Cell culture method

Abstract

The present invention relates to the use of limited amounts of cysteine and tryptophan in cell culture medium during the production of recombinant proteins, in particular antibodies. The proteins and antibodies produced under such controlled conditions exhibit reduced heterogeneity, in particular reduced charge variant heterogeneity.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201880035870.9, whose application date is May 29, 2018 and whose invention name is “Cell Culture Method”. Field of the Invention

[0002] The present invention belongs to the field of preparation of recombinant proteins, especially antibodies. More specifically, it relates to a cell culture method for expressing proteins with reduced heterogeneity in a commercial-scale manufacturing process. Background of the Invention

[0004] The development of recombinant proteins as therapeutic proteins (such as therapeutic antibodies) requires the production of recombinant proteins on an industrial scale. To achieve this, different expression systems, both prokaryotic and eukaryotic, can be used. However, in the past two decades, most therapeutic proteins approved for use as therapeutic agents have been produced by mammalian cell culture, and this system remains the preferred expression system for producing large quantities of recombinant polypeptides for human use.

[0005] However, mammalian cell culture presents significant challenges. Compared to other eukaryotic production systems (e.g., yeast and insect cell-based systems), the titers of recombinant proteins produced are often very low.

[0006] Over the past 30 years, people have been committed to establishing the basic parameters of cell culture and recombinant polypeptide expression, and the research focus has been on achieving optimal cell growth and the development of large-scale bioreactors by changing the composition of cell culture medium (see, for example, Hecklau C et al., J Biotech 218 (2016) 53-63; Zang Li. et al. Anal. Chem 83 (2011) 5422-5430) and operating conditions.

[0007] Although yield remains a very important aspect of mammalian cell culture, in recent years, the focus has shifted to controlling product quality and process consistency at all stages of development and production scale. Therapeutic proteins produced by mammalian cell culture show different levels of heterogeneity. Such heterogeneity includes, but is not limited to, different glycosylation patterns, differences caused by deamidation or oxidation, different charge or size variations. The heterogeneity of recombinant proteins may also lead to differences in product color, such as color differences between different batches of the same protein produced by the same manufacturing process. When therapeutic proteins are formulated at high concentrations, this heterogeneity, especially the color difference of the target recombinant protein, becomes more obvious. In recent years, there has been a steady trend towards the subcutaneous delivery of therapeutic proteins that require the formulation of therapeutic proteins at high concentrations. High concentrations are associated with increased levels of aggregates (Purdie J. et al., Biotechnology Progress, 2016, 32, 998-1008). Increased charge variants, such as elevated levels of acidic species, can affect protein stability (Banks DD et al. Journal of pharmaceutical sciences, 2009, 98, 4501-10), while the color of concentrated therapeutic proteins can be more intense.

[0008] Cell culture conditions, such as the composition of the culture medium (Kshirsagar R. et al., Biotechnology and Bioengineering, 109: 10, 2523-2532 (2012); US2013 / 0281355; WO 2013 / 158275) and growth conditions, including pH and temperature (US 8,765,413) have been shown to affect the quality attributes of therapeutic proteins. However, there is still a need to provide further improved cell culture methods for producing therapeutic proteins, particularly therapeutic antibodies with minimal heterogeneity. SUMMARY OF THE INVENTION

[0010] The present invention meets the above needs by reducing the total amount of cysteine ​​or cystine and tryptophan in the cell culture medium during the production stage of recombinant proteins.

[0011] The following specific embodiments are described as numbered below:

[0012] Embodiment 1: A method for producing a recombinant protein, comprising:

[0013] a. culturing host cells capable of producing recombinant proteins in a culture medium;

[0014] b. allowing the culture to proceed through a production phase, wherein the recombinant protein is produced by the cells, wherein during the production phase, the culture is supplemented with

[0015] ● Cysteine ​​or cystine in a total amount of 10 wt% to 30 wt% of the expected total amount of recombinant protein produced; and / or

[0016] tryptophan in a total amount of 8 wt % to 35 wt % of the expected total amount of recombinant protein produced,

[0017] c. and optionally, recovering the recombinant protein from the cell culture medium.

[0018] Embodiment 2. A method according to embodiment 1, wherein cysteine ​​or cystine is supplemented in the culture to a total amount of 12 wt % to 28 wt % of the expected total amount of recombinant protein produced, such as 12 wt % to 25 wt %, for example 12 wt % to 20 wt % of the expected total amount of recombinant protein produced.

[0019] Embodiment 3. A method according to embodiment 1 or 2, wherein tryptophan is supplemented in the culture in a total amount of 8wt% to 30wt% of the expected total amount of recombinant protein produced, such as 8wt% to 25wt%, for example 8wt% to 20wt% of the expected total amount of recombinant protein produced.

[0020] Embodiment 4. The method according to any one of the preceding embodiments, wherein the total amount of cysteine ​​or cystine provided in the method is 2.9 to 12 g / (10 12 cells), for example 2.9 to 7 g / (10 12 cells), for example 5.6 to 7 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase.

[0021] Embodiment 5. The method according to any one of the preceding embodiments, wherein the total amount of tryptophan provided in the method is 2.5 to 7 g / (10 12 cells), for example 2.5 to 3.5 g / (10 12 cells / L), where cells refers to the expected intact viable cell count at the end of the production phase.

[0022] Embodiment 1a: A method for producing a recombinant protein, comprising:

[0023] a. culturing host cells capable of producing recombinant proteins in a culture medium;

[0024] b. allowing the culture to proceed through a production phase, wherein the recombinant protein is produced by the cells, wherein during the production phase, the culture is supplemented with

[0025] ● Cysteine ​​or cystine in a total amount of 10 wt% to 30 wt% of the total amount of recombinant protein produced; and / or

[0026] tryptophan in an amount of 8 wt % to 35 wt % of the total amount of the produced recombinant protein,

[0027] c. and optionally, recovering the recombinant protein from the cell culture medium.

[0028] Embodiment 2a. A method according to embodiment 1, wherein the culture is supplemented with cysteine ​​or cystine in a total amount of 12 wt% to 28 wt% of the total amount of recombinant protein produced, such as 12 wt% to 25 wt%, for example 12 wt% to 20 wt% of the total amount of recombinant protein produced.

[0029] Embodiment 3a: A method according to embodiment 1 or 2, wherein tryptophan is supplemented to the culture in a total amount of 8wt% to 30wt% of the total amount of recombinant protein produced, such as 8wt% to 25wt%, for example 8wt% to 20wt% of the total amount of recombinant protein produced.

[0030] Embodiment 4a. A method according to any one of the preceding embodiments, wherein the total amount of cysteine ​​or cystine provided in the method is 2.9 to 12 g / (10 12 cells), such as 2.9 to 7 g / (10 12 cells), for example 5.6 to 7 g / (10 12 cells), where cells refers to the complete viable cell count at the end of the production phase.

[0031] Embodiment 5a: A method according to any one of the preceding embodiments, wherein the total amount of tryptophan provided in the method is 2.5 to 7 g / (10 12 cells), for example 2.5 to 3.5 g / (10 12 cells / L), where cells refers to the intact viable cell count at the end of the production phase.

[0032] Embodiment 6. The method according to any one of the preceding embodiments, wherein the total amount of cysteine ​​or cystine and / or tryptophan in the culture is achieved by adding cysteine ​​or cystine and / or tryptophan to the cell culture medium at:

[0033] a. at the beginning of the said production phase,

[0034] b. once or more times at any point in the production phase,

[0035] c. added continuously throughout the production phase, or

[0036] d. Any combination of a, b and c.

[0037] Embodiment 7. The method according to any one of the preceding embodiments, wherein the method is a batch method, such as a fed-batch method.

[0038] Embodiment 8. The method according to any of the preceding embodiments, wherein cysteine ​​or cystine and / or tryptophan are provided by daily addition during the production phase.

[0039] Embodiment 9. The method according to embodiment 8, wherein on the second day before adding cysteine ​​or cystine, for example by reducing the cysteine ​​or cystine addition to 5.6 to 7 g / (10 12 The depletion of cysteine ​​or cystine in the culture is carried out by reducing the level of cysteine ​​per cell (cells) where cells refers to the intact viable cell count expected at the end of the production phase.

[0040] Embodiment 9a: A method according to embodiment 8, wherein before adding cysteine ​​or cystine on the second day, the amount of cysteine ​​or cystine added is reduced to 5.6 to 7 g / (10 12 Depletion of cysteine ​​or cystine in culture is accomplished by reducing the level of cysteine ​​per cell (cells) where cells refers to the intact viable cell count at the end of the production phase.

[0041] Embodiment 10. A method according to embodiment 8 or 9, wherein tryptophan is depleted in the culture at a late stage of production, ie when the cells have reached a maximum viable cell density, before tryptophan is added the next day.

[0042] Embodiment 11. A method according to any of the preceding embodiments, wherein at any time point in the production phase, the concentration of cysteine ​​or cystine in the cell culture medium does not exceed 0.9 g / L, preferably at any time point in the production phase, the concentration of cysteine ​​or cystine in the cell culture medium does not exceed 0.3 g / L.

[0043] Embodiment 12. A method according to any of the preceding embodiments, wherein the tryptophan concentration in the cell culture medium does not exceed 0.6 g / L at any time point in the production phase, and preferably the tryptophan concentration in the cell culture medium does not exceed 0.3 g / L at any time point in the production phase.

[0044] Embodiment 13: A process wherein the production phase is carried out for at least 7 days, preferably at least 14 days.

[0045] Embodiment 14. The method according to any of the preceding embodiments, wherein at any point in time during the second half of the production phase:

[0046] ● the amount of cysteine ​​or cystine in the culture is 10 wt % to 30 % of the expected amount of recombinant protein produced; and / or

[0047] - The amount of tryptophan in the culture is between 8 wt% and 35% of the expected amount of recombinant protein produced.

[0048] Embodiment 15, a method according to any of the preceding embodiments, wherein at any point in time during the production phase:

[0049] ● the amount of cysteine ​​or cystine in the culture is 10 wt % to 30 % of the expected amount of recombinant protein produced; and / or

[0050] - The amount of tryptophan in the culture is between 8 wt% and 35% of the expected amount of recombinant protein produced.

[0051] Embodiment 14a. The method according to any of the preceding embodiments, wherein at any point in time during the second half of the production phase:

[0052] ● the amount of cysteine ​​or cystine in the culture is 10 wt% to 30% of the amount of recombinant protein produced; and / or

[0053] • The amount of tryptophan in the culture is 8 wt% to 35% of the amount of recombinant protein produced.

[0054] Embodiment 15a. A method according to any of the preceding embodiments, wherein at any point in the production phase:

[0055] ● the amount of cysteine ​​or cystine in the culture is 10 wt% to 30% of the amount of recombinant protein produced; and / or

[0056] • The amount of tryptophan in the culture is 8 wt% to 35% of the amount of recombinant protein produced.

[0057] Embodiment 16. A method according to any one of the preceding embodiments, wherein the host cell is a mammalian cell, preferably a CHO cell.

[0058] Embodiment 17. A method according to any of the preceding embodiments, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof.

[0059] Embodiment 18. The method of embodiment 17, wherein the antibody or antigen-binding fragment thereof is:

[0060] 1) an antibody or an antigen-binding fragment thereof,

[0061] a. comprising a CDR-H1 having a sequence as defined in SEQ ID NO: 1; a CDR-H2 having a sequence as defined in SEQ ID NO: 2; a CDR-H3 having a sequence as defined in SEQ ID NO: 3; a CDR-L1 having a sequence as defined in SEQ ID NO: 4; a CDR-L2 having a sequence as defined in SEQ ID NO: 5 and a CDR-L3 having a sequence as defined in SEQ ID NO: 6; or

[0062] b. comprising a light variable region having a sequence as defined in SEQ ID NO: 7 and a heavy variable region having a sequence as defined in SEQ ID NO: 8; or

[0063] c. comprising a light variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7 and a heavy variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

[0064] d. comprising a light variable region having a sequence as defined in SEQ ID NO: 7 and a heavy chain having a sequence as defined in SEQ ID NO: 11; or

[0065] e. comprising a light variable region having at least 80% identity or similarity, preferably 90% identity or similarity to a sequence as defined in SEQ ID NO: 7 and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to a sequence as defined in SEQ ID NO: 11; or

[0066] 2) an antibody comprising a light chain having a sequence as defined in SEQ ID NO: 9 and a heavy chain having a sequence as defined in SEQ ID NO: 10; or

[0067] 2) An antibody comprising a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO:9 and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO:10.

