the relevant databases (Chinese hamster genome; CH PICRH (GCF_003668045.3) status January 27, 2021, see https: / / www.chogenome.org / about.php) are also represented by the corresponding abbreviation of LPL. All Uniprot IDs for any protein listed in Tables 1, 2, and 3 correspond to the Uniprot ID of each protein as of the date of filing of this application. Tables 1, 2, and 3 should be understood so that they also include all homologs, orthologs, or paralogs of all proteins explicitly included in any of Tables 1, 2, and 3.

[0072] Biotechnological engineering of CHO cells began with the use of traditional gene targeting strategies based on homologous recombination (Yamane-Ohnuki et al., 2004, doi:10.1002 / bit.20151). However, such strategies have proven to be quite inefficient because the frequency of homologous recombination (HR) in mammalian cells is several orders of magnitude lower than that of non-homologous end-joining (NHEJ) (Sedivy and Sharp, 1989). NHEJ is particularly well-suited to generating gene disruptions because it is an incomplete repair process, often inducing insertions or deletions at double-strand break (DSB) sites. However, to generate gene disruptions in a site-specific manner, endonucleases suitable for sequence-specific targeting are required, such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZNFs), CRISPR / Cas effectors, and meganucleases, all of which have been successfully used in mammalian cells, i.e., human or CHO cells (Galetto et al., 2009 DOI: 10.1517 / 14712590903213669; Santiago et al., 2008 DOI: 10.1073 / pnas.0800940105; Miller et al., 2010 DOI: 10.1038 / nbt.1755; Hillary and Ceasar, Mol Biotechnol 65, 311–325 (2023). https: / / doi.org / 10.1007 / s12033-022-00567-0 ).

[0073] To identify relevant HCP knockout targets, multiple clones producing monoclonal antibodies (mAbs) were processed under industrial USP (ambr250 fed-batch processing) and DSP (standard ProA capture, viral inactivation, cation exchange (CEX) and anion exchange (AEX) polishing steps). Samples were taken on harvest days -5, -4, -3, -2, -1, and on the day of harvest. Purification was performed in a standard cascade, starting with Protein A, followed by viral inactivation, CEX, and finally AEX polishing.

[0074] In the first round, a total of 1,254 proteins were identified and classified. Of these, 351 were characterized as acHCPs. From these 351 candidates, 67 were identified that were consistently above the limit of quantification (see Table 4 below). These were further grouped into different functional clusters based on protein localization: membrane (2), cytoplasmic (39), extracellular (16), and unclear or mixed (10) (see Figure 2 C) The panel was expanded in a stepwise manner to fully cover the CHO HCP spectrum.

[0075] By systematically testing the acHCP and drHCP repertoires, relevant targets and their combinations were identified, and for each target, test knockouts were generated to evaluate the suitability of the protein as a knockout target, avoiding lethal or USP-blocking knockouts.

[0076] The term "recombinant," as used herein and as generally defined above, generally refers to molecules (e.g., proteins or nucleic acid molecules) that are not endogenous to the cells in which they are expressed. For example, genetic material can be introduced into a cell, which can then express (transcribe and / or translate) the recombinant molecule of interest.

[0077] In one embodiment of all aspects and embodiments disclosed herein, the modified CHO cell may be selected from or may be derived from (meaning derived from) a CHO cell variant selected from the group consisting of: CHO-K1, CHO-DXB11 (synonymously referred to as CHO-DUKX), CHO-DG44, CHO-S and CHO-Pro minus, or CHO GS knockout. Alternatively, the modified CHO cell according to the present invention may also be derived from other CHO cell variants.

[0078] In a preferred embodiment, the modified CHO cell may be CHO-DG44, or may be derived from CHO-DG44.

[0079] In another preferred embodiment, the modified CHO cell may be CHO-K1, or may be derived from CHO-K1.

[0080] In yet another preferred embodiment, the modified CHO cell may be CHO-DXB11 (synonymously referred to as CHO-DUKX), or may be derived from CHO-DXB11 (synonymously referred to as CHO-DUKX).

[0081] In yet another preferred embodiment, the CHO cell may be a CHO GS knockout cell, or may be derived from a CHO GS knockout cell.

[0082] In a preferred embodiment, the modified CHO cell may be derived from CHO-S, or it may be derived from CHO-Pro minus.

[0083] In one embodiment, the at least one modification may be present in at least one endogenous coding sequence encoding an HCP selected from the group consisting of the HCPs listed in Table 1 having SEQ ID 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, and 89, or any homolog, ortholog thereof.

[0084] In one embodiment, the at least one modification may be present in at least one endogenous coding sequence encoding a HCP selected from the group consisting of SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, or any homolog, ortholog, or paralog thereof having a sequence identical to that corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, any homolog, ortholog, or paralog thereof having a sequence identical to that corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 98%, 99%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, The sequence of NO:2 has an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0085] In some preferred embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, or any homologs, orthologs or paralogs thereof, can be combined with each other or with any knockout of the HCPs shown in Table 1, preferably, wherein at least one modification or at least 2, 3, 4, 5, 6, or 7 simultaneous mutations are of SEQ ID NO: 1, 2, 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 or more modifications are of SEQ ID NO: 1, 2, 4, 5, 6, or 7 simultaneous mutations are of SEQ ID NO: 1, Knockout of the sequences shown in ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, or any homologs, orthologs or paralogs thereof.

[0086] In a second aspect, a CHO cell having a modified host cell protein (HCP) profile is provided, characterized by a reduced load or reduced expression level of at least one endogenous protein, the at least one endogenous protein being a difficult to remove HCP (drHCP) and / or an abundant core HCP (acHCP), wherein the CHO cell: comprises two or more modifications in two or more coding sequences encoding two or more drHCPs and / or acHCPs defined / shown in Tables 2, 3, 4 or 5, or any homologues, orthologues or paralogues thereof, preferably, wherein at least one of the two or more modifications is a modification of THBS-1 and / or FN-1 corresponding to SEQ ID NOs: 1 and 2, respectively, or any homologues, orthologues or paralogues thereof, optionally the homologues, orthologues or paralogues have the same or similar modifications as the THBS-1 and / or FN-1 corresponding to SEQ ID NOs: 1 and 2, respectively. The sequence of NO: 1 or 2 has an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0087] Table 2

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] In one embodiment, the two or more modifications may be independently selected from insertions, deletions and substitutions.

[0095] In another embodiment of the present invention, at least one of the two or more modifications, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven, preferably at least twelve, preferably at least thirteen, preferably at least fourteen, preferably at least fifteen, preferably at least sixteen, preferably at least seventeen, preferably at least eighteen, preferably at least nineteen, preferably all can be a deletion.

[0096] In certain embodiments, at least five modifications of CHO cell drHCP and / or acHCP are provided in one cell, wherein the modifications comprise at least two, preferably all, modifications of THBS1, FN1, LP1, Nid1, Pcolce and / or Pxdn, or any orthologs, paralogs or homologs thereof, optionally wherein at least one additional modification is selected from LGALS3BP, HSP90AA1, CSPG4, PPIB, CLU, ACTB, ENO3, GSTP1, TKT, HTRA1, YWHAZ, CTSA, PPIA, GSTM6, HSP90B1, PGK1, PLBD2, H2B, TINAGL1, NUDIK, QSOX1, NUCB2, CTSZ, ANXA2, FLNA, H1.4, AKR1B1, CALR and / or LGMN, or any orthologs, paralogs or homologs thereof.

[0097] In one embodiment, at least eleven modifications of CHO cell drHCP and / or acHCP are provided in one cell, preferably knockout modifications, wherein the modifications comprise or consist of THBS1, FN1, LPl, Nid1, Pcolce, Pxdn, LGALS3BP, HSP90AA1, CSPG4, PPIB and / or CLU.

[0098] In a preferred embodiment of the present invention, at least one of the two or more modifications, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven, preferably at least twelve, preferably at least thirteen, preferably at least fourteen, preferably at least fifteen, preferably at least sixteen, preferably at least seventeen, preferably at least eighteen, preferably at least nineteen, preferably all can be insertions.

[0099] In another preferred embodiment of the present invention, at least one of the two or more modifications, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven, preferably at least twelve, preferably at least thirteen, preferably at least fourteen, preferably at least fifteen, preferably at least sixteen, preferably at least seventeen, preferably at least eighteen, preferably at least nineteen, preferably all can be replacements.

[0100] In an exemplary embodiment of the present invention, the modified CHO cells may be characterized by a reduced load or reduced expression level of fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2) and at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven, preferably at least twelve, preferably at least thirteen, preferably at least fourteen, preferably at least fifteen, preferably at least sixteen, preferably at least seventeen, preferably at least eighteen, preferably at least nineteen such other endogenous proteins independently selected from the group consisting of fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2) and at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven ... IDNO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 ,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,50,51,52,53,54,55,56,57,58,59,6 0, 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, 127 or 128, or any homolog, ortholog or paralog thereof, or a protein selected from any one of Tables 1, 2, 3, 4 or 5, or any homolog, ortholog or paralog thereof.

[0101] In a preferred embodiment of all aspects disclosed herein relating to the CHO cells according to the invention, the at least one modification or the two or more modifications may affect a drHCP, preferably, wherein the at least one modification or the two or more modifications affect one, two or more proteins classified as both a drHCP and an acHCP.

[0102] In a preferred embodiment of all aspects of the invention, the two or more modifications may affect one, two or more proteins classified as both drHCPs and acHCPs, independently selected from all proteins listed in Table 3.

[0103] Table 3

[0104]

[0105]

[0106]

[0107] In one embodiment of all aspects disclosed herein, at least one modification may be present in at least one endogenous coding sequence encoding an HCP selected from the group consisting of thrombospondin-1 (THBS1, Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (FN / FN1 / FN-1, Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (PSAP, Uniprot ID: G3I1Y9; SEQ ID NO: 9) and / or chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), or an ortholog, homolog or paralog of the aforementioned sequences. These targets are acHCPs and knocking out at least one or all four of them advantageously reduces the overall HCP load in the final purified preparation. In one embodiment, THBS1 and / or FN1 and / or PSAP and / or CSPG4 represent knockout targets, alone or in double, triple or quadruple combinations. In addition, THBS1 and / or FN1 and / or PSAP and / or CSPG4m, alone or in combination with each other, represent a group of suitable basic HCP modifications that can be combined with at least one additional HCP knockout, preferably with at least one additional acHCP and / or drHCP knockout.

[0108] In some preferred embodiments, at least one modification may be present in at least one endogenous coding sequence that encodes a high abundance HCP (acHCP). Table 4 below summarizes a list of high abundance acHCPs, and for each individual HCP, its average relative concentration (referred to as "MRC" in Table 4 below) is calculated by determining the average of all measured concentrations (in ng / mL) relative to the sum of all measurement probes. All averages are added together, and for each HCP, the average is divided by the average of the total HCP concentration. The resulting value corresponds to the "average relative concentration" or MRC used herein.