[0068] Embodiment 19. The method according to any of the preceding embodiments, wherein the production phase is carried out in a bioreactor, preferably having a volume of 50 L or more, 100 L or more, 500 L or more, 1000 L or more, 2,000 L or more, 5,000 L or more, 10,000 L or more, or 20,000 L or more.

[0069] Embodiment 20. A method according to any one of the preceding embodiments, wherein the method comprises a step of recovering the recombinant protein from the cell culture medium and an additional step of purifying the recombinant protein.

[0070] Embodiment 21. A method according to embodiment 20, wherein the purification comprises protein A chromatography.

[0071] Embodiment 22. The method according to embodiment 20 or 21, further comprising the step of formulating the purified recombinant protein.

[0072] Embodiment 23. A method according to embodiment 22, wherein the recombinant protein is formulated in a liquid formulation comprising one or more amino acids and a surfactant.

[0073] Embodiment 24. The method of embodiment 23, wherein the formulation comprises histidine and / or proline.

[0074] Embodiment 25. A method according to embodiment 24, wherein the formulation comprises histidine in a concentration of 5 mM to 100 mM, for example, in a concentration of 10 mM to 50 mM and / or proline in a concentration of 100 mM to 500 mM at a pH of 5 to 7.4 (such as 5 to 6.5, for example 5 to 6).

[0075] Embodiment 26. The method according to embodiment 25, wherein the formulation comprises histidine at a concentration of 30 mM and proline at a concentration of 250 mM at a pH of 5.2 to 6.0, preferably at a pH of about 5.6.

[0076] Embodiment 27. A method according to any one of embodiments 23 to 26, wherein the surfactant is polysorbate 80, preferably at a concentration of 0.001% to 0.1% (w / v), for example 0.005% to 0.1% (w / v), such as 0.01% to 0.1%, for example 0.01% to 0.05%, such as 0.03%.

[0077] Embodiment 28. The method of any one of Embodiments 23 to 27, wherein the recombinant protein is an antibody and the antibody is formulated at a concentration of 10 mg / ml to 250 mg / ml, for example 20 mg / ml to 250 mg / ml, such as 50 mg / ml to 250 mg / ml, for example 120 mg / ml to 160 mg / ml, such as about 140 mg / ml.

[0078] Embodiment 29. The method according to any one of the preceding embodiments, wherein the method reduces the heterogeneity of the recombinant protein produced, wherein the reduction of heterogeneity comprises reducing:

[0079] a. Charge heterogeneity, preferably acidic peak group (APG); and / or

[0080] b. amino acid oxidation, isomerization, fragmentation, glycosylation, deamidation, cysteinylation of other covalent adducts; and / or

[0081] c. color or color intensity, for example between different batches of the recombinant protein; and / or

[0082] d. high molecular weight species (HMWS); and / or

[0083] e. Recombinant protein instability.

[0084] Embodiment 30. A method for producing a recombinant protein, comprising:

[0085] a. culturing host cells capable of producing recombinant proteins in a culture medium;

[0086] b. allowing the culture to proceed through a production phase, wherein the recombinant protein is produced by the cells and the cell culture medium is supplemented with cysteine ​​or cystine and / or tryptophan, wherein

[0087] The total amount of cysteine ​​or cystine provided in the process is 2.9 to 7 g / (10 12 cells), for example 5.6 to 7 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase, and / or

[0088] The total amount of tryptophan provided in the method is 2.5 to 3.5 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase,

[0089] c. and optionally, recovering the recombinant protein from the cell culture medium.

[0090] Embodiment 30a. A method for producing a recombinant protein, comprising:

[0091] a. culturing host cells capable of producing recombinant proteins in a culture medium;

[0092] b. allowing the culture to proceed through a production phase, wherein the recombinant protein is produced by the cells and the cell culture medium is supplemented with cysteine ​​or cystine and / or tryptophan, wherein

[0093] The total amount of cysteine ​​or cystine provided in the method is 2.9 to 7 g / (10 12 cells), for example 5.6 to 7 g / (10 12 cells), where cells refers to the complete viable cell count at the end of the production phase, and / or

[0094] The total amount of tryptophan provided in the method is 2.5 to 3.5 g / (10 12 cells), where cells refers to the complete viable cell count at the end of the production phase,

[0095] c. and optionally, recovering the recombinant protein from the cell culture medium.

[0096] Embodiment 31. A method according to Embodiment 30, wherein the method has one or more of the additional features of any one of Embodiments 2 to 29.

[0097] Embodiment 32. A method for reducing the heterogeneity of a recombinant protein population in a batch produced by a recombinant host cell during a production phase, comprising limiting the total amount of the following substances present in the cell culture medium during the production phase of the recombinant protein.

[0098] a. Cysteine ​​or cystine and / or

[0099] b.Tryptophan.

[0100] Embodiment 33. A method according to Embodiment 32, wherein the method has one or more of the additional features of any one of Embodiments 2 to 29.

[0101] Embodiment 34. A recombinant protein preparation obtainable or obtained by the method of any one of the preceding embodiments.

[0102] Embodiment 35. A pharmaceutical composition comprising an antibody, wherein the composition has one or more of the additional features of any one of embodiments 23 to 28, preferably, wherein the antibody is the antibody described in embodiment 18. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 : Description of a computational analysis measuring the total amount of the amino acids cysteine ​​or cystine and tryptophan added during the entire production phase / weight percentage of the recombinant protein produced performed in a biosensor.

[0105] Figure 2 : Effect of total tryptophan and cysteine ​​or cystine addition as a percentage of the total mAb1 weight produced in wt% (g / g), respectively, on b* values ​​(a) and acidic peak group (APG) variants (b) normalized to 40 mg / mL, and maximum tryptophan and cysteine ​​or cysteine ​​concentrations did not affect b* values ​​(c) or APG% (d).

[0106] Figure 3 : Effect of total addition of tryptophan and cysteine ​​or cystine as wt% (g / g) of total mAb1 weight produced on b* values ​​(a) normalized to 40 mg / mL and acidic peak group (APG) variants (b), respectively, and lack of correlation of maximum concentrations of cysteine ​​or cystine (c) and tryptophan (d) on APG.

[0107] Figure 4 : Multiple linear regression model of acidic peak group (APG) variants of recombinant monoclonal antibody mAb1 as a function of wt% (g / g) of total added cysteine ​​or cystine and tryptophan of total mAb1 produced.

[0108] Figure 5 : Multiple linear regression model of the main peak group variants of the recombinant monoclonal antibody mAb1 as a function of the total added amount of cysteine ​​or cystine and tryptophan in wt% (g / g) of the total recombinant mAb1 produced.

[0109] Figure 6 : Multiple linear regression model of high molecular weight species (HMWS) variants of recombinant monoclonal antibody mAb1 as a function of the total amount of cysteine ​​or cystine added in wt % (g / g) of the total recombinant mAb1 produced.

[0110] Figure 7 : Multiple linear regression model of b* values ​​normalized to 40 mg / mL of variants of the recombinant monoclonal antibody mAb1 as a function of the total added amount of cysteine ​​or cystine and tryptophan in wt% (g / g) of the total recombinant mAb1 produced.

[0111] Figure 8: Contour plots of the effect of the total amount of cysteine ​​or cystine and tryptophan added as wt% (g / g) of the total recombinant mAb1 produced on (a) high molecular weight species (HMWS) and (b) b* values ​​normalized to 40 mg / mL, (c) acidic peak groups (APG) and (d) main peak group variants. The black dashed square corresponds to the ideal total amount of cysteine ​​or cystine and tryptophan added as wt% (g / g) of the total recombinant mAb1 produced to reduce APG, HMWS, b* values ​​normalized to 40 mg / mL and increase main peak group variants, which for cysteine ​​or cystine corresponds to 12.06 wt% and 28.03 wt% of the total mAb1 produced, and for tryptophan, which corresponds to 8.84 wt% and 32.06 wt% of the total mAb1 produced.

[0112] Fig. 9 : Effect of the total wt% of added cysteine ​​or cystine and tryptophan by weight of cell culture volume (CSV) on the intact viable cell count (IVCC) normalized to CSV. (a) shows the multiple linear regression model of the cumulative IVCC normalized to CSV as a function of the total wt% of added cysteine ​​or cystine and tryptophan by weight of CSV. (b) shows a contour plot of the effect of the total wt% of added cysteine ​​or cystine and tryptophan by weight of CSV on the cumulative IVCC normalized to CSV.

[0113] Fig.10 : Effect of the total amount of cysteine ​​or cystine and tryptophan added to the cell culture medium during the production phase in wt% of CSV weight on the final mAb1 titer determined by HPLC method (mAb HPLC). (a) shows the multiple linear regression model of the final mAb1 HPLC titer as a function of the total amount of cysteine ​​or cystine and tryptophan added in wt% of CSV weight. (b) shows a contour plot of the effect of the total amount of cysteine ​​or cystine and tryptophan added in wt% of CSV weight on the final mAb HPLC titer.

[0114] Fig.11 : (a) shows the total amount of cysteine ​​or cystine and tryptophan added to the cell culture medium during the production phase / IVCC*10 at the end of the production phase -12 Contour plots showing the effect of IVCC on 14-day production. (b) shows the total amount of cysteine ​​or cystine and tryptophan added to the cell culture medium during the production phase / IVCC at the end of the production phase*10 -12The effect of the addition of cysteine ​​or cystine and tryptophan on the final mAb HPLC titer is shown in (c). The amount of amino acids cysteine ​​or cystine and tryptophan added throughout the production phase in the bioreactor / IVCC*10 at the end of the production phase is measured. -12 Description of the computational analysis.

[0115] Fig.12 : Contour plot of the effect of the maximum concentration of cysteine ​​or cystine and tryptophan reached in the cell culture medium during the entire production phase on IVCC normalized to CSV.

[0116] Fig.13 : Total amount of cysteine ​​or cystine added and total amount of cysteine ​​or cystine added / IVCC for the conditions described in Table 6.

[0117] Fig.14 Figure 2: Effect of cysteine ​​or cystine depletion on cell growth and mAb titer. Viable cell concentration (VCC) profiles (a), mAb titer (b) and Cys concentration before feed addition (c) are shown as a function of three experimental conditions: no cysteine ​​or cystine depletion during the entire production phase [no depletion - (Cys in feed was 34.35 g / L)] and two cases: daily depletion of cysteine ​​or cystine from day 6 until the end of fed-batch production, and the concentration of Cys in the feed was 17.17 g / L and 6.87 g / L.

[0118] Fig.15 : Effect of total addition of cysteine ​​or cystine and tryptophan as wt % of total recombinant antibody produced by weight on: (a) b* values ​​normalized to 40 mg / mL variants for mAb2, APG and (b) for mAb3, APG variants and (c) for mAb4, APG, BPG (Basic Peak Group) and main group variants.

[0119] Fig.16 : Effect of the total addition of cysteine ​​or cystine and tryptophan as a wt% of the total recombinant mAb1, mAb2, mAb3 and mAb4 produced on APG. In (a), APG variants of mAb1, mAb2, mAb3 and mAb4 are plotted relative to the total cysteine ​​or cystine addition as a wt% of the total recombinant mAb1, mAb2, mAb3 and mAb4 produced by weight. In (b), multiple linear regression models of APG variants of recombinant monoclonal antibodies mAb1, mAb2 and mAb3 and recombinant multispecific antibody mAb4 are plotted as a function of the total addition of cysteine ​​or cystine and tryptophan as a wt% of the total recombinant mAb by weight.

[0120] Fig.17 : An equation was developed to predict the acidic peak group (APG) levels based on data from the DG44 CHO cell line expressing the mAbl antibody (Table 3).

[0121] Fig.18 : Comparison of experimental acidic peak group (APG) levels (APG exp) with predicted APG levels (APG pred) for the DG44 CHO cell line expressing the mAbl antibody. Data were generated in a perfusion production process using alternating tangential flow (ATF) technology in a 2L bioreactor. APG predictions were based on Fig.17 The equation in . DETAILED DESCRIPTION OF THE INVENTION

[0123] The present invention is based on the discovery that by limiting the total amount of cysteine ​​or cystine and / or tryptophan used in the cell culture medium during the production phase in a method for producing a recombinant protein, the heterogeneity of the produced recombinant protein can be reduced. Thus, the present invention teaches the use of limited amounts of cysteine ​​or cystine and / or tryptophan in the cell culture medium to reduce the heterogeneity of the antibodies or antigen-binding fragments thereof expressed in the culture medium.

[0124] Reduced heterogeneity is preferably with respect to:

[0125] a. Charge, preferably acidic peak group (APG) heterogeneity; and / or

[0126] b. Amino acid oxidation, isomerization, fragmentation, other covalent adducts, glycosylation, deamidation, cysteinylation; and / or

[0127] c. Color or color intensity (b* value normalized to 40 mg / mL); and / or

[0128] d. formation of high molecular weight species (HMWS); and / or

[0129] e. Recombinant protein instability and / or

[0130] F. Their combinations.

[0131] As used herein, the term "heterogeneity" refers to the differences between individual molecules, such as recombinant proteins, in a population of molecules produced by the same production method or within the same production batch. Heterogeneity can be caused by, for example, incomplete or inhomogeneous modifications of recombinant proteins caused by post-translational modifications of expressed proteins. Such modifications can be the result of deamination reactions and / or oxidation reactions and / or covalent additions of small molecules (such as glycosylation reactions) and / or isomerization reactions and / or fragmentation reactions and / or other reactions, and also include changes in glycosylation patterns. The chemical and physical manifestations of this heterogeneity lead to various characteristics in the resulting recombinant protein products, including but not limited to charge variation spectra, color or color intensity and molecular weight spectra.

[0132] The term "production phase" according to the present invention includes the stage of cell culture in the method for preparing recombinant protein when cells express (i.e. produce) recombinant protein. When the titer of the desired product increases, the production phase begins, and ends with the harvesting of cells or cell culture fluid or supernatant. Usually, at the beginning of the production phase, the cell culture is transferred to a bioreactor. Harvesting is the step of removing the cell culture fluid (so as to recycle and purify recombinant proteins such as recombinant antibodies) from, for example, a production vessel during this period. When used in this article, the term "initial cell culture weight" refers to the weight of the culture at the beginning of the production phase, typically the weight when inoculating a bioreactor.

[0133] In a first aspect, the present invention provides a method for producing a recombinant protein, the method comprising:

[0134] a. culturing host cells capable of producing recombinant proteins in a culture medium;

[0135] b. allowing the culture to proceed through a production phase, wherein the recombinant protein is produced by the cells, wherein during the production phase, the culture is supplemented with

[0136] a. cysteine ​​or cystine in an amount of 10 wt % to 30 wt % of the expected total amount of the recombinant protein produced; and / or

[0137] b. tryptophan, the total amount of which reaches 8wt% to 35wt% of the expected total amount of the recombinant protein produced,

[0138] and optionally, recovering the recombinant protein from the cell culture medium.

[0139] It will be apparent from the following description of the invention that the culture is supplemented with cysteine ​​or cystine and / or tryptophan; this supplementation may be carried out using:

[0140] 1. Cysteine; or

[0141] 2. Cystine; or

[0142] 3. Cysteine ​​and cystine; or

[0143] 4. Cysteine ​​and tryptophan; or

[0144] 5. Cystine and tryptophan; or

[0145] 6. Cysteine, cystine and tryptophan; or

[0146] 7. Tryptophan.

[0147] When used herein, the expression "the total amount of cysteine ​​or cystine" or "the total amount of cysteine ​​or cystine to ..." refers to: a) the total amount of cysteine ​​alone if cysteine ​​is not used in the process, b) the total amount of cysteine ​​alone if cysteine ​​is not used in the process; or c) the total amount of cysteine ​​+ cystine if both compounds are used in the process. Cysteine ​​and cystine in the cell culture medium are in a constant equilibrium, in which two cysteine ​​molecules are oxidized to one cystine molecule, and one cystine molecule is reduced to two cysteine ​​molecules.

[0148] The total amount of cysteine ​​or cystine and / or tryptophan may be expressed herein as a percentage of the total amount of recombinant protein produced. As used herein, the term "wt%" refers to weight percentage. "Total" refers to the total amount determined at the end of the production phase, i.e., the total amount of cysteine ​​or cystine and / or tryptophan added during the production phase, and the total amount of recombinant protein produced during the production phase, wherein the total amount of recombinant protein produced is measured at the end of the production phase.

[0149] Figure 1 It is shown how to calculate the total amount of cysteine ​​or cystine and / or tryptophan per wt% of recombinant protein. The total amount of cysteine ​​or cystine or tryptophan added is calculated as a function of the feed rate (or feed volume) and the concentration of cysteine ​​or cystine or tryptophan in the feed and the concentration of cysteine ​​or cystine or tryptophan in the culture medium, where the feed is added by volume of feed added ( Figure 1 , A). The amount of recombinant protein produced was calculated as a function of the final volume of cell culture medium and the final recombinant protein titer ( Figure 1 , B). The ratio of these two calculated parameters is the total amount of cysteine ​​or cystine and / or tryptophan added / the amount of recombinant protein produced ( Figure 1 , C).

[0150] Initially (in step a) the host cells may be grown in a cell culture medium that may or may not contain cysteine ​​or cystine and tryptophan. If the cell culture medium already contains an initial amount of cysteine ​​or cystine and / or tryptophan, the total amount will include that initial amount.

[0151] In one embodiment of the method of the present invention, cysteine ​​or cystine is supplemented in the culture to a total amount of 12.06wt% to 28.03wt% of the expected total amount of recombinant protein produced, such as a total amount of 12wt% to 28wt%, such as 12wt% to 25wt%, such as 12wt% to 20wt% of the expected total amount of recombinant protein produced.

[0152] In another embodiment of the method of the present invention, tryptophan is supplemented in the culture in a total amount reaching 8.84 wt % to 32.06 wt % of the expected total amount of recombinant protein produced, such as 8 wt % to 30 wt %, such as 8 wt % to 25 wt %, such as 8 wt % to 20 wt % of the expected total amount of recombinant protein produced.

[0153] Alternatively, the total amount of cysteine ​​or cystine and / or tryptophan may be expressed as the total amount added in the process relative to the intact viable cell count at the end of the production phase. In one embodiment, the total amount of cysteine ​​and / or cystine provided in the process is 2.9 to 12 g / (10 12 cells), such as 2.9 to 7 g / (10 12 cells), for example 5.6 to 7 g / (10 12 cells), wherein cells refers to the expected complete viable cell count at the end of the production phase. In another embodiment, the total amount of tryptophan provided in the method is 2.5 to 7 g / (10 12 cells), for example 2.5 to 3.5 g / (10 12 cells / L), where cells refers to the expected intact viable cell count at the end of the production phase.

[0154] It should be understood that those skilled in the art will know how to measure the amount of cysteine ​​or cystine and / or tryptophan added to and / or present in cell culture at a particular stage (such as a production stage). For example, this can be performed as described in the embodiments herein. Similarly, those skilled in the art will know how to measure the total amount of recombinant protein produced by cell culture, and therefore apply the teachings of the present invention to achieve the desired technical effects. For example, this can be performed as described in the embodiments herein, such as using aForteBio Octet model analyzer (ForteBio, Inc., Menlo Park, CA) or protein A high pressure liquid chromatography (HPLC) and cell culture supernatant samples stored at -80°C before analysis.

[0155] In order to design a method according to the invention, wherein the amount of cysteine ​​or cystine and / or tryptophan / the expected total amount of recombinant protein produced is kept within a certain range, it may be necessary to perform one or more initial experiments to determine the approximate level of recombinant protein produced by a specific host cell under specific culture conditions. Once the approximate total level of recombinant protein produced is known, a method according to the invention can be designed, wherein the amount of cysteine ​​or cystine and / or tryptophan / the expected total amount of recombinant protein produced is kept within a specified range.

[0156] Various strategies can be used to reach the total amount of cysteine ​​or cystine and / or tryptophan in the cell culture medium during the production phase. In one embodiment, the total amount can be reached by adding cysteine ​​or cystine and / or tryptophan just at the beginning of the production phase, for example only when contained in the production cell culture medium or when already contained in the production cell culture medium. In another embodiment, the total amount can be reached by summing the additions (e.g., daily additions or continuous additions) during the production phase. In yet another embodiment, the total amount can be reached by a combination of the initial cysteine / cysteine ​​and / or tryptophan concentration in the cell culture fluid at the beginning of the production phase and by additions.

[0157] Thus, in one embodiment of the method of the invention, the total amount of cysteine ​​or cystine and / or tryptophan in the cell culture medium is achieved by adding cysteine ​​or cystine and / or tryptophan to the cell culture medium:

[0158] a. At the beginning of the production phase,

[0159] b. once or more times at any point in the production phase,

[0160] c. continuously added during the production phase, or

[0161] d. Any combination of a, b and c.

[0162] In a preferred embodiment, cysteine ​​or cystine and / or tryptophan are added at the beginning of the production phase and added by daily large doses during the production phase. Preferably, the production phase lasts at least 7 days, more preferably more than 7 days, such as 10 days, more preferably 14 days or more days.

[0163] In a preferred embodiment, the cysteine ​​or cystine concentration in the cell culture medium does not exceed 0.9 g / L at any time point during the production phase, preferably, the cysteine ​​or cystine concentration in the cell culture medium does not exceed 0.3 g / L at any time point during the production phase.

[0164] Furthermore, in a preferred embodiment, the tryptophan concentration in the cell culture medium does not exceed 0.6 g / L at any time point during the production phase, and preferably, the tryptophan concentration in the cell culture medium does not exceed 0.3 g / L at any time point during the production phase.

[0165] In one embodiment, cysteine ​​or cystine is added to the cell culture medium every day, and on day 6, the cysteine ​​or cystine in the cell culture medium increases to a maximum concentration of 0.3 g / L, and from day 7 to day 14, the cysteine ​​or cystine in the cell culture medium increases to a maximum concentration of 0.9 g / L.

[0166] In some embodiments, the amount of cysteine ​​or cystine and / or tryptophan is within the specified range not only at the end of the production phase, calculated for the entire production phase, but also at any time point during a portion of the production phase or even at any time point throughout the production phase. Thus, in one embodiment, at any time point in the second half of the production phase (e.g., day 7 to day 14 of a 14-day production phase):

[0167] ● The amount of cysteine ​​or cystine in the culture is 10% of the expected amount of recombinant protein produced.

[0168] wt% to 30%; and / or

[0169] • The amount of tryptophan in the culture is between 8 wt% and 35% of the expected amount of recombinant protein produced.

[0170] In another embodiment, at any point during the production phase:

[0171] The amount of cysteine ​​or cystine in the culture is between 10 wt% and 30% of the expected amount of recombinant protein produced; and / or

[0172] • The amount of tryptophan in the culture is between 8 wt% and 35% of the expected amount of recombinant protein produced.

[0173] When cysteine ​​or cystine is provided to the cells by daily addition, the cysteine ​​or cystine in the culture may be depleted before the next daily addition is provided. In one embodiment, prior to adding cysteine ​​or cystine the next day, for example, by reducing the amount of cysteine ​​or cystine added to 5.6 to 7 g / [10 12 In a second embodiment, tryptophan is depleted in the culture during the cell culture in the late production phase, i.e., when the cells have reached a maximum viable cell density, e.g., starting on day 8 or later in a 14-day production phase.

[0174] Without wishing to be bound by theory, it is believed that despite the depletion of cysteine ​​or cystine, the cells in the production phase are not deprived of cysteine ​​or cystine, but are thought to have an internal mechanism that stores the cysteine ​​or cystine available in the cell culture medium (via addition) as an inactive metabolite that is converted to cysteine ​​or cystine once cysteine ​​or cystine is depleted.