[0109] Modifying (i.e., reducing) the amount of at least one acHCP expressed in cells at a high average relative concentration (meaning a concentration above 0.05%, preferably above 0.1%, even more preferably 0.15% and above) will result in a significant reduction in the overall HCP load. This is particularly true for acHCPs at very high average relative concentrations (meaning a concentration above 0.5%, preferably above 1%, even more preferably an average relative concentration of 1.5% and above).

[0110] Table 4

[0111]

[0112]

[0113]

[0114]

[0115] Furthermore, in some specific embodiments, it may be preferred that at least one modification is present in at least one endogenous coding sequence encoding a difficult-to-remove HCP (drHCP).

[0116] Table 5 below summarizes a list of drHCPs, whose mean relative concentrations (referred to as "MRC" in Table 5 below) were determined as detailed in Table 4 above. As defined above, drHCP is the one present at least after protein A column purification (ProA), after virus inactivation (VI), after cation exchange chromatography (CEX), or even after anion exchange chromatography (AEX) when PrA, VI, CEX, and AEX are performed as a series of subsequent purification steps.

[0117] Therefore, modification of at least one drHCP (preferably simultaneously an acHCP) or a mixture of at least two drHCPs and acHCPs or both can significantly reduce the amount of HCPs in a targeted manner by specifically defining a set of acHCPs and / or drHCPs in a first step to select and modify a suitable set of candidates.

[0118] Table 5

[0119]

[0120]

[0121]

[0122] In certain embodiments, it is preferred to modify more than one acHCP and / or drHCP in the same setting in a multiplexed approach. First, this allows for the reliable elimination of certain abundant and difficult-to-remove HCPs in the final biopharmaceutical independent of any upstream or downstream processing methods, while maintaining the high performance of CHO cells with respect to their growth and titer capabilities to produce a high yield of the functional therapeutic agent of interest. Second, when targeting multiple HCPs in the same experiment, knockout in the same assay reduces the number of iterative modifications to the host cells to be modified. Generally speaking, all target HCPs specified in any one of Tables 1 to 3 above, alone or in combination, qualify as suitable knockout targets.

[0123] In a specific embodiment, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty or more, or all of the HCP targets according to Tables 1, 2, or 3 above, can be simultaneously knocked out to obtain modified cells according to the present disclosure. This is due to the fact that it can be experimentally shown that the effect of single knockout of the exemplary HCPs selected from Tables 1 and 2 on the growth / viability and productivity / titer of the engineered cells was evaluated under bioprocess conditions by generating stable mAb expressing cell clones followed by fed-batch operation in shake flasks and was not negative, and therefore these targets are suitable for knockout alone or in combination.

[0124] Figure 5A matrix showing preferred combinations of HCP targets suitable for multiple knockdown and / or knockout is shown in Specifically, as detailed for certain targets in Example 5, the multiple knockdown and / or knockout of HCPs includes at least one or a specific combination of FN, LPL, THBS, BGN, NID-1, PCOLCE, PXDN, THBS, PKM, CSPG4, LDHA, VIM, HSPA5, PPIB, HSP90AA1, LAMA5, FSTL1 and / or AEBP1, or any orthologs, homologs or paralogs thereof, and / or additionally includes at least one of PSAP, PRDX, HSPG2, CTSB, LGALS3BP, TNX, LAMB1, ITIH5 or TIMP.

[0125] In the preferred embodiment of some combinations knocking out, at least one of the knockout targets is FN, LPL or THBS. By upper tables 4 and 5, it is obvious that the inventors can, in the case of the excessive protein present in the host cell, provide a reliable scheme for classifying HCP by limiting the subset of acHCP and drHCP under the relevant setting, and this selection scheme itself is used as the basis for limiting the knockout or modification group that is intended to remove more than a kind of HCP from the host cell in a convenient manner. In certain embodiments of knocking out in combination, at least two of the knockout targets are selected from FN, LPL and THBS. In some other embodiments of knocking out in combination, at least three of the knockout targets are selected from FN, LPL and THBS, wherein other target, particularly as defined in any one of above tables 1, 2 and 3 and preferred tables 4 and 5, with FN, LPL, THBS knockout group combination.

[0126] In one embodiment, at least one modification may therefore be present in at least one endogenous coding sequence encoding thrombospondin-1 (UniprotID: G3HHV4; SEQ ID NO: 1).

[0127] In another embodiment, at least one modification may be present in at least one endogenous coding sequence encoding fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2).

[0128] In yet another embodiment, at least one modification may be present in at least one endogenous coding sequence encoding sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9).

[0129] In a further embodiment of the present invention, at least one modification may be present in at least one endogenous coding sequence encoding chondroitin sulfate proteoglycan 4 (=CSPG4) (UniprotID: G3H0E4; SEQ ID NO: 10).

[0130] In one embodiment of the present invention, the modified CHO cell may be derived from CHO-DG44, and at least one modification may be present in at least one endogenous coding sequence encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1).

[0131] In yet another embodiment of the present invention, the modified CHO cell may be derived from CHO-DG44, and at least one modification may be present in at least one endogenous coding sequence encoding fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2).

[0132] In one embodiment of the present invention, the modified CHO cell may be derived from CHO-DG44, and at least one modification may be present in at least one endogenous coding sequence encoding sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9).

[0133] In one embodiment of the present invention, the modified CHO cell may be derived from CHO-K1 and at least one modification may be present in at least one endogenous coding sequence encoding chondroitin sulfate proteoglycan 4 (=CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10).

[0134] In another embodiment of the present invention, the modified CHO cell may be derived from CHO-K1, and at least one modification may be present in at least one endogenous coding sequence encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1).

[0135] In yet another preferred embodiment of the present invention, the modified CHO cell may be derived from CHO-K1, and at least one modification may be present in at least one endogenous coding sequence encoding fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2).

[0136] In another embodiment of the present invention, the modified CHO cell may be derived from CHO-K1, and at least one modification may be present in at least one endogenous coding sequence encoding sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9).

[0137] In another embodiment of the present invention, the modified CHO cells may be derived from CHO-K1 and at least one modification may be present in at least one endogenous coding sequence encoding chondroitin sulfate proteoglycan 4 (=CSPG4) (UniprotID: G3H0E4; SEQ ID NO: 10).

[0138] In yet another embodiment of the present invention, the modified CHO cells may be derived from CHO-S, and at least one modification may be present in at least one endogenous coding sequence encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1).

[0139] In one embodiment of the present invention, the modified CHO cell may be derived from CHO-S, and at least one modification may be present in at least one endogenous coding sequence encoding fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2).

[0140] In another embodiment of the present invention, the modified CHO cell may be derived from CHO-S, and at least one modification may be present in at least one endogenous coding sequence encoding sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9).

[0141] In yet another embodiment of the present invention, the modified CHO cells may be derived from CHO-S and at least one modification may be present in at least one endogenous coding sequence encoding chondroitin sulfate proteoglycan 4 (=CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10).

[0142] In yet another embodiment of the present invention, the modified CHO cells may be derived from CHO-DXB11 (synonymously referred to as CHO-DUKX), and at least one modification may be present in at least one endogenous coding sequence encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1).

[0143] In another embodiment of the present invention, the modified CHO cell may be derived from CHO-DXB11 (synonymously referred to as CHO-DUKX), and at least one modification may be present in at least one endogenous coding sequence encoding fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2).

[0144] In yet another embodiment of the present invention, the modified CHO cell may be derived from CHO-DXB11 (synonymously referred to as CHO-DUKX), and at least one modification may be present in at least one endogenous coding sequence encoding sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9).

[0145] In another embodiment of the present invention, the modified CHO cells may be derived from CHO-DXB11 (synonymously referred to as CHO-DUKX), and at least one modification may be present in at least one endogenous coding sequence encoding chondroitin sulfate proteoglycan 4 (=CSPG4) (UniprotID: G3H0E4; SEQ ID NO: 10).

[0146] In yet another embodiment of the present invention, the modified CHO cell may be derived from CHO-Prominus, and at least one modification may be present in at least one endogenous coding sequence encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1).

[0147] In another embodiment of the present invention, the modified CHO cell may be derived from CHO-Prominus, and at least one modification may be present in at least one endogenous coding sequence encoding fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2).

[0148] In a preferred embodiment of the present invention, the modified CHO cell may be derived from CHO-Prominus, and at least one modification may be present in at least one endogenous coding sequence encoding sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9).

[0149] In a preferred embodiment of the present invention, the modified CHO cells may be derived from CHO-Prominus and at least one modification may be present in at least one endogenous coding sequence encoding chondroitin sulfate proteoglycan 4 (=CSPG4) (UniprotID: G3H0E4; SEQ ID NO: 10).

[0150] In another embodiment of the present invention, the modified CHO cells may be derived from CHO GS cells, and at least one modification may be present in at least one endogenous coding sequence encoding: thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), or any homolog, ortholog, or paralog thereof, and / or an HCP selected from the group consisting of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, individually, or any homolog, ortholog, or paralog thereof, or any combination of these HCPs with each other or with other HCPs.

[0151] In another embodiment of the present invention, the modified CHO cells may be derived from CHO GS cells, and at least one modification may be present in at least one endogenous coding sequence encoding: fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2) or any homolog, ortholog or paralog thereof, and / or a HCP selected from SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, individually, or any homolog, ortholog or paralog thereof, or any combination of these HCPs with each other or with other HCPs.

[0152] In another embodiment of the present invention, the modified CHO cells may be derived from CHO GS cells, and at least one modification may be present in at least one endogenous coding sequence encoding: sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9), or any homolog, ortholog, or paralog thereof, and / or an HCP selected from the group consisting of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, individually, or any homolog, ortholog, or paralog thereof, or any combination of these HCPs with each other or with other HCPs.

[0153] In another embodiment of the present invention, the modified CHO cells may be derived from CHO GS cells, and at least one modification may be present in at least one endogenous coding sequence encoding: chondroitin sulfate proteoglycan 4 (= CSPG4) (UniprotID: G3H0E4; SEQ ID NO: 10) or any homologue, orthologue or paralogue thereof, and / or an HCP selected from the group consisting of SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, individually, or any homologue, orthologue or paralogue thereof, or any combination of these HCPs with each other or with other HCPs.

[0154] In a preferred embodiment, the modified CHO cells according to the present invention may comprise one or more exogenously added genes encoding one or more recombinant molecules (including at least one recombinant protein or peptide of interest, RNA or DNA). Preferably, the one or more recombinant molecules are recombinant proteins or peptides of interest representing biopharmaceutical proteins selected from the exemplary group consisting of recombinant proteins or peptides, including monoclonal antibodies or single-chain Fvs, or any other antibody formats, including monospecific, bispecific and multispecific antibodies, chimeric antigen receptors, virus-like proteins, vaccines, and any type of immunogenic compounds.