[0175] In a further independent aspect, the present invention relates to a method for producing a recombinant protein, the method comprising:

[0176] a. culturing host cells capable of producing recombinant proteins in a culture medium;

[0177] b. allowing the culture to proceed through a production phase, wherein the recombinant protein is produced by the cells and the cell culture medium is supplemented with cysteine ​​or cystine and / or tryptophan, wherein

[0178] The total amount of cysteine ​​or cystine provided in the method is 2.9 to 7 g / (10 12 cells), for example, from 5.6 to 7 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase, and / or

[0179] The total amount of tryptophan provided in the method is 2.5 to 3.5 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase,

[0180] c. and optionally, recovering the recombinant protein from the cell culture medium.

[0181] In one embodiment of the method, cysteine ​​or cystine is supplemented in the cell culture medium in a total amount of 12 wt % to 28 wt % of the expected total amount of recombinant protein produced, such as 12 wt % to 25 wt %, such as 12 wt % to 20 wt % of the expected total amount of recombinant protein produced.

[0182] In another embodiment of the method, tryptophan is supplemented in the cell culture medium in a total amount of 8 wt % to 30 wt % of the expected total amount of recombinant protein produced, such as 8 wt % to 25 wt %, for example 8 wt % to 20 wt % of the expected total amount of recombinant protein produced.

[0183] In another embodiment of the method, the total amount of cysteine ​​or cystine and / or tryptophan in the cell culture medium is achieved by adding cysteine ​​or cystine and / or tryptophan to the cell culture medium:

[0184] a. At the beginning of the production phase,

[0185] b. once or more times at any point in the production phase,

[0186] c. continuously added during the production phase, or

[0187] d. Any combination of a, b and c.

[0188] In another embodiment of the method, the method is a batch method, such as a fed-batch method.In another embodiment of the method, cysteine ​​or cystine and / or tryptophan are provided by daily addition during the production phase.

[0189] In another embodiment of the method, before adding cysteine ​​or cystine on the second day, for example by reducing the amount of cysteine ​​or cystine added to 5.6 to 7 g / [10 12 per cell] to deplete cysteine ​​or cystine in the cell culture medium.

[0190] In another embodiment of the method, tryptophan is depleted from the cell culture medium during late production, ie, when the cells have reached a maximum viable cell density, prior to adding tryptophan the next day.

[0191] In another embodiment of the method, the cysteine ​​or cystine concentration in the cell culture does not exceed 0.9 g / L at any time point in the production phase, preferably wherein the cysteine ​​or cystine concentration in the cell culture does not exceed 0.3 g / L at any time point in the production phase.

[0192] In another embodiment of the method, the tryptophan concentration in the cell culture does not exceed 0.6 g / L at any time point during the production phase, preferably wherein the tryptophan concentration in the cell culture does not exceed 0.3 g / L at any time point during the production phase.

[0193] In another embodiment of the process, the production phase is carried out for at least 7 days, preferably at least 14 days.

[0194] In one embodiment of the method, at any point in time during the second half of the production phase:

[0195] a. the amount of cysteine ​​or cystine in the cell culture medium is 10 wt % to 30 % of the expected amount of recombinant protein produced; and / or

[0196] b. The amount of tryptophan in the cell culture medium is 8 wt % to 35 % of the expected amount of recombinant protein produced.

[0197] In another embodiment of the method, at any point during the production phase:

[0198] a. the amount of cysteine ​​or cystine is 10 wt % to 30 % of the expected amount of recombinant protein produced; and / or

[0199] b. The amount of tryptophan is 8 wt% to 35% of the expected amount of recombinant protein produced.

[0200] In another embodiment of the method, the host cell is a mammalian cell, preferably a CHO cell.

[0201] In another embodiment of this method, the recombinant protein is an antibody or an antigen-binding fragment thereof.

[0202] In another embodiment of the method, the production phase is carried out in a bioreactor, preferably having a volume of 50 L or more, 100 L or more, 500 L or more, 1000 L or more, 2,000 L or more, 5,000 L or more, 10,000 L or more, or 20,000 L or more.

[0203] In one embodiment of the method, the method comprises the step of recovering the recombinant protein from the cell culture medium and the further step of purifying the recombinant protein.

[0204] In another embodiment of the method, the purification comprises Protein A chromatography.

[0205] In another embodiment of the method, the method further comprises the step of formulating the purified recombinant protein.

[0206] In one embodiment of the method, the recombinant protein is formulated in a liquid formulation comprising one or more amino acids and a surfactant.

[0207] In yet another embodiment of the method, the formulation comprises histidine and / or proline.

[0208] In another embodiment of the method, the formulation comprises histidine at a concentration of 5 mM to 100 mM, for example at a concentration of 10 mM to 50 mM, and / or proline at a concentration of 100 mM to 500 mM at a pH of 5 to 7.4 (such as 5 to 6.5, for example 5 to 6, such as 5.5 to 6).

[0209] In yet another embodiment of the method, the formulation comprises histidine at a concentration of 30 mM and proline at a concentration of 250 mM at a pH of 5.2 to 6.0, such as about 5.6.

[0210] In another embodiment of the method, the surfactant is polysorbate 80, preferably at a concentration of 0.001% to 0.1% (w / v), for example 0.005% to 0.1%, such as 0.01% to 0.1%, for example 0.01% to 0.05%, such as 0.03%.

[0211] In another embodiment of the method, the recombinant protein is an antibody and the antibody is formulated at a concentration of 10 mg / ml to 250 mg / ml, for example 20 mg / ml to 250 mg / ml, such as 50 mg / ml to 250 mg / ml, for example 120 mg / ml to 160 mg / ml, such as about 140 mg / ml.

[0212] In another embodiment of the method, the method reduces the heterogeneity of the produced recombinant protein, wherein the reduction of heterogeneity comprises reducing:

[0213] a. Charge heterogeneity, preferably acidic peak group (APG); and / or

[0214] b. amino acid oxidation, isomerization, fragmentation, glycosylation, deamidation, cysteinylation of other covalent adducts; and / or

[0215] c. Color or color intensity, for example between different batches of recombinant protein; and / or

[0216] d. high molecular weight species (HMWS); and / or

[0217] e. Recombinant protein instability.

[0218] In another independent aspect, the present invention relates to a method for reducing the heterogeneity of a population of recombinant proteins in a batch produced by a recombinant host cell during a production phase, the method comprising limiting the total amount of the following substances present in the cell culture medium during the production phase of the recombinant protein:

[0219] a. Cysteine ​​or cystine and / or

[0220] b.Tryptophan.

[0221] In one embodiment, the method comprises:

[0222] a. culturing host cells capable of producing recombinant proteins in a culture medium;

[0223] b. allowing the culture to proceed through a production phase, wherein the recombinant protein is produced by the cells, wherein during the production phase, the culture is supplemented with

[0224] ● Cysteine ​​or cystine in a total amount of 10 wt% to 30 wt% of the expected total amount of recombinant protein produced; and / or

[0225] tryptophan in a total amount of 8 wt % to 35 wt % of the expected total amount of recombinant protein produced,

[0226] c. and optionally, recovering the recombinant protein from the cell culture medium.

[0227] In one embodiment of the method, cysteine ​​or cystine is supplemented in the culture in a total amount of 12 wt % to 28 wt % of the expected total amount of recombinant protein produced, such as 12 wt % to 25 wt %, for example 12 wt % to 20 wt % of the expected total amount of recombinant protein produced.

[0228] In another embodiment of the method, tryptophan is supplemented in the culture in a total amount of 8 to 30 wt % of the expected total amount of recombinant protein produced, such as 8 to 25 wt %, for example 8 to 20 wt % of the expected total amount of recombinant protein produced.

[0229] In another embodiment of the method, the total amount of cysteine ​​or cystine provided in the method is 2.9 to 12 g / (10 12 cells), such as 2.9 to 7 g / (10 12 cells), for example 5.6 to 7 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase.

[0230] In another embodiment of the method, the total amount of tryptophan provided in the method is 2.5 to 7 g / (10 12 cells), such as 2.5 to 3.5 g / (10 12 cells / L), where cells refers to the expected intact viable cell count at the end of the production phase.

[0231] In another embodiment of the method, the total amount of cysteine ​​or cystine and / or tryptophan in the cell culture medium is achieved by adding cysteine ​​or cystine and / or tryptophan to the cell culture medium:

[0232] a. At the beginning of the production phase,

[0233] b. once or more times at any point in the production phase,

[0234] c. by continuous addition during the production phase, or

[0235] d. Any combination of a, b and c.

[0236] In another embodiment of the method, the method is a batch method, such as a fed-batch method.

[0237] In another embodiment of the method, cysteine ​​or cystine and / or tryptophan are provided by daily addition during the production phase.

[0238] In another embodiment of the method, before adding cysteine ​​or cystine on the second day, for example by reducing the amount of cysteine ​​or cystine added to 5.6 to 7 g / [10 12 cells] to deplete cysteine ​​or cystine in culture.

[0239] In another embodiment of the method, tryptophan is depleted from the cell culture medium during late production, ie, when the cells have reached a maximum viable cell density, prior to tryptophan addition the next day.

[0240] In another embodiment of the method, the cysteine ​​or cystine concentration in the cell culture medium does not exceed 0.9 g / L at any time point during the production phase, preferably wherein the cysteine ​​or cystine concentration in the cell culture medium does not exceed 0.3 g / L at any time point during the production phase.

[0241] In another embodiment of the method, the tryptophan concentration in the cell culture medium does not exceed 0.6 g / L at any time point during the production phase, preferably wherein the tryptophan concentration in the cell culture medium does not exceed 0.3 g / L at any time point during the production phase.

[0242] In another embodiment of the process, the production phase is carried out for at least 7 days, preferably at least 14 days.

[0243] In another embodiment of the method, at any point during the second half of the production phase:

[0244] The amount of cysteine ​​or cystine in the culture is between 10 wt% and 30% of the expected amount of recombinant protein produced; and / or

[0245] • The amount of tryptophan in the culture is between 8 wt% and 35% of the expected amount of recombinant protein produced.

[0246] In another embodiment of the method, at any point during the production phase:

[0247] The amount of cysteine ​​or cystine in the culture is between 10 wt% and 30% of the expected amount of recombinant protein produced; and / or

[0248] • The amount of tryptophan in the culture is between 8 wt% and 35% of the expected amount of recombinant protein produced.

[0249] In another embodiment of the method, the host cell is a mammalian cell, preferably a CHO cell.

[0250] In another embodiment of this method, the recombinant protein is an antibody or an antigen-binding fragment thereof.

[0251] In another embodiment of the method, the production phase is carried out in a bioreactor, preferably having a volume equal to or greater than 50 L, equal to or greater than 100 L, equal to or greater than 500 L, equal to or greater than 1000 L, equal to or greater than 2,000 L, equal to or greater than 5,000 L, equal to or greater than 10,000 L or equal to or greater than 20,000 L.

[0252] In one embodiment of the method, the method comprises the step of recovering the recombinant protein from the cell culture medium and the further step of purifying the recombinant protein.

[0253] In another embodiment of the method, the purification comprises Protein A chromatography.

[0254] In another embodiment of the method, the method further comprises the step of formulating the purified recombinant protein.

[0255] In one embodiment of the method, the recombinant protein is formulated in a liquid formulation comprising one or more amino acids and a surfactant.

[0256] In another embodiment of the method, the formulation comprises histidine and / or proline.

[0257] In another embodiment of the method, the formulation comprises histidine at a concentration of 5 mM to 100 mM, for example at a concentration of 10 mM to 50 mM and / or proline at a concentration of 100 mM to 500 mM at a pH of 5 to 7.4, such as 5 to 6.5, for example 5 to 6, such as 5.5 to 6.

[0258] In yet another embodiment of the method, the formulation comprises histidine at a concentration of 30 mM and proline at a concentration of 250 mM at a pH of 5.2 to 6.0, such as about 5.6.

[0259] In another embodiment of the method, the surfactant is polysorbate 80, preferably at a concentration of 0.001% to 0.1% (w / v), for example 0.005% to 0.1%, such as 0.01% to 0.1%, for example 0.01% to 0.05%, such as 0.03%.

[0260] In another embodiment of the method, the recombinant protein is an antibody and the antibody is formulated at a concentration of 10 mg / ml to 250 mg / ml, e.g. 20 mg / ml to 250 mg / ml, such as 50 mg / ml to 250 mg / ml, e.g. 120 mg / ml to 160 mg / ml, such as about 140 mg / ml.

[0261] In another embodiment of the method, the method reduces the heterogeneity of the produced recombinant protein, wherein the reduction of heterogeneity comprises reducing:

[0262] a. Charge heterogeneity, preferably acidic peak group (APG); and / or

[0263] b. amino acid oxidation, isomerization, fragmentation, glycosylation, deamidation, cysteinylation of other covalent adducts; and / or

[0264] c. Color or color intensity, for example between different batches of recombinant protein; and / or

[0265] d. high molecular weight species (HMWS); and / or

[0266] e. Recombinant protein instability.

[0267] In yet another aspect, the present invention relates to a recombinant protein preparation that can be obtained or obtained by the method of the present invention. In one embodiment, the preparation is a bulk preparation. In other embodiments, for example, when the method includes other steps of preparing a protein product, the preparation obtained is a prepared protein preparation, for example, a preparation suitable for use in a patient.

[0268] The recombinant protein (preferably an antibody or antigen-binding fragment thereof) in the preparation obtained herein exhibits reduced heterogeneity relative to the same recombinant protein obtained by the same method, but wherein the total amount of cysteine ​​or cystine and / or tryptophan in the production stage is not limited as described in the present invention.

[0269] In yet another aspect, the invention relates to a pharmaceutical composition comprising an antibody, wherein the composition is a liquid formulation comprising one or more amino acids and a surfactant.

[0270] In one embodiment, the pharmaceutical composition comprises histidine and / or proline.

[0271] In yet another embodiment, the pharmaceutical composition comprises histidine and / or proline.

[0272] In yet another embodiment, the pharmaceutical composition comprises histidine at a concentration of 5 mM to 100 mM, for example at a concentration of 10 mM to 50 mM and / or proline at a concentration of 100 mM to 500 mM at a pH of 5 to 7.4, such as 5 to 6.5, for example 5 to 6, such as 5.5 to 6.

[0273] In yet another embodiment, the pharmaceutical composition comprises histidine at a concentration of 30 mM and proline at a concentration of 250 mM at a pH of 5.2 to 6.0, such as about 5.6.

[0274] In another embodiment of the pharmaceutical composition, the surfactant is polysorbate 80, preferably at a concentration of 0.001% to 0.1% (w / v), for example 0.005% to 0.1%, such as 0.01% to 0.1%, for example 0.01% to 0.05%, such as 0.03%.

[0275] In further embodiments of the pharmaceutical composition, the antibody is formulated at a concentration of 10 to 250 mg / ml, e.g. 20 to 250 mg / ml, such as 50 to 250 mg / ml, e.g. 120 to 160 mg / ml, such as about 140 mg / ml.

[0276] In another embodiment of the pharmaceutical composition, the antibody is

[0277] 1) an antibody or an antigen-binding fragment thereof,

[0278] a. comprising a CDR-H1 having a sequence as defined in SEQ ID NO: 1; a CDR-H2 having a sequence as defined in SEQ ID NO: 2; a CDR-H3 having a sequence as defined in SEQ ID NO: 3; a CDR-L1 having a sequence as defined in SEQ ID NO: 4; a CDR-L2 having a sequence as defined in SEQ ID NO: 5 and a CDR-L3 having a sequence as defined in SEQ ID NO: 6; or

[0279] b. comprising a light variable region having a sequence as defined in SEQ ID NO: 7 and a heavy variable region having a sequence as defined in SEQ ID NO: 8; or

[0280] c. comprising a light variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7 and a heavy variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

[0281] d. comprising a light variable region having a sequence as defined in SEQ ID NO: 7 and a heavy chain having a sequence as defined in SEQ ID NO: 11; or

[0282] e. comprising a light variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7 and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 11; or

[0283] 2) an antibody comprising a light chain having a sequence as defined in SEQ ID NO: 9 and a heavy chain having a sequence as defined in SEQ ID NO: 10; or

[0284] 3) An antibody comprising a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO:9 and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO:10.

[0285] Host cells and culture conditions

[0286] Recombinant proteins, antibodies or antigen-binding fragments thereof may preferably be produced by culturing mammalian host cells, most preferably Chinese Hamster Ovary (CHO) cells.

[0287] The term "cell culture" or grammatical variations thereof includes, but is not limited to, a plurality of host cells, preferably mammalian host cells, maintained or grown in a cell culture medium for a specific period of time (e.g., a production phase) that are appropriately engineered and / or manipulated to express (i.e., produce) one or more recombinant proteins.

[0288] Mammalian cells, particularly CHO cells, can be cultured in any medium that supports their growth and recombinant protein expression, preferably a medium that does not contain animal-derived products (such as animal serum and peptone). Different cell culture media can be used by those skilled in the art, each of which contains different combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose or equivalent energy sources, which are present in appropriate concentrations to allow cell growth and protein production. Suitable culture media have been described, for example, in WO98 / 08934 and US2006 / 0148074 (both of which are incorporated herein in their entirety). Other suitable commercially available culture media that can be used in the present invention or modified to meet the requirements of cysteine / cystine and / or tryptophan include AmpliCHO CD medium, Dynamis TM Culture medium, Advanced TM CHO Fed-Batch System, CD FortiCHO TM Culture medium, CP OptiCHO TM Culture medium, minimum essential medium (MEM), CHOGrowth A medium, ActiPro TM culture medium, DMEM–Dulbecco's Modified Eagle's Medium, and RPMI-1640 medium.

[0289] Cell culture can be carried out in any suitable container (such as shake flask or bioreactor), which may or may not be operated in fed-batch mode, depending on the desired production scale. These bioreactors can be stirred tank reactors or gas stripping reactors. Various large bioreactors with a capacity greater than 1,000L to 50,000L, preferably between 5,000L to 20,000L or to 10,000L can be used. Alternatively, smaller-scale bioreactors (such as 2L to 100L) can also be used to produce antibodies or antibody fragments.

[0290] In a preferred embodiment of the invention, the production phase is carried out in a bioreactor or any other suspension culture container (such as a shake flask or a rotary bottle), no matter where any previous stage (i.e., the amplification phase) is carried out. The production phase is preferably operated in a fed-batch mode, but any other mode may also be used, such as batch, perfusion or chemical constant mode. In the case of perfusion or chemical constant, the ratio of the total amount of cysteine ​​or cystine and / tryptophan used is calculated according to the perfusion flow rate compared to the removal rate of the recombinant protein produced from the production container.

[0291] In one embodiment, the method comprises the step of recovering the recombinant protein from the cell culture medium and the further step of purifying the recombinant protein.

[0292] In yet another embodiment, purification comprises Protein A chromatography.

[0293] In yet another embodiment, the method comprises the further step of formulating the purified recombinant protein.

[0294] In one embodiment, the recombinant protein is formulated in a liquid formulation comprising one or more amino acids and a surfactant.

[0295] In yet another embodiment, the formulation comprises histidine and / or proline.

[0296] In yet another embodiment, the formulation comprises histidine at a concentration of 5 mM to 100 mM, such as at a concentration of 10 mM to 50 mM, and / or proline at a concentration of 100 mM to 500 mM at a pH of 5 to 7.4, such as 5 to 6.5, for example 5 to 6, such as 5.5 to 6.

[0297] In yet another embodiment, the formulation comprises histidine at a concentration of 30 mM and proline at a concentration of 250 mM at a pH of 5.2 to 6.0, such as about 5.6.

[0298] In yet another embodiment, the surfactant is polysorbate 80, preferably at a concentration of 0.001% to 0.1% (w / v), for example 0.005% to 0.1%, such as 0.01% to 0.1%, for example 0.01% to 0.05%, such as 0.03%.

[0299] In yet another embodiment, the recombinant protein is an antibody and the antibody is formulated at a concentration of 10 to 250 mg / ml, e.g. 20 to 250 mg / ml, such as 50 to 250 mg / ml, e.g. 120 to 160 mg / ml, such as about 140 mg / ml.

[0300] Recombinant proteins, such as antibodies or their antigen-binding fragments are typically present in the supernatant of mammalian cell cultures (typically CHO cell cultures). For CHO host cells, antibodies or their antigen-binding fragments are secreted in the supernatant, and the supernatant can be collected by methods known in the art, typically by centrifugation.

[0301] Recombinant proteins produced using the methods of the present invention can be

[0302] The method of the present invention can be used to produce any type of recombinant polypeptide or protein, including for example peptides or larger proteins with significant tertiary structure and for example glycoproteins and multimeric proteins. However, preferably, the recombinant protein produced in the method according to the present invention is an antibody or its antigen-binding fragment. As used herein, the term "antibody" or "antibodies" includes for example monoclonal antibodies and polyclonal antibodies and monospecific and multispecific (such as bispecific) antibodies.

[0303] "Antibodies" include antibodies of any species, particularly antibodies of mammalian species (which generally have two heavy chains and two light chains), human antibodies of any isotype (including IgA1, IgA2, IgD, IgG1, IgG 2a IgG 2b, IgG3, IgG4IgE and IgM and modified variants thereof), non-human primate antibodies (e.g., from chimpanzees, baboons, rhesus monkeys or cynomolgus monkeys), rodent antibodies (e.g., from mice, rats or rabbits); goat or horse antibodies and derivatives thereof, or antibodies of bird species (such as chicken antibodies) or antibodies of fish species (such as shark antibodies). The term "antibody" also refers to a "chimeric" antibody in which the first portion of at least one heavy and / or light chain antibody sequence is from a first species and the second portion of the heavy and / or light chain is from a second species. The chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, such as baboons, rhesus monkeys or cynomolgus monkeys) and human constant region sequences. A "humanized" antibody is a chimeric antibody comprising sequences derived from a non-human antibody. In most cases, humanized antibodies are human antibodies (recipient antibodies) in which the residues of the hypervariable region of the recipient are replaced by residues of the hypervariable region or complementary determining region (CDR) of a non-human species (donor antibody) (such as mouse, rat, rabbit, chicken or non-human primate), and the hypervariable region or complementary determining region of the non-human species has the required specificity, affinity and activity. In most cases, the residues of the human (recipient) antibody outside the CDR, i.e., the residues in the framework region (FR), are additionally replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Humanization reduces the immunogenicity of non-human antibodies in humans, thereby facilitating the application of antibodies in the treatment of human diseases. Humanized antibodies and several different techniques for producing them are well known in the art. "Antibodies" also refer to human antibodies, which can be produced instead of humanization. For example, it is possible to produce transgenic animals (e.g., mice) that can produce a full set of human antibodies without endogenous mouse antibody production when immunized. For example, it has been described that the homozygous deletion of the antibody heavy chain hinge region (JH) gene in chimeric and germline mutant mice leads to the complete inhibition of endogenous antibody production. In this germline mutant mouse, the transfer of human germline immunoglobulin gene arrays will result in the production of human antibodies specific to the antigen when the transgenic animals carrying human germline immunoglobulin genes are immunized with specific antigens. The technology for producing such transgenic animals and the technology for separating and producing human antibodies from such transgenic animals are known in the art. Alternatively, in transgenic animals (such as mice), only the immunoglobulin genes encoding the variable regions of mouse antibodies are replaced by the corresponding human variable immunoglobulin gene sequences. The mouse germline immunoglobulin genes encoding the constant regions of antibodies remain unchanged. In this way, the antibody effector works in the immune system of transgenic mice, so the development of B cells is basically unchanged, which may lead to improved antibody response during antigen attack in vivo. Once the gene encoding a specific target antibody is isolated from such transgenic animals, the gene encoding the constant region can be replaced with the human constant region gene to obtain a fully human antibody.As used herein, the term "antibody" also refers to an aglycosylated antibody.