[0155] In one embodiment of the present invention, the modified CHO cells may be derived from CHO-DG44, and at least one modification may be present in at least one endogenous coding sequence encoding a protein selected from the group consisting of: thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9), and chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), and the modified CHO cells may comprise an exogenously added gene encoding a recombinant protein of interest, wherein the recombinant protein of interest is a monoclonal antibody or a fragment or domain thereof, or any other therapeutic protein (including bispecific antibodies, fusion proteins, peptibodies and peptides), or any prophylactic agent (including vaccines or fragments or portions thereof).

[0156] In one embodiment, at least one of the proteins to be knocked out is selected from the group consisting of: at least one or a specific combination of knockouts of HCP FN, LPL, THBS, BGN, NID-1, PCOLCE, PXDN, THBS, PKM, CSPG4, LDHA, VIM, HSPA5, PPIB, HSP90AA1, LAMA5, FSTL1 and / or AEBP1, or any orthologs, homologs or paralogs thereof, and / or additionally includes at least one of PSAP, PRDX HSPG2, CTSB, LGALS3BP, TNX, LAMB1, ITIH5 or TIMP.

[0157] In a preferred embodiment of all aspects and embodiments disclosed herein, at least one of FN, LPL, THBS, BGN, NID-1, PCOLCE, PXDN, THBS, PKM, CSPG4, LDHA, VIM, HSPA5, PPIB, HSP90AA1, LAMA5, FSTL1, AEBP1, PSAP, PRDX HSPG2, CTSB, LGALS3BP, TNX, LAMB1, ITIH5 and / or TIMP, or any ortholog, homolog or paralog thereof, is knocked out.

[0158] In one embodiment of all aspects as disclosed herein, the modified CHO cell may be derived from CHO-DG44, and the at least one modification may be present in at least one endogenous coding sequence encoding a protein selected from the group consisting of: at least one or two or more proteins classified as both drHCP and acHCP, or wherein the at least one endogenous protein has an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, or any homolog, ortholog, or paralog thereof having an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, respectively. NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of amino acids, including, for example, thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (Uniprot ID: G3I1V4; SEQ ID NO: 3), The modified CHO cells may contain at least one of: a protein encoding a recombinant protein of interest (Uniprot ID: G3I1Y9; SEQ ID NO: 9) and a chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), and the modified CHO cells may contain more than one, preferably two, three, four or five exogenously added genes encoding the recombinant protein of interest.

[0159] In another embodiment of all aspects as disclosed herein, the modified CHO cell may be derived from CHO-DG44, and the at least one modification may be present in at least one endogenous coding sequence selected from the group consisting of: at least one or two or more proteins classified as both drHCP and acHCP, or wherein the at least one endogenous protein has an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, or any homolog, ortholog, or paralog thereof having an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, respectively. NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of amino acids 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, including, for example, amino acid sequences encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9) and / or chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), or any other HCP, preferably as listed in Tables 3, 4 and 5 or any homologs, orthologs or paralogs thereof, and the modified CHO cells may contain an exogenously added gene encoding the recombinant protein of interest.

[0160] In a further embodiment of all aspects as disclosed herein, the modified CHO cell may be derived from CHO-K1, and the at least one modification may be present in at least one endogenous coding sequence selected from the group consisting of: at least one or two or more proteins classified as both drHCP and acHCP, or wherein the at least one endogenous protein has an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, or any homolog, ortholog, or paralog thereof having an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, respectively. NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of amino acids 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, including, for example, amino acid sequences encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9) and / or chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), or any other HCP, preferably as listed in Tables 3, 4 and 5 or any homologs, orthologs or paralogs thereof, and the modified CHO cells may contain an exogenously added gene encoding the recombinant protein of interest.

[0161] In yet another embodiment of all aspects as disclosed herein, the modified CHO cell may be derived from CHO-K1, and the at least one modification may, for example, be present in at least one endogenous coding sequence encoding fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), and the modified CHO cell may contain an exogenously added gene encoding a recombinant protein of interest, wherein the recombinant protein of interest is a monoclonal antibody or a fragment or domain thereof.

[0162] In a further embodiment of all aspects of the present disclosure, the modified CHO cell may be derived from CHO-S, and the at least one modification may be present in at least one endogenous coding sequence selected from the group consisting of: at least one or two or more proteins classified as both drHCP and acHCP, or wherein the at least one endogenous protein has an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, or any homolog, ortholog, or paralog thereof having an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49, and 55, respectively. NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of amino acids 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, including, for example, amino acid sequences encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9) and / or chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), or any other HCP, preferably as listed in Tables 3, 4 and 5, and the modified CHO cells may contain an exogenously added gene encoding the recombinant protein of interest.

[0163] In a preferred embodiment, the modified CHO cells may be derived from CHO-S, and at least one modification may be present in at least one endogenous coding sequence encoding a protein selected from, for example, the group consisting of: thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9), chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), and the modified CHO cells may contain more than one, preferably two, three, four or five exogenously added genes encoding recombinant proteins of interest, wherein at least one, preferably two, three, four, five or all of the recombinant proteins of interest are monoclonal antibodies or fragments or domains thereof.

[0164] In yet another embodiment of all aspects of the invention, the modified CHO cell may be derived from CHO-DXB11 (synonymously referred to as CHO-DUKX), and the at least one modification may be present in at least one endogenous coding sequence selected from the group consisting of: at least one or two or more proteins classified as both drHCP and acHCP, or wherein the at least one endogenous protein has an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, or any homologue, orthologue or paralogue thereof having an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, respectively. NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of amino acids 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, including, for example, amino acid sequences encoding thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9) and / or chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), or any other HCP, preferably as listed in Tables 3, 4 and 5, and the modified CHO cells may contain an exogenously added gene encoding the recombinant protein of interest.

[0165] In a preferred embodiment of the present invention, the modified CHO cells may be derived from CHO-DXB11 (synonymously referred to as CHO-DUKX), and at least one modification may be present in at least one endogenous coding sequence encoding a protein selected from the group consisting of, for example, thrombospondin-1 (Uniprot ID: G3HHV4; SEQ ID NO: 1), fibronectin (Uniprot ID: G3I1V3; SEQ ID NO: 2), sulfated glycoprotein 1 (Uniprot ID: G3I1Y9; SEQ ID NO: 9) and chondroitin sulfate proteoglycan 4 (= CSPG4) (Uniprot ID: G3H0E4; SEQ ID NO: 10), and the modified CHO cells may contain more than one, preferably two, three, four or five exogenously added genes encoding recombinant proteins of interest, wherein at least one, preferably two, three, four, five or all of the recombinant proteins of interest are monoclonal antibodies or fragments or domains thereof.

[0166] In a particularly preferred embodiment of the present invention, the modified CHO cells may be characterized by a reduced load or reduced expression level of at least fibronectin (UniprotID: G3I1V3; SEQ ID NO: 2) and at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven, preferably at least twelve, preferably at least thirteen, preferably at least fourteen, preferably at least fifteen, preferably at least sixteen, preferably at least seventeen, preferably at least eighteen, preferably at least nineteen independently selected from the group consisting of the proteins according to SEQ ID NOs as listed in Tables 3, 4 and / or 5. Proteins of NO: 1, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55 or other endogenous proteins of any homologs, orthologs or paralogs thereof.

[0167] In a preferred embodiment of the CHO cell according to the invention, said at least one modification results in reduced transcription and / or reduced functional expression of said endogenous protein, thereby reducing the total content of drHCP and / or acHCP.

[0168] In one embodiment of the invention, at least one modification can result in premature transcriptional termination, resulting in a truncated and therefore inactive form of the corresponding protein. Preferably, at least one modification can result in premature transcriptional termination in exon 1 of the corresponding protein. In another preferred embodiment, the mutation can be in another exon other than exon 3, for example, where another exon is more accessible for successful and efficient editing.

[0169] Preferably, premature transcription termination may occur in the first 50%, preferably the first 40%, preferably the first 30%, preferably the first 20%, preferably the first 10%, preferably the first 5%, preferably the first 2% of the corresponding genomic coding sequence, viewed from its transcription start point (i.e., its start codon).

[0170] By prematurely terminating the transcription of a given target gene as close as possible to its respective transcription start point, the amount of transcript formed is kept to a minimum and the overall load of the corresponding protein in the modified CHO cells according to the invention is minimized, thereby maximizing the positive effect on optimizing downstream processing efficiency.

[0171] Preferably, the CHO cell according to the present invention comprises at least one recombinant gene encoding at least one recombinant protein of interest and / or encoding at least one recombinant RNA molecule of interest.

[0172] In a preferred embodiment of all aspects and embodiments disclosed herein, the at least one recombinant protein of interest may be selected from the group consisting of a therapeutic protein, a monoclonal antibody, a bispecific antibody, a fusion protein, a peptibody and a peptide.

[0173] In one embodiment of all aspects and embodiments disclosed herein, at least one recombinant RNA molecule of interest can be selected from mRNA therapeutics, including mRNA vaccines, non-coding single-stranded RNA species, such as antisense RNA and microRNA (also known as miR), interfering RNA (RNAi), such as small interfering RNA (siRNA) or microRNA (miRNA), and RNA aptamers.

[0174] In a preferred embodiment of the CHO cell according to the present invention, at least one recombinant protein of interest is a therapeutic molecule.

[0175] In a preferred embodiment of the CHO cell according to the present invention, the at least one modification is selected from at least one insertion, at least one deletion and at least one substitution, including base editing, or any combination thereof, preferably, wherein the at least one modification is present in exon 1 of the corresponding endogenous coding sequence, and / or wherein the at least one modification in the coding sequence is a frameshift mutation or a point mutation, which point mutation generates a stop codon.

[0176] In a preferred embodiment of the CHO cells according to the invention, the at least one modification in the endogenous coding sequence allows for optimized downstream processing and / or ensures a reduced load or a reduced expression level of total HCPs, (i) wherein the CHO cells comprise at least one recombinant non-endogenous gene encoding at least one recombinant protein or RNA of interest, and wherein the optimized downstream processing is characterized by a reduced load or a reduced expression level of total HCPs in the recombinant protein or RNA of the product of interest, wherein the total load of HCPs is reduced by at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, preferably at least 1%, at least 1.25%, at least 1.5%, at least 1.75%, at least 2%, at least 2.25%, at least 2.5%, at least 2.75%, at least 3%, at least 3.25%, at least 3.5%, at least 3.75%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at at least 4.5%, more preferably at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, and even more preferably at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75%, as determined by measuring the relative concentration (in ng / mL) of total HCPs per sample obtained in the modified CHO cells compared to a reference sample obtained from CHO wild-type cells, and / or (ii) wherein said at least one modification in said endogenous coding sequence results in an improved upstream process performance, wherein said improved upstream process performance is characterized by an increased viable cell concentration and / or an increased specific productivity and / or an increased final titer and / or an increased process duration compared to a wild-type cell not carrying the at least one modification in its genome.