[0304] As used herein, the term "antigen-binding fragment thereof" or grammatical variations thereof refers to an antibody fragment. A fragment of an antibody comprises at least one heavy or light chain immunoglobulin domain known in the art and binds to one or more antigens. Examples of antibody fragments according to the invention include Fab, Fab', F(ab')2, and Fv and scFv fragments; as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies (dAbs), such as sdAbs, VHHs, or camelid antibodies (e.g., from camels or llamas, such as Nanobodies TM ) and VNAR fragments, single-chain antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies or multispecific antibodies formed by antibody fragments or antibodies, including but not limited to Fab-Fv or Fab-Fv-Fv constructs. Antibody fragments as defined above are known in the art.

[0305] In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof produced by the method according to the invention is (Table 1):

[0306] 1) An antibody or antigen-binding fragment thereof,

[0307] a. comprising a CDR-H1 having a sequence as defined in SEQ ID NO: 1; a CDR-H2 having a sequence as defined in SEQ ID NO: 2; a CDR-H3 having a sequence as defined in SEQ ID NO: 3; a CDR-L1 having a sequence as defined in SEQ ID NO: 4; a CDR-L2 having a sequence as defined in SEQ ID NO: 5 and a CDR-L3 having a sequence as defined in SEQ ID NO: 6; or

[0308] b. comprising a light variable region having a sequence as defined in SEQ ID NO: 7 and a heavy variable region having a sequence as defined in SEQ ID NO: 8; or

[0309] c. comprising a light variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7 and a heavy variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

[0310] d. comprising a light variable region having a sequence as defined in SEQ ID NO: 7 and a heavy chain having a sequence as defined in SEQ ID NO: 11; or

[0311] e. comprising a light variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7 and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 11; or

[0312] 2) an antibody comprising a light chain having a sequence as defined in SEQ ID NO: 9 and a heavy chain having a sequence as defined in SEQ ID NO: 10; or

[0313] 3) An antibody comprising a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO:9 and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO:10.

[0314] Throughout the specification, complementarity determining regions ("CDRs") are defined according to the Kabat definition. The Kabat definition is a standard for numbering residues in antibodies and is often used to identify CDR regions (Kabat et al., (1991), 5th edition, NIH publication number 91-3242).

[0315] Table 1

[0316]

[0317]

[0318]

[0319] Recombinant proteins or preferably antibodies or antigen-binding fragments thereof can generally be produced by host cells containing a vector containing a nucleotide sequence encoding the protein or antibody.

[0320] The antibody or antigen-binding fragment thereof may contain only heavy chain or light chain proteins, in which case only the heavy chain or light chain protein coding sequence is used to transfect the cell. In order to produce a product containing both heavy and light chains, two vectors may be used to transfect the cell, the first vector encoding the light chain protein and the second vector encoding the heavy chain protein. Alternatively, a single vector may be used that includes sequences encoding both light and heavy chain proteins.

[0321] In a preferred embodiment, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, the method comprising:

[0322] a. Culturing CHO cells capable of producing antibodies or antigen-binding fragments thereof in a culture medium;

[0323] b. allowing the culture to proceed through a production phase, wherein the antibody or antigen-binding fragment thereof is produced by the cells, wherein during the production phase, cysteine ​​or cystine is supplemented in the culture to a total amount of 10 wt % to 30 wt % of the expected total amount of the antibody or antibody-binding fragment thereof produced

[0324] c. and optionally, recovering the antibody or antigen-binding fragment thereof from the cell culture medium,

[0325] The total amount of cysteine ​​or cystine provided in the method is 2.9 to 12 g / (10 12 cells), such as 2.9 to 7 g / (10 12 cells), for example 5.6 to 7 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase, and

[0326] wherein cysteine ​​or cystine is provided by daily addition during the production phase, and

[0327] wherein the concentration of cysteine ​​or cystine in the cell culture medium does not exceed 0.9 g / L at any time point during the production phase, preferably wherein the concentration of cysteine ​​or cystine in the cell culture medium does not exceed 0.3 g / L at any time point during the production phase, and

[0328] The antibody or antigen-binding fragment thereof is preferably:

[0329] 1) comprising a CDR-H1 having a sequence as defined in SEQ ID NO: 1; a CDR-H2 having a sequence as defined in SEQ ID NO: 2; a CDR-H3 having a sequence as defined in SEQ ID NO: 3; a CDR-L1 having a sequence as defined in SEQ ID NO: 4; a CDR-L2 having a sequence as defined in SEQ ID NO: 5 and a CDR-L3 having a sequence as defined in SEQ ID NO: 6; or

[0330] 2) comprising a light variable region having a sequence as defined in SEQ ID NO:7 and a heavy variable region having a sequence as defined in SEQ ID NO:8.

[0331] In another preferred embodiment, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, the method comprising:

[0332] a. Culturing CHO cells capable of producing antibodies or antigen-binding fragments thereof in a culture medium;

[0333] b. allowing the culture to proceed through a production phase, wherein the antibody or antigen-binding fragment thereof is produced by the cells, wherein during the production phase, the culture medium is supplemented with:

[0334] ● Cysteine ​​or cystine in a total amount of 10 wt% to 30 wt% of the expected total amount of antibody or antigen-binding fragment thereof produced; and / or

[0335] tryptophan in a total amount of 8 wt % to 35 wt % of the expected total amount of antibody or antigen-binding fragment thereof produced,

[0336] c. and optionally, recovering the antibody or antigen-binding fragment thereof from the cell culture medium,

[0337] The total amount of cysteine ​​or cystine provided in the method is 2.9 to 12 g / (10 12 cells), such as 2.9 to 7 g / (10 12 cells), for example 5.6 to 7 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase, and

[0338] The total amount of tryptophan provided in the method is 2.5 to 7 g / (10 12 cells), such as 2.5 to 3.5 g / (10 12 cells), where cells refers to the expected complete viable cell count at the end of the production phase, and

[0339] wherein cysteine ​​or cystine and / or tryptophan are provided by daily addition during the production phase, and

[0340] wherein the concentration of cysteine ​​or cystine in the cell culture medium does not exceed 0.9 g / L at any time point during the production phase, preferably wherein the concentration of cysteine ​​or cystine in the cell culture medium does not exceed 0.3 g / L at any time point during the production phase, and

[0341] wherein the tryptophan concentration in the cell culture medium does not exceed 0.6 g / L at any time point during the production phase, preferably wherein the tryptophan concentration in the cell culture medium does not exceed 0.3 g / L at any time point during the production phase, and

[0342] The antibody or antigen-binding fragment thereof is preferably:

[0343] 1) comprising a CDR-H1 having a sequence as defined in SEQ ID NO: 1; a CDR-H2 having a sequence as defined in SEQ ID NO: 2; a CDR-H3 having a sequence as defined in SEQ ID NO: 3; a CDR-L1 having a sequence as defined in SEQ ID NO: 4; a CDR-L2 having a sequence as defined in SEQ ID NO: 5 and a CDR-L3 having a sequence as defined in SEQ ID NO: 6; or

[0344] 2) comprising a light variable region having a sequence as defined in SEQ ID NO:7 and a heavy variable region having a sequence as defined in SEQ ID NO:8.

[0345] The invention will now be further described by way of example with reference to the embodiments shown in the accompanying drawings. Example

[0346] abbreviation

[0347] mAb: monoclonal antibody; MFCS: multi-fermentation control system; Cys: cysteine ​​or cystine; Trp: tryptophan

[0348] Materials and methods

[0349] Cell lines, cell culture, and experimental procedures

[0350] The CHO-DG44 cell line was used. Cells were cultured in a chemically defined animal component-free inoculum medium containing cystine (0.05 g / L) and tryptophan (0.2 g / L) in a 2 L stirred tank glass bioreactor under standard operating conditions (pH 7, temperature 36.8 ° C), and the bioreactor had a supply tower (C-DCUII, Sartorius Stedim Biotech) controlled by a multi-fermentation control system (Sartorius Stedim Biotech). Four different production cell lines were used, each of which produced monoclonal antibodies (mAbs), referred to as mAb1, mAb2, mAb3 and mAb4. mAb1 is an anti-FcRn antibody comprising a light chain having a sequence as defined in SEQ ID NO:9 and a heavy chain having a sequence as defined in SEQ ID NO:10.

[0351] The production was run for 14 days in a fed-batch experimental mode. At this stage, the monoclonal antibody was secreted into the culture medium. Samples were taken every day to determine viable cell density (VCD), vigor, off-line pH, pCO , weight-gram molecular osmotic pressure concentration, glucose-lactate concentration, amino acid concentration and mAb concentration (stored at -80°C). Defoamers were added manually as needed every day to control the formation of foam. After inoculation for 72 hours, continuous nutrient feeding was started at a predetermined rate. The continuous nutrient feed culture medium did not contain cysteine / cystine or tryptophan. At this point, cysteine / cystine and tryptophan were added every day as a batch feed (bolus feed) with the amount described in the following examples within 10 days. The amount of cysteine / cystine and tryptophan described in the example is the total amount of large doses added starting 72 hours after inoculation considering the initial amount of these amino acids already present in the inoculation culture medium. When the glucose concentration dropped to below 6g / L (from the 6th day), the glucose large dose feed was added to the culture, and the glucose concentration was measured every day. The sample for amino acid analysis was collected before the feed was added. Post-feed concentrations were calculated based on feed composition and nutrient concentrations measured before feed addition.

[0352] Analytical methods

[0353] By using Cells were counted using a Beckman-Coulter XR (Beckman-Coulter, Inc., Brea, CA) automated cell counter based on the trypan blue exclusion method.

[0354] Glucose and lactate levels in the culture medium were determined using a NOVA 400 BioProfile automated analyzer (Nova Biomedical, Waltham, MA).

[0355] Osmotic pressure measurements were performed using a 2020 Freezing Point Osmometer (Advanced Instruments, Inc., Norwood, MA). Offline gas and pH measurements were performed using a blood gas analyzer (Nova Biomedical Corporation, Waltham, MA).

[0356] Metabolite concentrations were determined daily using the CedexBioHT system (Roche).

[0357] Product titer analysis was performed using a ForteBio Octet Model Analyzer (ForteBio, Inc., Menlo Park, CA) or Protein A high pressure liquid chromatography (HPLC) and cell culture supernatant samples stored at -80°C prior to analysis.

[0358] After ultrafiltration using an Amicon Ultra-0.5 mL centrifugal filter (Merck Millipore, Billerica, MA), amino acids were analyzed by reverse phase UPLC (Waters AccQ·Tagultra method).

[0359] Protein A purification ( System) was used to purify mAb in cell culture supernatant samples. The relative percentages of the major acidic (APG for acidic peak groups) and basic (BPG for basic peak groups) isoforms of the purified mAb were determined by imaging capillary electrophoresis (ProteinSimple iCE3). The levels of high molecular weight species (HMWS), monomers, and low molecular weight species (LMWS) of the purified mAb were determined by size exclusion chromatography (SE-UPLC).

[0360] The color intensity of concentrated mAbl and mAb2 preparations was measured in concentrated Protein A eluate using a transmission spectrophotometer (UltrascanPro) and compared to the Commission Internationale de L'éclairage (CIL) scale. Numerical results were normalized to a concentration of 40 mg / mL.

[0361] Electrospray ionization mass spectrometry (ESI-MS) was performed on the purified mAb1. Peptide mapping was performed to identify post-translational modifications of the antibody. Statistical analysis was performed using SAS software JMP 11.

[0362] Example 1

[0363] CHO cells producing mAb1 were cultured at 0.35×10 6 The 2-liter bioreactor was inoculated with an inoculation density of 10 cells / mL. As described in the Materials and Methods section, eight experimental conditions were tested in a fed-batch method with various maximum concentrations of cysteine ​​or cystine and tryptophan reached throughout the cell culture process and various total amounts of cysteine ​​or cystine (Cys) and tryptophan (Trp) accounting for the wt% of the total mAb1 weight produced (Table 2a). The first goal was to evaluate the impact of cysteine ​​or cystine and tryptophan on the heterogeneity of recombinant mAb1. The second goal was to determine whether the impact was due to the high concentration of cysteine ​​or cystine and / or tryptophan reached throughout the cell culture process and / or whether the total amount added accounted for the wt% of the total weight of the mAb produced.

[0364] Table 2a

[0365]

[0366] Recombinant protein charge variants and color intensity were measured as described in the Materials and Methods section. Data were analyzed by one-way Anova statistical analysis for linear fit, and p values ​​< 0.05 were considered acceptable.

[0367] like Figure 2 As shown in b, there is a correlation between the increase in acidic peak group charge variants (APG%) in mAb1 and the increase in the total amount of cysteine ​​or cystine added / total mAb1 weight % (g / g) produced.

[0368] Regarding the color intensity of mAb1, there was a correlation between the increased color intensity of mAb1 (b* values ​​normalized to 40 mg / mL) and the increase in the total amount of tryptophan added / total mAb1 wt% (g / g) produced ( Figure 2 a).

[0369] However, when the data were analyzed for the maximum concentration of tryptophan or cysteine ​​or cystine, there was no effect on color or APG ( Figure 2 c and Figure 2 d).

[0370] To confirm that it was the total amount of cysteine ​​or cystine and tryptophan / total mAb1 weight produced that affected the heterogeneity of mAb1, rather than the maximum concentration of cysteine ​​or cystine and / or tryptophan reached during the entire production phase in the fed-batch case, 8 experimental conditions were tested with various large doses of cysteine ​​or cystine and tryptophan added on day 3 to reach high concentrations of both amino acids (Table 2b). The feeding strategy was adjusted to have the same amount of added cysteine ​​or cystine and tryptophan as a percentage of the total mAb1 weight produced (wt% (g / g)). As shown in the fed-batch conditions, there was a significant difference between the increased acidic peak group charge variants (APG%) in mAb1 and the increase in the total amount of cysteine ​​or cystine added / total mAb1 wt% (g / g) produced ( Figure 3 b) and the relationship between the increasing mAbl color intensity (b* value normalized to 40 mg / mL) and the increasing total amount of tryptophan added / total mAbl wt% (g / g) produced ( Figure 3 However, there was no correlation with APG charge variants and the maximum concentrations (g / L) of cysteine ​​or cystine and tryptophan (respectively Figure 3 c and Figure 3 d). These results demonstrate that the total amount of cysteine ​​and tryptophan added during cell culture / wt% of total mAbl weight produced (g / g) affects APG charge variants and color intensity. The maximum concentrations of cysteine ​​or cystine and tryptophan do not affect APG charge variants and color intensity.

[0371] Table 2b

[0372]

[0373] in conclusion

[0374] The total addition of Cys and Trp as wt% (g / g) of the total recombinant mAb1 produced had an impact on mAb1 charge variants and color intensity. In contrast, the maximum concentration of cysteine ​​or cystine and tryptophan in the cell culture medium did not affect mAb1 quality.

[0375] Example 2

[0376] To further investigate the effect of cysteine ​​or the total amount of cystine and tryptophan added during cell culture on the wt% (g / g) of total mAb produced, 48 experimental conditions in a 2L bioreactor run were prepared as described in the Methods section (Table 3). mAb1 charge variants, aggregates (HMWS), color intensity, titer, and viable cell growth were analyzed.

[0377] Table 3

[0378]

[0379]

[0380]

[0381] like Figure 4 As shown, the total amount of cysteine ​​or cystine and tryptophan added throughout the production phase as a percentage of the wt% (g / g) of the total mAb1 produced affects the acidic charge variant group (APG%). There is a saturation effect of about 50 wt% for the total amount of cysteine ​​or cystine added throughout the 14-day production phase as a percentage of the weight% (g / g) of the total mAb1 produced. The effects of cysteine ​​or cystine and tryptophan are cumulative and there is no interaction. Reducing the total amount of cysteine ​​or cystine and tryptophan added throughout the 14-day production phase as a percentage of the wt% (g / g) of the total mAb1 produced will reduce the percentage of acidic peaks on the mAb1 produced.

[0382] Figure 5 The effect of the total amount of cysteine ​​or cystine and tryptophan (added throughout the 14 days of cell culture) as wt% of the total mAbl weight produced on the main peak group is shown. As seen for APG%, the effects of cysteine ​​or cystine and tryptophan are cumulative and do not interact. Reducing the total amount of cysteine ​​or cystine and tryptophan added as wt% of the total mAbl weight produced throughout the 14 day production phase increases the percentage of the main peak on the recombinant mAb produced.

[0383] Figure 6 The effect of the total amount of cysteine ​​or cystine added over the entire 14-day production phase as a percentage of the wt% (g / g) of the total mAbl produced on high molecular weight species (HMWS) is shown. There was a saturation effect of approximately 50% for the total amount of cysteine ​​or cystine added over the entire 14-day production phase as a percentage of the wt% (g / g) of the total mAbl produced. Reducing the total amount of cysteine ​​or cystine decreased the percentage of HMWS on the recombinant mAb produced. No effect of the total amount of Trp added on HMWS was observed.

[0384] Figure 7 The results shown in illustrate the effect of the total amount of cysteine ​​or cystine and tryptophan added throughout the 14-day production phase as a percentage of the wt % (g / g) of the total mAb1 produced on the color intensity (b-value normalized to 40 mg / mL) of the recombinant mAb1. Reducing the total amount of cysteine ​​or cystine and tryptophan added throughout the 14-day production phase reduces the color intensity of the recombinant mAb1 produced. There is an interaction between the total amount of cysteine ​​or cystine added throughout the 14-day production phase as a percentage of the wt % (g / g) of the total mAb1 produced and the total amount of tryptophan as a percentage of the wt % (g / g) of the total mAb1 produced.

[0385] Figure 8 A contour plot is shown illustrating the optimal range of the total amount of cysteine ​​or cystine and tryptophan added as a percentage of the wt% (g / g) of the total mAb-1 produced during the entire 14-day production period for obtaining the lowest values ​​of APG, HMWS, color intensity and the highest values ​​of the main peak groups; the total amount of cysteine ​​or cystine and tryptophan added as a percentage of the weight% (g / g) of the total mAb-1 produced is 12.06 to 28.03 wt% for cysteine ​​or cystine and 8.84 to 32.06 wt% for tryptophan.

[0386] The cumulative intact viable cell count (IVCC) was calculated throughout the 14-day production phase and normalized by cell culture volume (CSV). Fig. 9 The results shown show the effect of the total amount of cysteine ​​or cystine and tryptophan added to the cell culture medium / percentage of the initial CSV weight on IVCC. There was an optimal range of the total amount of cysteine ​​or cystine and tryptophan added to the cell culture medium / percentage of the initial CSV weight, ranging from 0.08% to 0.24% for cysteine ​​or cystine and from 0.07% to 0.15% for tryptophan. No synergistic effect was observed, only a cumulative effect ( Fig. 9 a and Fig. 9 b).

[0387] Fig.10 a and 10b show the effect of the total amount of cysteine ​​or cystine and tryptophan added to the cell culture medium / percentage of the initial CSV weight on the titer of mAb1. There is an optimal range of the total amount of cysteine ​​or cystine and tryptophan added to the cell culture medium / percentage of the CSV weight, which is 0.08% to 0.24% for cysteine ​​or cystine and 0.07% to 0.15% for tryptophan. There is no interaction.

[0388] Fig.11 The contour plots shown in a and 11b show the total amount of cysteine ​​or cystine and tryptophan added to the cell culture medium at the end of the production phase / IVCC*10 -12 The optimal ranges are 2.9 g to 12 g for Cys and 2.5 g to 7 g for Trp.

[0389] Example 3

[0390] As described in Materials and Methods, recombinant monoclonal antibodies were characterized for three fed-batch experimental conditions using various wt % of added cysteine ​​or total amount of cystine and tryptophan relative to total recombinant mAbl produced (Table 4).

[0391] Table 4

[0392]

[0393] Mass spectrometry analysis showed a mass shift in the most intense peak observed in the mass spectra for glycosylated mAb1 under both native and denaturing conditions due to increasing concentrations of cysteine ​​or cystine and tryptophan. These observations led to the conclusion that the modifications were not linked to changes in the glycosylation pattern. Mass spectrometry analysis of the light chain, heavy chain and hemimer (one heavy chain plus one light chain) after manual deconvolution showed that glycosylation of the mAb may occur when the total amount of cysteine ​​or cystine and tryptophan added during the entire 14-day production period is higher in wt% relative to the total mAb1 weight produced. More adducts, i.e., addition of small molecules on mAb1, can be observed. When the total amount of cysteine ​​or cystine and tryptophan added was also increased, the cysteine ​​adduction on the light chain increased. Table 5 shows an overview of the mAb1 characteristics obtained by peptide mapping under the three conditions tested. The results showed that increasing the total wt% of cysteine ​​or cystine and tryptophan added throughout the 14-day production phase relative to the total mAb weight produced resulted in increased methionine oxidation at threonine 19 of the heavy chain and deamidation at threonine 33 of the heavy chain. In addition, the APG% and BPG% variants of mAb1 increased dramatically with increasing the total wt% of cysteine ​​or cystine and tryptophan added throughout the 14-day production phase relative to the total mAb weight produced, while the main peak increased with decreasing the total wt% of cysteine ​​or cystine and tryptophan added throughout the 14-day production phase relative to the total mb1 weight produced.

[0394] Table 5

[0395]

[0396] Example 4

[0397] To identify inhibitory concentrations of cysteine ​​or cystine and tryptophan on the growth of the DG44 CHO cell line expressing mAb1, various large dose additions of cysteine ​​or cystine and tryptophan on day 3 were tested, aiming to reach high concentrations of these amino acids (Table 2b). The feeding strategy was adjusted so that the amount of cysteine ​​or cystine and tryptophan added was the same wt % of the total mAb1 weight produced. Fig.12 It was shown that high concentrations of cysteine ​​or cystine and tryptophan, 0.3 g / L to 0.9 g / L and 0.6 g / L, respectively, significantly reduced cell growth (cumulative IVCC over the entire 14-day production phase normalized by CSV).

[0398] Example 5

[0399] It was hypothesized that depletion of cysteine ​​or cystine may have an impact on the growth and productivity of the CHO cell line expressing mAb1. Nine experimental conditions in a 2L bioreactor were analyzed (Table 6a): three control conditions: cysteine ​​or cystine was not depleted during the entire production phase; two experimental conditions: daily depletion starting from day 6 and continuing until the end of the fed-batch production process, where the cysteine ​​or cystine concentration in the feed was 6.87 g / L, and 4 experimental conditions: cysteine ​​or cystine was depleted on day 6, where the cysteine ​​or cystine concentration in the feed was 17.17 g / L. Depletion was periodic due to the daily addition of cysteine ​​or cystine. The feeding strategy is described in Table 6b. The total amount of cysteine ​​or cysteine ​​added and the total amount of cysteine ​​or cysteine ​​added per IVCC are described in Fig.13 The cysteine ​​concentration before adding the feed is shown in Fig.14 c. If Fig.14 As shown in Figure 1, if the concentration of cysteine ​​or cystine in the feed is about 17.17 g / L, the depletion of cysteine ​​or cystine on day 6 does not affect cell growth. Without wishing to be bound by theory, it is believed that cysteine ​​or cystine-related metabolites accumulate and are stored in the cell and can be utilized when cysteine ​​or cystine is depleted. However, the productivity of the mAb1 cell line was affected by the depletion of cysteine ​​or cystine ( Fig.14 b).

[0400] Table 6a

[0401]

[0402] Table 6b

[0403]

[0404]

[0405] Example 6

[0406] The heterogeneity reduction effect caused by controlling the total wt% of cysteine ​​or cystine and tryptophan added throughout the production stage is not unique to mAb1, and experiments on the total amount of cysteine ​​or cystine and tryptophan added were tested with three other CHO cell lines that also produce recombinant antibodies (Table 7). Fig.15As shown in Figure 15a, the increase in APG charge variants and color intensity correlated with the increase in the total amount of added cysteine ​​or cystine and tryptophan as a percentage of the total wt% of mAb2. Similar results were obtained when the APG charge variants of mAb3 were analyzed (15b). Finally, the increase in APG and BPG charge variants and the decrease in the main peak correlated with the increase in the total amount of added cysteine ​​or cystine and tryptophan as a percentage of the total wt% of mAb4 (15c). These results confirm the results obtained for mAb1.