[0177] In a preferred embodiment, the total load of drHCP is reduced by at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, preferably at least 1%, at least 1.25%, at least 1.5%, at least 1.75%, at least 2%, at least 2.25%, at least 2.5%, at least 2.75%, at least 3%, at least 3.25%, at least 3.5%, at least 3.75%, at least 4%, at least 4.5%, more preferably at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7.6%, at least 8.75%, at least 9.8%, at least 10. At least 7%, at least 7.5%, at least 8%, and even more preferably at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, as determined by measuring the relative concentration (in ng / mL) of total drHCPs per sample obtained in the modified CHO cells compared to a reference sample obtained from CHO wild-type cells. In particular, for drHCPs, it is advantageous to increase the amount of drHCP removed, preferably above a certain threshold, because such drHCPs cannot be easily removed by conventional purification strategies, so that impurities will remain in the biopharmaceutical. This can be avoided by focusing on individual drHCPs to be knocked out as defined herein, and preferably certain combinations thereof, to significantly improve the purity of the biopharmaceutical product obtained after manufacture in the modified host cells of the invention.

[0178] In a preferred embodiment of the present invention, at least one modification in the endogenous coding sequence allows for optimized downstream processing, wherein the optimized downstream processing is characterized in that the number of downstream processing steps required to obtain the recombinantly produced molecule in purified form is reduced compared to the corresponding wild-type (i.e., unmodified) CHO cells. For example, at least one modification may have the advantage that at least one purification step, in particular an ion exchange chromatography step, i.e., anion or cation exchange chromatography, or even both, can be omitted because the total HCP load is significantly increased by the method of the present invention, making these steps no longer necessary to achieve the final purity grade. This represents a significant advantage because additional column chromatography steps require resources and each additional step leads to a reduced yield of the recombinant protein of interest to be purified.

[0179] Thus, the modified CHO cells according to the present invention allow downstream processing to be optimized in terms of cost efficiency, time efficiency, energy efficiency and sustainability.

[0180] In certain embodiments related to CHO cells according to the present invention, all alleles of the at least one endogenous coding sequence comprise at least one or more of the modifications. In certain embodiments, where a complete knockout may be detrimental to the cells, or at least detrimental in terms of cell growth, and where the ability to produce recombinant protein is significantly reduced, only one allele may be targeted to achieve a favorable balance between reducing the load of the corresponding HCP to be knocked out in one allele and the integrity and stability of the host cell.

[0181] In a preferred embodiment of the CHO cell according to the invention, at least one endogenous protein has an amino acid sequence corresponding to the amino acid sequence according to SEQ ID NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41 ,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128, or any of the amino acid sequences corresponding to the proteins mentioned in any one of Tables 1, 2, 3, 4 or 5 herein, or any homolog, or ortholog or paralog thereof,or corresponding to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 1 , 127, and 128 have an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0182] Preferably, the CHO cells according to the present invention comprise at least one modification in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty or more different HCP encoding sequences, wherein each HCP is preferably selected from acHCP and / or drHCP.

[0183] In another aspect, the present invention relates to a method for producing at least one modified CHO cell according to the present invention, said method comprising the steps of: (i) providing at least one CHO cell comprising at least one endogenous target nucleic acid segment; (ii) providing at least one genome or transcriptome editing agent comprising at least one RNAi agent or at least one editing agent comprising at least one site-directed endonuclease, said site-directed endonuclease preferably being selected from the group consisting of a mega-nuclease, a ZFN, a TALEN, a CRISPR nuclease, or a nickase or nuclease-inactive variant thereof, or a nucleic acid molecule encoding these, and optionally in the presence of a CRISPR nuclease. (iv) obtaining at least one modified CHO cell comprising at least one modification in the at least one endogenous target nucleic acid segment according to step (i); (v) optionally: selecting another target nucleic acid segment and repeating steps (i) to (iv) at least once to obtain at least one modified CHO cell comprising at least one additional modification in another endogenous target nucleic acid segment.

[0184] In a preferred embodiment of the method according to the present invention, the at least one endogenous target nucleic acid segment may be a nucleic acid sequence, preferably a DNA sequence, encoding: according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128.

[0185] Preferably, the target nucleic acid segment may be a nucleic acid sequence, preferably a DNA sequence, encoding an amino acid sequence, wherein the amino acid sequence is contained in the top 50%, preferably the top 40%, preferably the top 30%, preferably the top 20%, preferably the top 10%, preferably the top 5%, preferably the top 2% of: NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37 ,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72 , 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128.

[0186] In a preferred embodiment of the method according to the present invention, the CRISPR / Cas system can be used as a genome editing system. This clustered regularly interspaced short palindromic repeats (clustered regularly interspaced shortpalindromic repeat, CRISPR) and CRISPR-associated protein (CRISPR / Cas) system has significantly changed the research and development of molecular biology in the past decade, providing huge possibilities for modifying target cells (including complex eukaryotic cells) in a highly targeted manner. According to the present disclosure, any CRISPR / Cas system suitable for eukaryotic cell genome editing can be used. The CRISPR / Cas system may include a CRISPR / Cas9 system, a CRISPR / Cas13 system, a CRISPR / Cas12a system (also known as a CRISPR / Cas12a system), a CRISPR / C2C2 system, a CRISPR / CasX system (also known as a CRISPR / Cas12e system), a CRISPR / CasY system, a CRISPR / Cmr system, a CRISPR / Csm system, a CRISPR / MAD2 system, a CRISPR / MAD7 system, a CRISPR / CasZ system, a CRISPR / Cas14a, b or c system, a CRISPR / Cas phi system, a CRISPR / Cas12f system, or a catalytically active fragment or variant thereof. A variety of CRISPR systems and optimized mutation systems (including nickase- or inactivated nuclease-based systems, CRISPR mutants with optimized PAM specificity and temperature tolerance), as well as a variety of methods using these CRISPR variants and mutants, alone or in combination with other effectors (e.g., methods suitable for base editing and lead editing), have been developed and are suitable for use in accordance with the present disclosure (see

[0187] Jinek,et al.,A Programmable Dual-RNA-Guided DNA Endonuclease inAdaptive Bacterial lmmunity.Science 2012,337,816-821;Hillary and Ceasar,MolBiotechnol.2022Sep 27; 1-15, doi:10.1007 / s12033-022-00567-0; Zetsche, et al. Cpf1Is a Single RNA-Guided Endonuclease of a Class 2CRISPR-Cas System. Cell 2015,163,759-771; Kim et al. Nat Biotechnol.(2022);40(1):94-102;doi:10.1038 / s41587-021-01009-z;Liu et al. al., CRISPR J. April 2020; 3(2):97-108.; Karvalis et al. Nucleic Acids Res. (2020); 48(9): 5016-5023. doi:10.1093 / nar / gkaa208, Selkova et al. RNA Biol.(2020);17(10):1472-1479;doi:10.1080 / 15476286.2020.1777378,Reesand Liu,2018.Nat.Rev.Genet.;doi:10.1038 / s41576-018-0059-1,Komoret al., Nature533,420-460,2016; Komor et al.,2017,Science Advances,doi:10.1126 / sciadv.aao4774M; Gaudelli et al.,Nature,551:464-471,2017;Lu et al.,nt.J.Mol.Sci.2022,23,9862.https: / / doi.org / 10.3390 / ijms23179862).

[0188] In certain embodiments, CRISPR nucleases or variants thereof that are suitable for multiplexing applications may be preferred, meaning targeting more than one target site of interest in the genome to be modified in a site-directed manner. In certain embodiments, Class 2 V-type CRISPR / Cas nucleases or mutants or variants thereof may be preferred (see

[0189] Koonin&Makarova.Mobile genetic elements and evolution of CRISPR-Cassystems: All the way there and back.Genome Biology and Evolution.2017;9(10):2812-2825.doi:10.1093 / gbe / evx192,Tong et al.,Front.Cell Dev.Biol 2021,Sec.Cellular Biochemistry doi.389 / fcell.2020.622103.

[0190] As known to those skilled in the art, CRISPR / Cas effectors require guiding molecules to exert their activity." Guiding molecules", particularly "guide RNA" (gRNA) may include trans-activating CRISPR RNA (trans-activating CRISPR RNA, tracrRNA) and CRISPR RNA (CRISPR RNA, crRNA). Certain CRISPR nucleases, such as Cas12a, only require crRNA. TracrRNA and crRNA can be combined in one molecule to produce a single guide RNA (single guide RNA, sgRNA), which includes targeting sequence information of the genomic sequence for cutting by the nuclease. For the purpose of multiplexing, more than one gRNA that allows identification and modification of more than one target site in the genome can be designed and used in the same experiment or in one experiment after another.

[0191] In a preferred embodiment of the method according to the invention, the CRISPR nuclease may be a class 2 V-type nuclease, or a variant or mutant thereof, including Cas12a (formerly known as Cpf1) or MAD7 or Cas14 effectors. Suitable vectors and plasmids expressing relevant CRISPR effectors and cognate gRNAs are available to those skilled in the art and can be readily adapted by designing gRNAs in silico.

[0192] In another embodiment, RNAi agents can be used to knock down or silence at least one RNA encoding at least one HCP target of interest. For RNAi, small RNAs function to guide specific effector proteins to target nucleotide sequences through complementary base pairing, resulting in target degradation. "Gene silencing constructs" or "RNAi agents" typically include so-called "sense" and "antisense" sequences. Sense and antisense sequences are complementary sequences that exist in opposite directions in the nucleic acid sequence. If the nucleic acid construct contains a sense sequence and a corresponding antisense sequence, the two complementary sequences form an RNA duplex when transcribed, which produces an "RNA hairpin." In certain embodiments, more than one HCP target can be knocked down simultaneously using these RNA hairpin sequences.

[0193] In some embodiments, knockdown of at least one HCP (e.g., by an RNAi agent) and knockout mediated by genome editing (including CRISPR / Cas) can be combined to reduce expression of at least one target HCP.

[0194] In another aspect of the present invention, a method for producing at least one recombinant protein of interest is provided, comprising the following steps: (a) providing at least one modified CHO cell according to the present invention, wherein the at least one modified CHO cell comprises at least one recombinant gene encoding at least one recombinant protein of interest; (b) culturing the at least one target cell in a culture medium such that the at least one recombinant protein of interest is expressed; (c) harvesting the at least one recombinant protein of interest; and (d) purifying and / or purifying the at least one recombinant protein of interest.

[0195] In a preferred embodiment of the method according to the present invention, at least one recombinant gene is exogenously expressed from at least one expression vector (i.e., at least one plasmid). The expression vector can be particularly selected from mammalian expression vectors, such as those disclosed herein, or those known to those skilled in the art. Thus, the skilled person can easily define expression vectors that are compatible with the present disclosure and suitable for the intended host cell in their respective technical field.

[0196] In a preferred embodiment of the method according to the present invention, culturing the at least one target cell can be performed by a method selected from batch culture, fed-batch culture, perfusion culture, etc., and combinations thereof. In a preferred embodiment of the method according to the present invention, the culture medium for culturing the at least one target cell can be selected from, for example, non-chemically defined or chemically defined cell culture media.

[0197] In one embodiment of the method according to the invention, the purification and / or cleanup may comprise one or more process steps selected from affinity purification, ion exchange, hydrophobic interaction chromatography and size exclusion chromatography.

[0198] In a preferred embodiment of the method according to the present invention, the at least one recombinant protein of interest may be selected from, but not limited to, therapeutic proteins, monoclonal antibodies, bispecific antibodies, fusion proteins, peptibodies and peptides.

[0199] In another aspect of the present invention, there is provided at least one modified CHO cell according to any one of the present invention for producing at least one drug or a portion thereof. The drug can be any biomolecule, preferably a recombinant molecule, including a recombinant protein, peptide, RNA, DNA or any combination, a fusion molecule, including molecules of covalent or non-covalent (e.g., hybridization; antigen:: antibody complexes, etc.) tethers, which can be produced in the CHO cells of the present disclosure. The drug will preferably be purified. In addition, the drug can be further modified, for example, by chemical synthesis to connect another part, or two molecules are connected to each other. In addition, the drug can be stable, lyophilized, and provided together with suitable buffers and reagents that are all pharmaceutically acceptable. Galenic technology for compounding drugs is well known to those skilled in the art.

[0200] In another aspect of the present invention, a kit is provided comprising: (a) at least one modified CHO cell according to the present invention, said CHO cell or said kit comprising: (bi) at least one nucleic acid molecule suitable for expressing at least one recombinant protein or peptide of interest; optionally means for introducing it into the genome of said CHO cell, or alternatively (b.ii) at least one nucleic acid molecule encoding at least one recombinant protein of interest; optionally means for introducing it into the genome of said CHO cell; or alternatively (b.iii) at least one nucleic acid molecule suitable for transcribing at least one recombinant RNA molecule of interest; optionally means for introducing it into the genome of said CHO cell; or or alternatively (b.iv) at least one nucleic acid molecule encoding at least one recombinant RNA molecule of interest; optionally means for introducing it into the genome of said CHO cell; and optionally (c) a culture medium suitable for the at least one modified CHO cell according to step (a), (i) endogenously or exogenously replicating said at least one nucleic acid molecule according to any one of steps (bi), (b.ii), (b.iii) and (b.iv); and (ii.a) expressing said at least one recombinant protein of interest according to steps (bi) and / or (b.ii), and / or transcribing said at least one recombinant RNA molecule of interest according to steps (b.iii) and / or (b.iv).

[0201] Also provided are methods for using the purified recombinant molecules produced by the modified CHO cells of the present invention, alone or in combination with other pharmaceutically acceptable ingredients and buffers, in methods of treating subjects in need thereof. Given the high purity based on the reduced amount of HCP, the recombinant molecules are particularly suitable for safe use in humans.

[0202] In yet another aspect of the present disclosure, a method is provided for identifying and optionally selecting a cell, preferably a CHO cell, for a favorable modified host cell protein (HCP) profile, preferably a CHO cell characterized by a defined load of at least one endogenous protein that is a difficult-to-remove HCP (drHCP) and / or an abundant core HCP (acHCP), wherein the method comprises: (i) providing at least one CHO cell, optionally a CHO cell expressing at least one recombinant molecule of interest; (ii) providing at least two, preferably at least three, four, five, six, seven, eight, eight, nine ... , 6, 7, 8, 9, 10 or even more expression profiles; (iii) comparing and thereby evaluating the data obtained from the expression profile analysis and associating the data with at least one quantifiable parameter for each protein or RNA of interest; (iv) classifying the data by assigning the values ​​obtained for at least one quantifiable parameter to at least one state or group; and (v) identifying a favorable modified host cell protein (HCP) profile; the method optionally comprising preparing a cell, preferably a CHO cell, having a favorable modified HCP profile by modifying at least one endogenous HCP, and optionally selecting and thereby obtaining a modified cell, preferably a modified CHO cell, having a favorable modified HCP profile.

[0203] Correlation can be performed using computational analysis methods. Typically, correlation will mean comparing the obtained data with publicly available genomic, transcriptomic, and protein expression databases.

[0204] In one embodiment, the expression profile can be a transcriptome profile that determines and / or quantifies RNA transcripts. In another embodiment, the expression profile can be a profile that determines and / or quantifies protein or peptide profiles, preferably high throughput methods including LC-MS and SWATHLC-MS.

[0205] In certain embodiments, expression profiles can be obtained by taking a series of sample measurements over time, for example, by collecting and analyzing samples at certain time points before harvest, or by collecting and analyzing samples after a post-harvest purification step, or by collecting and analyzing samples after stimulating a culture comprising at least one CHO cell of interest or a population thereof with at least one stimulatory or inducing agent, such as a transcriptomic profile, to assess the effect of the stimulatory or inducing agent (e.g., a chemical agent, a biological agent, or any abiotic stress factor) on the cell or cell population.

[0206] The modification of at least one cell, preferably at least one CHO cell, may be performed as defined herein for the methods for providing a CHO cell with a modified HCP profile.

[0207] Therefore, the above methods integrate and thus promote high-throughput cell culture, screening and modification methods to accelerate the identification of HCP targets and establish a pipeline for rapid identification of HCP targets of interest for the desired environment, followed by rapid modification of the desired cells, preferably in a multiplexed manner, to obtain optimized production CHO host cells in a short period of time.

[0208] In another embodiment, a recombinant molecule, preferably a recombinant protein or peptide, obtained from the modified CHO cells according to the present invention as a pharmaceutical agent, comprising at least one monoclonal antibody, is provided for use in a method of treating a subject in need thereof, including preventing a disease and curing a disease, wherein the treatment is associated with fewer side effects due to the reduced amount of HCP in the recombinant molecule as the active pharmaceutical agent.

[0209] All individual embodiments and aspects disclosed herein can be combined with one another within the framework and context of the present disclosure.

[0210] The present invention will now be further illustrated by the following non-limiting examples.

[0211] Example

[0212] Example 1: Generation of HCP Profiles

[0213] To identify those HCPs to be knocked out to obtain CHO cells with advantageously reduced HCP content, HCPs were first analyzed in a systematic manner in multiple samples. To this end, the Uniprot Chinese hamster reference proteome was used as the basis for preliminary classification to identify targets of significant interest.

[0214] HCP profiles were generated for a number of CHO production clones expressing mAbs processed under industrial USP (ambr250 fed-batch process) and DSP (standard ProA capture, viral inactivation [VI], cation exchange [CEX], and anion exchange [AEX] polishing steps) conditions. Figure 1 Shown in.

[0215] All samples were analyzed by SWATH LC-MS (for methods, see Krasny et al., Journal of Proteomics Vol. 189, 2018, Sim, et al., SciData 7, 263, 2020), and for each sample, the HCPs present were identified and quantified using the Uniprot Chinese hamster reference proteome.

[0216] Example 2: Systematic Identification of HCP Knockouts

[0217] Based on the HCP profiles generated in Example 1, two groups of relevant HCP knockout targets were identified:

[0218] (1) Abundant core HCPs (acHCPs), which comprise those HCPs detected in all samples collected before or on the day of harvest ( Figure 1 and marked with “abundant HCP” boxes in Table 4), and those HCPs present in quantifiable amounts (>LLOQ) in all these samples.

[0219] (2) Difficult to remove HCPs, including those detected in at least one of the samples collected during DSP processing ( Figure 1 and marked with the “difficult-to-remove HCP” box in Table 5).

[0220] After applying each standard purification protocol using cascade purification steps as detailed in Example 1, samples were taken after each step and also analyzed by SWATH LC-MS.

[0221] As a result of this extensive sample testing, a core set of abundant HCPs was identified (see Figure 1 middle column in ) and difficult-to-remove HCPs (see Figure 1 The relevant HCP knockout targets from the enriched core HCP (=acHCP) panel were defined based on the following criteria: (i) the HCP was present in Figure 1 in all samples marked in the middle column, and (ii) present in quantifiable amounts (>LLOQ, see Figure 2 ). The information was then aligned to the UniProt Chinese hamster reference genome.

[0222] Figure 2 The identification process, classification and quantity of abundant core HCP are shown: a total of 1,254 different HCPs were detected in the samples collected before or on the harvest day, but only 351 of them were present in all these samples. Of those, 67 were present in all samples with accurately quantifiable amounts (>LLOQ) and were defined as abundant core HCPs (acHCPs are shown in Table 4 below). The abundant core HCP species identified were further characterized in terms of subcellular localization and the average quantity measured: respectively, 39 HCPs were cytoplasmic proteins, present at an average concentration of approximately 230 mg / L, equivalent to approximately 32.6% of the total HCP amount, and 23 HCPs were extracellular (average concentration of approximately 163 mg / L, approximately 23.1% of the total HCP amount) and 5 membrane-bound proteins (27 mg / L, 3.8%).

[0223] Based on the criteria defined above, 49 proteins were classified as difficult-to-remove HCPs (drHCPs, see Table 5 above).

[0224] Thus, the generation of the list of acHCPs and drHCPs was the result of a targeted screening process using bioinformatics and high-throughput analysis steps of a large number of exemplary samples, the idea being to obtain guidance for the main HCPs of interest to be knocked out. This goal was achieved by limiting acHCPs and drHCPs to very important "core" HCPs, given their abundance and / or inherent difficulty in removal, following analysis (wet lab and in silico) of a pool of 1,254 HCPs (current status, further work in progress).

[0225] Table 6 below summarizes the Uniprot IDs for all 1,254 HCPs currently identified and tested (work in progress):

[0226] Table 6

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] After the initial data analysis characterization, a list of particularly interesting target HCPs was identified based on the above screening. Three subsets of HCP targets are defined as follows: First, all relevant HCPs that can be quantified are identified. These are summarized in Table 1. Next, HCPs are divided into subclasses acHCP and drHCP (see Table 4 and Table 5). All HCPs that follow the above-determined criteria of acHCP, drHCP or both are summarized in Table 2. Finally, a combined list of HCPs that meet the acHCP and drHCP qualifications is defined. A list of highly important targets is shown in Table 3.

[0233] To complete the initial classification of HCPs, which served as the basis for subsequent identification of a targetable knockout panel, relevant HCP knockout targets were defined from the difficult-to-remove HCP panel based on the following criteria: Figure 1 The right column shows at least one of the samples in which the HCP could not be removed by at least one of the used purification strategies.