[0407] Table 7

[0408]

[0409]

[0410] Example 7

[0411] Based on the data from the four different monoclonal antibodies tested herein, the effect of the total wt % of cysteine ​​or cystine and tryptophan added over the entire 14-day production period on the total recombinant mAb weight produced was analyzed (Tables 3 and 7). Fig.16 As shown in (a and b), for all four antibodies analyzed, the increase in APG charge variants correlated with an increase in the wt% of the total amount of added cysteine ​​or cystine and tryptophan relative to the weight of the total recombinant antibody produced. The results demonstrate that the relationship between the wt% of the total amount of added cysteine ​​or cystine and tryptophan relative to the weight of the total recombinant antibody produced and the heterogeneity of the antibody is not limited to a specific antibody, but is applicable to any antibody.

[0412] Example 8

[0413] Liquid pharmaceutical preparations

[0414] Under the standard operating conditions described in the materials and methods, the pharmaceutical preparation of the monoclonal antibody mAb1 was prepared on a large scale (i.e., in a 2000L stainless steel bioreactor) in a fed-batch mode using various cysteine ​​or cystine and tryptophan total additions specified in Table 9. The buffer of the antibody sample was replaced at least 7 times (7 dialysis volumes) with a dialysis buffer (33mM His and 250mM Pro, pH 5.6), and then ultrafiltration was performed using a membrane with a molecular weight cutoff (MWCO) of 30kDa. Once the concentration of the antibody (140mg / ml+ / -14mg / ml) was reached, polysorbate 80 was added at the desired concentration (based on a final concentration of 0.03% w / v). The concentration of the antibody was measured using UV A280.

[0415] Table 8 shows the occurrence of charge variants of formulated mAbl. In two products defined as "higher Cys addition" in Tables 8 and 9, the total cysteine ​​or cystine addition as a percentage of the wt% of total recombinant mAbl produced was higher than in the other three products defined as "lower Cys addition" in Tables 8 and 9. The increase in APG charge variants correlated with an increase in the total cysteine ​​or cystine addition as a percentage of the wt% of total recombinant mAbl produced.

[0416] Table 8

[0417]

[0418] Example 9

[0419] Based on the data of the DG44 CHO cell line expressing mAb1 antibody (Table 3), a model for predicting the acidic peak group (APG) level was developed ( Fig.17 APG was expressed as a function of the wt % of cysteine ​​or the total amount of cystine and tryptophan added over the entire 14 days of production using Michaelis-Menten kinetics, based on the weight of the total recombinant mAb produced.

[0420] To apply this model to perfusion production, each perfusion production day was defined as a new production batch. Therefore, the ratio of the total amount of cysteine ​​or cystine and / or tryptophan used was calculated based on the perfusion flow rate compared to the removal rate of the produced recombinant protein from the production vessel. Perfusion production was performed in a 2L bioreactor using alternating tangential flow (ATF) technology. Fig.18 As shown, the APG charge variant predictions are in good agreement with the experimental data. The results demonstrate that the relationship between the wt% of added cysteine ​​or the total amount of cystine and tryptophan relative to the total weight of the total recombinant antibody produced and antibody heterogeneity can be extended to other production modes, such as perfusion, batch or chemostat mode production.

Claims

1. A pharmaceutical composition comprising a recombinant protein, wherein the pharmaceutical composition is a liquid preparation, comprising the recombinant protein at a concentration of 50 mg / ml to 250 mg / ml, histidine at a concentration of 10 mM to 50 mM, proline at a concentration of 100 mM to 500 mM, and a surfactant at a concentration of 0.001% to 0.1% (w / v), wherein the recombinant protein is an antibody or an antigen-binding fragment thereof, which is: 1) An antibody or an antigen-binding fragment thereof, wherein: a. comprising a CDR-H1 having a sequence defined in SEQ ID NO: 1, a CDR-H2 having a sequence defined in SEQ ID NO: 2, a CDR-H3 having a sequence defined in SEQ ID NO: 3, a CDR-L1 having a sequence defined in SEQ ID NO: 4, a CDR-L2 having a sequence defined in SEQ ID NO: 5, and a CDR-L3 having a sequence defined in SEQ ID NO: 6; or b. comprising a light chain variable region having a sequence defined in SEQ ID NO: 7 and a heavy chain variable region having a sequence defined in SEQ ID NO: 8; or c. comprising a light chain variable region having a sequence defined in SEQ ID NO: 7 and a heavy chain having a sequence defined in SEQ ID NO: 11; or 2) An antibody comprising a light chain having the sequence defined in SEQ ID NO:9 and a heavy chain having the sequence defined in SEQ ID NO:

10.

2. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, wherein the pharmaceutical composition is a liquid preparation comprising the antibody or the antibody-binding fragment thereof at a concentration of 50 mg / ml to 250 mg / ml, histidine at a concentration of 10 mM to 50 mM, proline at a concentration of 100 mM to 500 mM, and a surfactant at a concentration of 0.001% to 0.1% (w / v), and wherein the antibody or the antigen-binding fragment thereof is: 1) The following antibody or antigen-binding fragment thereof, wherein: a. comprising a CDR-H1 having a sequence defined in SEQ ID NO: 1, a CDR-H2 having a sequence defined in SEQ ID NO: 2, a CDR-H3 having a sequence defined in SEQ ID NO: 3, a CDR-L1 having a sequence defined in SEQ ID NO: 4, a CDR-L2 having a sequence defined in SEQ ID NO: 5, and a CDR-L3 having a sequence defined in SEQ ID NO: 6; or b. comprising a light chain variable region having a sequence defined in SEQ ID NO: 7 and a heavy chain variable region having a sequence defined in SEQ ID NO: 8; or c. comprising a light chain variable region having a sequence defined in SEQ ID NO: 7 and a heavy chain having a sequence defined in SEQ ID NO: 11; or 2) An antibody comprising a light chain having the sequence defined in SEQ ID NO:9 and a heavy chain having the sequence defined in SEQ ID NO:

10.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pH is 5 to 7.4 or 5 to 6.5 or 5 to 6.

4. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of histidine is 30 mM, the concentration of proline is 250 mM, and the pH value is between 5.2 and 6.

0.

5. The pharmaceutical composition according to claim 1 or 2, wherein the surfactant is polysorbate 80 at a concentration of 0.01% to 0.05% or 0.03%.

6. The pharmaceutical composition according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is formulated at a concentration of 120 mg / ml to 160 mg / ml or 140 mg / ml.

7. A method for preparing the pharmaceutical composition according to any one of claims 1 to 6, comprising the following steps: a. culturing a host cell capable of producing the antibody or antigen-binding fragment thereof in a medium free of animal-derived products, wherein the host cell is a CHO cell; b. passing the culture through a production phase, wherein the antibody or antigen-binding fragment thereof is produced by the cells, wherein during the production phase, the culture is supplemented with: cysteine ​​or cystine in a total amount up to 10 wt% to 30 wt% of the expected total amount of recombinant protein produced, and Tryptophan, in a total amount up to 8 wt% to 35 wt% of the expected total amount of recombinant protein produced; c. recovering the antibody or antigen-binding fragment thereof from the cell culture medium; d. purifying the antibody or antigen-binding fragment thereof from the cell culture medium; and e. formulating the purified antibody or antigen-binding fragment thereof to obtain the pharmaceutical composition; One or more initial experiments are performed to determine the expected total amount of recombinant protein produced.

8. The method of claim 7, wherein the culture is supplemented with cysteine ​​or cystine in a total amount of up to 12 wt % to 28 wt % of the expected total amount of recombinant protein produced, for example, a total amount of 12 wt % to 25 wt %, for example 12 wt % to 20 wt % of the expected total amount of recombinant protein produced.

9. The method of claim 7 or 8, wherein the culture is supplemented with tryptophan in a total amount of up to 8 wt % to 30 wt % of the expected total amount of recombinant protein produced, for example, in a total amount of 8 wt % to 25 wt %, for example 8 wt % to 20 wt % of the expected total amount of recombinant protein produced.

10. The method according to claim 7 or 8, wherein the total amount of cysteine ​​or cystine and / or tryptophan in the culture is achieved by adding cysteine ​​or cystine and / or tryptophan to the cell culture medium at the following times: a. At the beginning of the production phase, b. once or more times at any point during the production phase, c. continuously added during the production phase, or Any combination of da, b and c.

11. The method according to claim 7 or 8, wherein the method is a batch process, such as a fed-batch process.

12. The method according to claim 7 or 8, wherein cysteine ​​or cystine and / or tryptophan are provided during the production phase by daily addition.

13. The method according to claim 7 or 8, wherein the production phase is carried out for at least 7 days, preferably at least 14 days.

14. The method according to claim 7 or 8, wherein at any point in time during the second half of the production phase: The amount of cysteine ​​or cystine in the culture is 10 wt % to 30 wt % of the expected amount of recombinant protein produced; and • The amount of tryptophan in the culture is between 8 wt% and 35 wt% of the expected amount of recombinant protein produced.

15. The method according to claim 7 or 8, wherein at any point in the production phase: The amount of cysteine ​​or cystine in the culture is 10 wt % to 30 wt % of the expected amount of recombinant protein produced; and • The amount of tryptophan in the culture is between 8 wt% and 35 wt% of the expected amount of recombinant protein produced.

16. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, wherein the pharmaceutical composition is a liquid preparation comprising the antibody or the antibody-binding fragment thereof at a concentration of 50 mg / ml to 250 mg / ml, histidine at a concentration of 10 mM to 50 mM, and a surfactant at a concentration of 0.001% to 0.1% (w / v), and wherein the antibody or the antigen-binding fragment thereof is: 1) The following antibody or antigen-binding fragment thereof, wherein: a. comprising a CDR-H1 having a sequence defined in SEQ ID NO: 1, a CDR-H2 having a sequence defined in SEQ ID NO: 2, a CDR-H3 having a sequence defined in SEQ ID NO: 3, a CDR-L1 having a sequence defined in SEQ ID NO: 4, a CDR-L2 having a sequence defined in SEQ ID NO: 5, and a CDR-L3 having a sequence defined in SEQ ID NO: 6; or b. comprising a light chain variable region having a sequence defined in SEQ ID NO: 7 and a heavy chain variable region having a sequence defined in SEQ ID NO: 8; or c. comprising a light chain variable region having a sequence defined in SEQ ID NO: 7 and a heavy chain having a sequence defined in SEQ ID NO: 11; or 2) An antibody comprising a light chain having the sequence defined in SEQ ID NO:9 and a heavy chain having the sequence defined in SEQ ID NO:

10.

17. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, wherein the pharmaceutical composition is a liquid preparation comprising the antibody or the antibody-binding fragment thereof at a concentration of 50 mg / ml to 250 mg / ml, proline at a concentration of 100 mM to 500 mM, and a surfactant at a concentration of 0.001% to 0.1% (w / v), and wherein the antibody or the antigen-binding fragment thereof is: 1) The following antibody or antigen-binding fragment thereof, wherein: a. comprising a CDR-H1 having a sequence defined in SEQ ID NO: 1, a CDR-H2 having a sequence defined in SEQ ID NO: 2, a CDR-H3 having a sequence defined in SEQ ID NO: 3, a CDR-L1 having a sequence defined in SEQ ID NO: 4, a CDR-L2 having a sequence defined in SEQ ID NO: 5, and a CDR-L3 having a sequence defined in SEQ ID NO: 6; or b. comprising a light chain variable region having a sequence defined in SEQ ID NO: 7 and a heavy chain variable region having a sequence defined in SEQ ID NO: 8; or c. comprising a light chain variable region having a sequence defined in SEQ ID NO: 7 and a heavy chain having a sequence defined in SEQ ID NO: 11; or 2) An antibody comprising a light chain having the sequence defined in SEQ ID NO:9 and a heavy chain having the sequence defined in SEQ ID NO:10.

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