[0234] Based on the above information, a complex matrix of single and multiple knockout strategies was defined and carried out as detailed below.

[0235] Example 3: Generation and evaluation of single cell clones with a single HCP KO

[0236] Seventeen genes identified as abundant core and / or difficult to remove HCPs (Lpl, Fn1, Prdx1, Bgn, Spp1, Thbs1, Ctsb, Psap, Tnx, Lamb1, Itih5, Nid1, Pxdn1, Lgals3bp, Pcolce, Timp, Hspg2, see Table 3) were selected as targets for generating CHO DG44 single cell clones with single knockouts. For all HCP genes, gRNAs targeting early exons were designed using Geneious Prime or Benchling software in combination with the annotated genome of the in-house CHO DG44 suspension cell line. For knockout, RNP complexes were formed by incubating 7.5 pmol of CRIPSR nuclease with 7.5 pmol of the corresponding target gRNA for 15 minutes. Subsequently, 2E5 CHO DG44 cells were transfected with the complex using the Neon transfection system (Thermo), transferred to 1 mL of CD DG44 medium, and incubated at 36.8°C and 7.5% CO2. On day 3 after transfection, single clones were isolated using CellCelector nanowell plates (Sartorius) and the CellCelector system (Sartorius). After a 4-day incubation period (36.8°C and 7.5% CO2), the CellCelector system was used to automatically identify overgrown colonies derived from single cells and 96 clones were transferred to 384-well plates. Cell growth was monitored using the CellMetric imaging system (Solentim), and clones that reached >17% confluence were transferred to 96-well plates. Genomic DNA was isolated using DNA QuickExtract TMAfter DNA extraction solution 1.0 (Lucigen) is separated, PCR reaction is performed with appropriate primers to produce 300 to 500bp amplicon covering the target area around the cleavage site. The DNA fragment obtained is subjected to Sanger sequencing (Microsynth Seqlab) and analyzed using open source software Inference of CRISPR edits (ICE) v3.0 (Synthego) to generate the InDel spectrum of the edited clone. For each target, 12 clones that only show InDel outside the frame and therefore completely functionally knock out the corresponding HCP are identified and amplified to the shake flask level. Afterwards, the effect of single knockout on the growth and productivity performance of the edited cell clones under bioprocess conditions was evaluated. For this reason, 1E6 cells of each knockout clone and unedited CHO DG44 (triplicate) for comparison reasons were transfected with 10 μg of proprietary expression plasmids encoding monoclonal antibodies and DHFR as a selective marker. In view of testing a large number of clones, the experiment was carried out in five rounds, always using CHO DG44 as a control. The five-part experimental design was carried out by Figure 3A to the dotted line in D.

[0237] 2 days after transfection, cells were transferred to proprietary selective culture medium, at 36.8°C, 7.5% CO , and cultivated under a linear oscillation of 110rpm, and subcultured every 3 to 4 days. About 10 to 14 days after transfection, when cell concentration and viability improved, the culture was used to run a fed-batch bioprocess in a 125mL shake flask: 3E5 cells / mL were inoculated in a 25mL proprietary production culture medium, and at 36.8°C, 7.5% CO , and cultivated under a linear oscillation of 110rpm, until the end of operation. From the 3rd day after inoculation, proprietary feed medium A and B and glucose of a limited amount were added every day according to the internal feed scheme. Viable cell concentration and viability, glucose and lactate concentrations and antibody titers were measured on the defined date of the process, respectively, using Vi-Cell cell counter (BeckmanCoulter), Biosen C-Line device (EKF) and Octet device (Sartorius). The run was terminated once the viability dropped below 70%. The highest viable cell concentration (=peak VCC), process duration, final titer and average specific productivity as key performance indicators were determined for each run and normalized to the corresponding mean values ​​obtained for the control (unedited CHO DG44 cells). Figure 3A As summarized in D, none of the single knockouts had a significant negative impact on growth, process duration, final titer, or specific productivity, whereas positive effects on process duration and final titer were observed in cells containing Fn1 knockout.

[0238] Example 4: Generation and evaluation of cell pools with single HCP knockouts

[0239] An additional 10 genes identified as abundant core and / or difficult to remove HCPs (Pkm, Cspg4, Ldha, Vim, Hspa5, Ppib, Hsp90aa1, Lama5, Fstl1, Aebp1) were selected as targets for generating CHO DG44 cell pools with single knockouts. For all HCP genes, gRNAs targeting early exons were designed using Geneious Prime or Benchling software in combination with the annotated genome of the in-house CHO DG44 suspension cell line. For knockout, RNP complexes were formed by incubating 3.75 pmol CRIPSR nuclease with 3.75 pmol of the corresponding target gRNA for 15 minutes. Subsequently, 2E5 CHO DG44 cells were transfected with the complex using the Neon transfection system (Thermo), transferred to 1 mL of LCD DG44 medium, and incubated at 36.8°C and 7.5% CO2. After 2 days, the transfected cell pool was transfected with RNP complexes for a second time as described above, and then a third round of transfection was performed after another 2 days. After the cell pool was expanded to the shake flask level, genomic DNA was extracted using DNA QuickExtract TMDNA extraction solution 1.0 (Lucigen) is separated, and PCR reaction is carried out with appropriate primer pairs, and 300 to 500bp amplicon of target region around covering cutting site is produced. The DNA fragment obtained is subjected to Sanger sequencing (Microsynth Seqlab), and is analyzed using open source software Inference of CRISPR edits (ICE) v3.0 (Synthego), to determine the ratio of InDel outside the frame of corresponding HCP in cell colony (and the ratio of functional knockout). Afterwards, the effect of single knockout on the growth and productivity performance of edited cell pools under bioprocess conditions was evaluated. For this reason, 1E6 cells of each kind of knockout pools and unedited CHO DG44 for comparison reasons are transfected (all transfections are completed in triplicate) with 10 μg of proprietary expression plasmids encoding purpose monoclonal antibodies and DHFR as a selective marker. 2 days after transfection, cells were transferred to proprietary selective culture medium, at 36.8°C, 7.5% CO and cultivated under a linear oscillation of 110rpm, and subcultured every 3 to 4 days. About 10 to 14 days after transfection, when cell concentration and viability improved and showed complete recovery from selective pressure, culture was used to run fed-batch bioprocess in 125mL shaking flasks: 3E5 cells / mL were inoculated in 25mL proprietary production culture medium, and at 36.8°C, 7.5% CO and cultivated under a linear oscillation of 110rpm, until the end of operation. From the 3rd day after inoculation, proprietary feed medium A and B and glucose of limited amount were added every day according to the internal feed scheme. Viable cell concentration and viability, glucose and lactate concentration and antibody titer were measured on the limited date of process using Vi-Cell cell counter (Beckman Coulter), Biosen C-Line device (EKF) and Octet device (Sartorius) respectively. Once viability dropped below 70%, the run was terminated.

[0240] The key performance indicators for each run were the maximum viable cell concentration (=peak VCC), process duration, final titer and average specific productivity. Figure 4A As summarized in D, none of the single knockouts had a significant negative impact on growth, process duration, final titer, or specific productivity.

[0241] Example 5: Generation and evaluation of single cell clones with multiple HCP KO

[0242] Based on the data generated in Example 3, 7 knockout targets (Bgn, Fn1, Lpl, Nid1, Pcolce, Pxdn, Thbs) were selected for the generation of CHO DG44 single cell clones with multiple knockouts. For each of the 7 targets, 1.1 pmol of CRIPSR nuclease was incubated with 1.1 pmol of the corresponding target gRNA for 15 minutes to form RNP complexes by using the identical gRNA as described in Example 3. Subsequently, all 7 RNP reactants were combined, and 2E5 CHO DG44 cells were transfected with RNP complexes using Neon transfection system (Thermo), transferred to 1 mL of CD DG44 culture medium, and incubated at 36.8 ° C and 7.5% CO . After 2 days, the transfected cell complexes were transfected for the second time with the RNP complexes merged as described above, and then the third round of transfection was performed after another 2 days. Three days after the third round of transfection, single clones were isolated using the CellCelector nanoplate (Sartorius) and the CellCelector system (Sartorius). After a 4-day incubation period (36.8°C and 7.5% CO2), overgrown colonies derived from single cells were automatically identified using the CellCelector system and 172 clones were transferred to 384-well plates. Cell growth was monitored using the CellMetric imaging system (Solentim) and 158 clones that reached >17% confluence were transferred to 96-well plates. TM After DNA extraction solution 1.0 (Lucigen) is used to separate genomic DNA, PCR reactions are performed with appropriate primers to generate 300 to 500bp amplicons covering the target region around the cleavage site. The obtained DNA fragments are subjected to Sanger sequencing (Microsynth Seqlab) and analyzed using open source software Inference of CRISPR edits (ICE) v3.0 (Synthego) to generate InDel profiles of edited clones. All clones that only show out-of-frame InDels and therefore complete functional knockouts are identified for at least 4 of the 7 proposed HCP targets and amplified to shake flask levels. Figure 5The observed knockout combinations and the number of clones present in each combination are summarized in . Afterwards, the effects of different knockout combinations on the growth and productivity performance of edited cell clones under bioprocess conditions were evaluated. To this end, 1E6 cells of each knockout clone and unedited CHO DG44 (quadruplicate) for comparison reasons were transfected with 10 μg of a proprietary expression plasmid encoding a monoclonal antibody and DHFR as a selective marker. Two days after transfection, the cells were transferred to a proprietary selective culture medium, cultured at 36.8°C, 7.5% CO2 and 110rpm linear shaking, and subcultured every 3 to 4 days. About 10 to 14 days after transfection, when cell concentration and viability increased, indicating complete recovery from selective pressure, the culture was used to run a fed-batch bioprocess in a 125 mL shake flask: 3E5 cells / mL were inoculated in 25 mL of proprietary production medium and cultured at 36.8°C, 7.5% CO2 and 110 rpm linear shaking until the end of the run. Starting on the 3rd day after inoculation, a limited amount of proprietary feed medium A and B and glucose were added every day according to the internal feed scheme. Viable cell concentration and viability, glucose and lactate concentrations, and antibody titers were measured on the defined dates of the process using a Vi-Cell cell counter (Beckman Coulter), a Biosen C-Line device (EKF), and an Octet device (Sartorius), respectively. Once viability dropped below 70%, the run was terminated. The highest viable cell concentration (=peak VCC), process duration, final titer, and average specific productivity as key performance indicators were determined each time.

[0243] As summarized in Figure 6, no significant negative impact on growth, process duration, final titer, or specific productivity was observed for any of the analyzed HCP knockout combinations compared to unedited CHO DG44 cells. This confirms that the targets represent suitable targets for reducing the overall HCP burden across multiple KOs while maintaining cellular integrity.

[0244] Example 6: Nuclease Test

[0245] Having confirmed that multiple HCP knockouts can be simultaneously inserted into target CHO cells without negatively impacting the overall viability and productivity of the target cells, several nucleases were selected for testing to define the optimal setup for multiple knockout experiments. To this end, several multiple targets (at least 5 simultaneously) were selected as shown in Table 1. In addition, suitable nucleases were provided, and guide RNAs were adjusted accordingly to meet the needs of the target nucleases and recognize the corresponding target sequences.

[0246] It can be shown that certain nucleases perform better in multiple knockout experiments. Generally speaking, it is preferable to knock out more than one HCP target in a single experiment to reduce the overall process time required, reduce cellular stress on the target cells, and better control and examine the efficiency and effectiveness of the knockout.

[0247] Since several nucleases could be shown to be suitable for generating multiple desired knockouts simultaneously, further experiments are currently underway to define alternative strategies.

[0248] Example 7: Other compositions and devices

[0249] Media, compositions, kits, and chemicals

[0250] For CHO cell culture, commercially available culture media from Thermo Fisher Scientific and Sartorius StedimCellca were used.

[0251] For other materials, Corning's 125 mL shake flasks, ALS's 24-well nanoplates, Corning's 384-well flat-bottom clear black microplates, 96-well PCR plates, OMNI Life Science's non-skirted BrandCASY cups, OMNI Life Science's CASY ton, and Sartorius Enhanced Qsol Cartridge Kit for Screening, Sartorius Marker (B / yellow), Sartorius Verification beads ( Validation Bead), NEON from Thermo Fisher Scientific TM Transfection system (NEON TM Transfection System) 10μl / 100μl kit, Macherey-Nagel miRNA, Nunc cell culture plates from Thermo Fisher Scientific, New England Biolabs High-fidelity DNA polymerase ( High-Fidelity DNAPolymerase), Lonza's Vector and Lucigen's QuickExtract DNA extraction solution (QuickExtract DNAExtraction Solution), but alternative materials can be used equally. For devices and equipment, the following devices and equipment were used, but of course alternative equipment available to those skilled in the art can be used equally: Beckman Coulter's cell counter, Roche's cell counter, Solentim's cell imager, Thermo Fisher Scientific's CO2 incubator, Sartorius's flow cytometer, EKF-diagnostics's glucose / lactate analyzer (Glucose / LactateAnalyzer), Sartorius's protein quantification system (ProteinQuantificationSystem), ThermoFisher Scientific's real-time PCR device, Automated Lab Solutions's single cell cloning device and Thermo Fisher Scientific's transfection device.

[0252] Example 8: sgRNA

[0253] To knockout the desired gene target, sgRNAs were designed targeting early exons present in all available transcript variants using the bioinformatics tool Geneious Prime 11.0.11 (Biomatters, Auckland, New Zealand) or cloud-based CRISPR Guide RNA design software (https: / / www.benchling.com). sgRNAs were then ordered and synthesized from different manufacturers, depending on the nuclease of interest for which the sgRNA was designed. To generate frameshift mutations in the desired HCP gene, at least three sgRNAs per target were typically evaluated for their KO efficiency regarding their ability to generate the desired inDel that resulted in a functional KO. Desired inDels were defined as those that were not a multiple of three, showed a high prevalence (>30%) in the heterologous sequence library, and resulted in an early stop codon in the translated protein sequence. In the first round of KO, a pool of CHO DG44 host cells was transfected with sgRNAs for each target gene as shown in Tables 1 to 3 above. The cells transfected with R- buffer were used as negative controls and pMaxGFP was used as positive controls to determine the feasibility of the transfection efficiency and the transfection settings of the evaluation device and reagent. 24 hours after transfection, transfection efficiency, gating and efficiency (data not shown here) were determined in two separate LPs with three technical replicates. The total mean value of transfection efficiency was measured to be 96.9% (data not shown). Genomic DNA was extracted 48 hours after transfection. By Sanger sequencing, the InDel events (i.e., insertions or deletions) of the corresponding site-directed endonucleases at the cleavage site were assessed in the previously amplified pools and cloned DNA fragments by PCR. Geneious Prime 11.0.11 (Biomatters, Auckland, New Zealand) was used to view the chromatogram from sequencing reaction. In addition, a quality score corresponding to the percentage of high-quality untrimmed bases was assigned for each sequencing reaction, where high quality was defined as a function of the percentage of ambiguity present in the Sanger trace. The result obtained by Sanger sequencing can, for example, be used to assess specific editing efficiency and InDel spectra. The selected gRNAs used (crRNA only, 5' to 3', w / o PAM) are shown as SEQ ID NOs: 129 to 153.

[0254] Wild-type CHO cells and all derivative clones are always handled under sterile conditions. CHO cell culture is always carried out under 36.8 ℃, 7.5% CO2 environment, while shaking at 110rpm. Only the culture in plate (i.e. nanopore plate, 384-well plate, 96-well plate, 24-well plate and 12-well plate) is hatched when not shaking. Use CASY cell counter to determine culture parameters, such as viable cell concentration (VCC), total cell concentration (TCC), viability and peak diameter. From the pool transfected with the target site-directed endonuclease, stable single cell clones are produced to be evaluated in fed-batch mode.

[0255] Single-cell cloning was typically performed 3 days after transfection with the corresponding site-directed endonuclease.

[0256] After genetic screening, knockout clones (KO clones) were resuspended and diluted, and the clones were further incubated and cell growth was continuously monitored by measuring confluence.

[0257] The KO clones transfected with the corresponding expression vectors (as described above) were expanded. Once good cell growth could be restored after the transfection procedure (approximately 8 to 10*10 5 cells / mL) and good cell viability of >90%, a fed-batch bioprocess was performed to evaluate the culture performance of each KO clone.

[0258] use Antibody titers of samples obtained from fed-batch cultures were determined using the Sartorius (Sartorius) system according to the manufacturer's instructions. Titers were determined by thawing (RT) supernatant samples collected during fed-batch cultures. Results were then analyzed using Forte Bio Data Analysis 9.0 software (Sartorius Lab Instruments GmbH & Co. KG, Goettingen, Germany).

[0259] To screen the HCPs present in the supernatant from the harvested samples, amino acid analysis and peptide fragment matching with a protein database were performed to identify the HCPs present using UniProt identifiers by external liquid chromatography (LC-MS) (Alphalyse A / S, 5230 Odense M, Denmark). In addition, SWATH LC-MS was used for HCP profiling screening (see Krasny et al., Journal of Proteomics Vol. 189, 2018).

[0260] The relative abundance of each HCP can then be calculated using the production titers provided by Sartorius. Additional data on the cellular localization, amino acid sequence, gene ID, and gene structure and function of each HCP were obtained from the UniProt database. The genetic sequences of all proteins were obtained and verified through NCBI.

[0261] Example 9: Generation and evaluation of cell pools with single HCP knockouts

[0262] An additional 25 genes identified as abundant core and / or difficult-to-remove HCPs (CLU, ACTB, ENO3, GSTP1, TKT, HTRA1, YWHAZ, CTSA, PPIA, GSTM6, HSP90B1, PGK1, PLBD2, H2B, TINAGL1, NUDIK, QSOX1, NUCB2, CTSZ, ANXA2, FLNA, H1.4, AKR1B1, CALR, LGMN) were selected as targets for generating CHO DG44 cell pools with single knockouts. For all HCP genes, gRNAs targeting early exons were designed using Geneious Prime or Benchling software in combination with the annotated genome of the in-house CHO DG44 suspension cell line. For knockout, RNP complexes were formed by incubating 3.75 pmol of CRIPSR nuclease with 3.75 pmol of the corresponding target gRNA for 15 minutes. Subsequently, 2E5 CHO DG44 cells were transfected with the complex using the Neon transfection system (Thermo), transferred to 1 mL of CDDG44 medium, and incubated at 36.8°C and 7.5% CO2. After 2 days, the transfected cell pool was transfected a second time with the RNP complex as described above, and in some cases, a third round of transfection was performed after another 2 days. After the cell pool was expanded to the shake flask level, genomic DNA was extracted using DNA QuickExtract TMDNA extraction solution 1.0 (Lucigen) is separated and PCR reaction is carried out with appropriate primer pairs to produce 300 to 500bp amplicon covering the target area around the cleavage site. The DNA fragment obtained is subjected to Sanger sequencing (Microsynth Seqlab) and analyzed using open source software Inference of CRISPR edits (ICE) v3.0 (Synthego) to determine the ratio of InDel outside the frame of the corresponding HCP in the cell colony (and the ratio of functional knockout). Afterwards, the impact of single knockout on the growth and productivity performance of the edited cell pool under bioprocess conditions was evaluated. For this reason, 1E6 cells of each knockout pool and unedited CHO DG44 for comparison reasons were transfected with 10 μg of proprietary expression plasmids encoding the monoclonal antibody of interest and DHFR as a selective marker (all transfections were completed in triplicate). 2 days after transfection, cells were transferred to proprietary selective culture medium, at 36.8°C, 7.5% CO and cultivated under a linear oscillation of 110rpm, and subcultured every 3 to 4 days. About 10 to 14 days after transfection, when cell concentration and viability improved and showed complete recovery from selective pressure, culture was used to run fed-batch bioprocess in 125mL shaking flasks: 3E5 cells / mL were inoculated in 25mL proprietary production culture medium, and at 36.8°C, 7.5% CO and cultivated under a linear oscillation of 110rpm, until the end of operation. From the 3rd day after inoculation, proprietary feed medium A and B and glucose of limited amount were added every day according to the internal feed scheme. Viable cell concentration and viability, glucose and lactate concentration and antibody titer were measured on the limited date of process using Vi-Cell cell counter (Beckman Coulter), Biosen C-Line device (EKF) and Octet device (Sartorius) respectively. Once viability dropped below 70%, the run was terminated.

[0263] The experiment was performed in four rounds, always using CHO DG44 as a control. This four-part experimental setup consisted of Figure 7A to the dotted line in D.

[0264] The key performance indicators for each run were the maximum viable cell concentration (=peak VCC), process duration, final titer and average specific productivity. Figure 7A As summarized in D, none of the single knockouts affected growth ( Figure 7A ), process duration( Figure 7B ), final titer ( Figure 7B ) or specific productivity ( Figure 7D ) have a significant negative impact.

[0265] Example 10: Generation and evaluation of single cell clones with 11 HCP KOs

[0266] A clone comprising 6 kinds of knockouts (Fn1, Lpl, Nid1, Pcolce, Pxdn, Thbs) derived from embodiment 5 is selected as the basis for producing a CHO DG44 single cell clone with a total of 11 kinds of knockouts. For this reason, 5 other knockouts (LGALS3BP, HSP90AA1, CSPG4, PPIB, CLU) derived from embodiment 3 are selected. For each of 5 targets, 1.1 pmol CRIPSR nuclease and 1.1 pmol of corresponding target gRNA are hatched for 15 minutes to form RNP complexes by using the identical gRNA as described in embodiment 3. Subsequently, all 5 RNP reactants are merged together, and 2E5 CHO DG44 cells are transfected with RNP complexes using Neon transfection system (Thermo), transferred to 1 mL CD DG44 culture medium, and incubated at 36.8 ° C and 7.5% CO2. After 3 days, the transfected cell complexes are transfected for the second time as described above with the RNP complexes merged. Subsequently, single clones were isolated using the CellCelector nanowell plate (Sartorius) and the CellCelector system (Sartorius). After a 5-day incubation period (36.8°C and 7.5% CO2), overgrown colonies derived from single cells were automatically identified using the CellCelector system and 192 clones were transferred to 96-well plates. Cell growth was monitored using the CellMetric imaging system (Solentim). Genomic DNA was extracted using DNA QuickExtract TMAfter DNA extraction solution 1.0 (Lucigen) is separated, PCR reaction is carried out with appropriate primers to produce 300 to 500bp amplicon covering the target region around the cleavage site. The DNA fragment obtained is subjected to Sanger sequencing (Microsynth Seqlab), and analyzed using open source software Inference of CRISPR edits (ICE) v3.0 (Synthego) to generate the InDel spectrum of the edited clone. For all 5 proposed HCP targets (addressed HCP target), all clones that only show InDel outside the frame and therefore complete functional knockout are identified and amplified to shake flask levels. Afterwards, the impact of different knockout combinations on the growth and productivity performance of edited cell clones under bioprocess conditions was evaluated. For this reason, 1E6 cells of each knockout clone and the unedited CHO DG44 (triplicate) for comparison reasons were transfected with 10 μg of the proprietary expression plasmid encoding monoclonal antibodies and DHFR as a selective marker. 2 days after transfection, cells were transferred to proprietary selective culture medium, at 36.8 ℃, 7.5% CO2 and 110rpm linear shaking, and subcultured every 3 to 4 days. About 10 to 14 days after transfection, when cell concentration and viability improved, the culture was used to run a fed-batch bioprocess in a 125mL shake flask: 3E5 cells / mL were inoculated in a 25mL proprietary production culture medium, and at 36.8 ℃, 7.5% CO2 and 110rpm linear shaking, cultivated until the end of the run. Starting on the 3rd day after inoculation, proprietary feed medium A and B and glucose of a limited amount were added every day according to the internal feed scheme. Viable cell concentration and viability, glucose and lactate concentrations and antibody titers were measured on the limited date of the process using Vi-Cell cell counter (Beckman Coulter), Biosen C-Line device (EKF) and Octet device (Sartorius) respectively. The run was terminated once the viability dropped below 70%.The highest viable cell concentration (=peak VCC), process duration, final titer and average specific productivity were determined each time as key performance indicators.

[0267] As summarized in Figure 8, no effect on growth was observed for any of the analyzed 11×HCP knockout clones compared to unedited CHO DG44 cells ( Figure 8A ), process duration( Figure 8B ), final titer ( Figure 8C ) or specific productivity ( Figure 8DThis confirms that the target represents a suitable target for reducing the overall HCP burden across multiple KOs while maintaining cellular integrity.

Claims

1. A CHO cell having a modified host cell protein (HCP) profile, characterized in that The CHO cells have a reduced load or reduced expression level of at least one endogenous protein that is a difficult-to-remove HCP (drHCP) and / or an abundant core HCP (acHCP), wherein the CHO cells: At least one modification is comprised in at least one endogenous coding sequence encoding at least one drHCP and / or acHCP selected from the group summarized in Table 1, or any homologue, orthologue or paralogue thereof, preferably wherein at least one HCP is THBS-1, preferably THBS-1 corresponding to SEQ ID NO: 1, or any homologue, orthologue or paralogue thereof, optionally the homologue, orthologue or paralogue has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence corresponding to SEQ ID NO:

1.

2. CHO cells with a modified host cell protein (HCP) profile, characterized in that The CHO cells have a reduced load or reduced expression level of at least one endogenous protein that is a difficult-to-remove HCP (drHCP) and / or an abundant core HCP (acHCP), wherein the CHO cells: Two or more modifications are comprised in two or more coding sequences encoding two or more drHCPs and / or acHCPs defined / shown in Tables 2, 3, 4 or 5, or any homologues, orthologues or paralogues thereof, preferably wherein at least one of the two or more modifications is a modification of THBS-1 and / or FN-1 corresponding to SEQ ID NOs: 1 and 2, respectively, or a modification of any homologue, orthologue or paralogue of said THBS-1 and / or FN-1, optionally the homologue, orthologue or paralogue has a sequence identity to that of the THBS-1 and / or FN-1 corresponding to SEQ ID NOs: 1 and 2, respectively. The sequence of NO: 1 or 2 has an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

3. The CHO cell of claim 1 or 2, wherein the at least one modification or the two or more modifications affect a drHCP, preferably wherein the at least one modification or the two or more modifications affect one, two or more proteins classified as both a drHCP and an acHCP, or wherein the at least one modification or the two or more modifications affect an endogenous protein having an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, or having an amino acid sequence corresponding to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, respectively. %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of NO: 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 127, 14, 16, 17, 19, 20, 21, 22, 23, 24, 27, 31, 34, 35, 41, 49 and 55, or any homologs, orthologs thereof.

4. The CHO cell according to any one of the preceding claims, wherein the at least one modification results in reduced transcription and / or reduced functional expression of the endogenous protein, thereby reducing the total content of drHCP and / or acHCP.

5. The CHO cell according to any one of the preceding claims, wherein the CHO comprises at least one recombinant gene encoding at least one recombinant protein of interest and / or encoding at least one recombinant RNA molecule of interest. The CHO cell of claim 5 , wherein the at least one recombinant protein of interest is a therapeutic molecule.

7. The CHO cell according to any one of the preceding claims, wherein the at least one modification is selected from at least one insertion, at least one deletion and at least one substitution, including base editing, or any combination thereof, Preferably, wherein said at least one modification is present in exon 1 of the corresponding endogenous coding sequence, and / or wherein said at least one modification in said coding sequence is a frameshift mutation or a point mutation, said point mutation generating a stop codon.

8. The CHO cell according to any one of the preceding claims, wherein the at least one modification in the endogenous coding sequence allows for optimized downstream processing and / or ensures a reduced load or reduced expression level of total HCPs, (i) wherein the CHO cells comprise at least one recombinant non-endogenous gene encoding at least one recombinant protein or RNA of interest, and wherein the optimized downstream processing is characterized by a reduced total HCP load or reduced expression level in the recombinant protein or RNA of the product of interest, wherein the total HCP load is reduced by at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, preferably at least 1%, at least 1.25%, at least 1.5%, at least 1.75%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least at least 2.25%, at least 2.5%, at least 2.75%, at least 3%, at least 3.25%, at least 3.5%, at least 3.75%, at least 4%, at least 4.5%, more preferably at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, and even more preferably at least 9%, at least 10%, at least 15%, at least 20% or at least 25%, as determined by measuring the relative concentration in ng / mL of total HCPs per sample obtained in the modified CHO cells compared to a reference sample obtained from CHO wild-type cells, and / or (ii) wherein said at least one modification in said endogenous coding sequence results in improved upstream processing performance, wherein said improved upstream processing performance is characterized by an increased viable cell concentration and / or an increased specific productivity and / or an increased final titer and / or an increased process duration compared to a wild-type cell not carrying said at least one modification in its genome.

9. The CHO cell of any one of the preceding claims, wherein all alleles of the at least one endogenous coding sequence comprise at least one or more of the modifications.

10. The CHO cell according to any one of the preceding claims, wherein the at least one endogenous protein has an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128,or corresponding to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 1 , 127, and 128 have an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

11. The CHO cell of any of the preceding claims, wherein the CHO cell comprises at least one modification in at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty or more different HCP coding sequences, wherein each HCP is preferably selected from acHCP and / or drHCP.

12. A method for producing at least one modified CHO cell according to any one of claims 1 to 11, comprising the following steps: (i) providing at least one CHO cell comprising at least one endogenous target nucleic acid segment; (ii) providing at least one genome editing agent or transcriptome editing agent comprising at least one RNAi agent or at least one site-directed endonuclease, preferably selected from a mega-nuclease, a ZFN, a TALEN, a CRISPR nuclease, or a nickase- or nuclease-inactive variant thereof, or nucleic acid molecules encoding these, and optionally in the case of a CRISPR nuclease: providing at least one suitable functional guide RNA molecule or a nucleic acid molecule encoding the same; (iii) introducing the at least one genome editing agent of step (ii) into the at least one CHO cell; (iv) obtaining at least one modified CHO cell comprising at least one modification in said at least one endogenous target nucleic acid segment according to step (i); (v) optionally: selecting another target nucleic acid segment and repeating steps (i) to (iv) at least once to obtain at least one modified CHO cell comprising at least one additional modification in another endogenous target nucleic acid segment.

13. A method for producing at least one recombinant molecule of interest, preferably at least one recombinant protein of interest, comprising the following steps: (a) providing at least one modified CHO cell according to any one of claims 1 to 11, wherein the at least one modified CHO cell comprises at least one recombinant gene encoding at least one recombinant protein, DNA or RNA of interest; (b) culturing the at least one target cell in a culture medium such that at least one recombinant molecule of interest is transcribed and / or translated; (c) harvesting the at least one recombinant molecule of interest; (d) purifying and / or purifying the at least one recombinant molecule, preferably the at least one recombinant protein of interest.

14. Use of at least one modified CHO cell according to any one of claims 1 to 11 for the production of at least one medicament.

15. A medicine kit comprising (a) at least one modified CHO cell according to any one of claims 1 to 11, the CHO cell or the kit comprising: (bi) at least one nucleic acid molecule suitable for expressing at least one recombinant molecule of interest, preferably at least one recombinant protein of interest; optionally means for introducing it into the genome of said CHO cell, or alternatively (b.ii) at least one nucleic acid molecule encoding at least one recombinant molecule of interest, preferably at least one recombinant protein of interest; optionally means for introducing the same into the genome of the CHO cell; or alternatively (b.iii) at least one nucleic acid molecule suitable for transcribing at least one recombinant RNA molecule of interest; optionally means for introducing it into the genome of said CHO cell; or alternatively (b.iv) at least one nucleic acid molecule encoding at least one recombinant RNA molecule of interest; optionally means for introducing the same into the genome of said CHO cell; and optionally (c) a culture medium suitable for the at least one modified CHO cell according to step (a) to (i) endogenously or exogenously replicate the at least one nucleic acid molecule according to any one of steps (bi), (b.ii), (b.iii) and (b.iv); and (ii.a) expressing the at least one recombinant protein of interest according to steps (bi) and / or (b.ii), and / or transcribing the at least one recombinant RNA molecule of interest according to steps (b.iii) and / or (b.iv).

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