EA044551B1 - CELL CULTURING METHODS - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of production of recombinant proteins, in particular antibodies. More particularly, it relates to methods for culturing cells on an industrial scale to express proteins with reduced heterogeneity.

State of the art

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, over the past two decades, the majority of therapeutic proteins approved as therapeutics have been produced using mammalian cell cultures, and this system remains the expression system of choice for producing large quantities of recombinant polypeptides for human use.

However, mammalian cell cultures pose significant challenges. The titer of recombinant protein produced is typically very low compared to other eukaryotic production systems, such as yeast and insect cell-based systems.

Over the past 30 years, much effort has been devoted to establishing the basic parameters of cell culture and the expression of recombinant polypeptides, with much attention given to research aimed at achieving optimal cell growth by changing the composition of the 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, and on the development of large bioreactors.

Although productivity is still a very important aspect of mammalian cell culture, in recent years the emphasis has shifted towards product quality control and process stability throughout all stages of development and production. Therapeutic proteins produced in mammalian cell culture exhibit varying levels of heterogeneity. Such heterogeneity includes, but is not limited to, different glycosylation profiles, differences resulting from deamidation or oxidation, variants differing in charge or size. Heterogeneity of recombinant proteins can also lead to differences in product color, for example between different batches of the same protein produced in the same manufacturing process. Such heterogeneity, and in particular differences in the color of the recombinant protein of interest, becomes more apparent when the therapeutic proteins are produced in high concentrations. In recent years, there has been a strong trend towards the use of subcutaneous delivery of therapeutic proteins, which requires the production of dosage forms with high concentrations of therapeutic proteins. High concentrations are associated with increased levels of aggregates (Purdie J., et al., Biotechnology Progress, 2016, 32, 9981008). Increased levels of charge variants, such as increased levels of acidic species, may affect protein stability (Banks D. D., et al., Journal of pharma Sciences, 2009, 98, 450110), and the color of the concentrated therapeutic protein may become more intense.

Cell culture conditions, such as medium composition (Kshirsagar R., et al., Biotechnology and Bioengineering, 109:10, 2523-2532 (2012); US 2013/0281355; WO 2013/158275) and growth conditions, including pH and temperature (US 8,765,413), affect the quality characteristics of therapeutic proteins. However, there remains a need to develop even more improved methods for culturing cells to produce therapeutic proteins and, in particular, therapeutic antibodies with minimal heterogeneity.

The essence of the invention

The present invention addresses the above need by reducing the total amount of cysteine or cystine and tryptophan in the cell culture medium during the recombinant protein production phase.

Specific embodiments are described below and are numbered.

Option 1.

A method for producing a recombinant protein, including:

a) culturing host cells capable of producing the recombinant protein into the medium;

b) passing the culture through a production phase, in which the cells produce the recombinant protein, and during said production phase the culture is fed:

cysteine or cystine to a total amount of from 10 to 30 wt.% of the expected total amount of recombinant protein produced; and/or tryptophan to a total amount of from 8 to 35 wt.% of the expected total amount of recombinant protein produced,

c) and optionally recovering the recombinant protein from the cell culture medium.

Embodiment 2.

The method of Embodiment 1, wherein the culture is fed with cysteine or cystine to a total amount of 12 to 28 wt% of the expected total amount of recombinant protein produced, for example a total of 12 to 25 wt%, for example 12 to 20 wt% , from the expected total amount of recombinant protein produced.

- 1 044551

Embodiment 3.

The method of Embodiment 1 or 2, wherein the culture is fed with tryptophan to a total amount of 8 to 30 wt% of the expected total amount of recombinant protein produced, for example a total of 8 to 25 wt%, for example 8 to 20 wt% , from the expected total amount of recombinant protein produced.

Embodiment 4.

The method according to any of the preceding embodiments, wherein the total amount of cysteine or cystine provided in the method is from 2.9 to 12 g/(10 ¹² cells), for example from 2.9 to 7 g/(10 ¹² cells), for example from 5.6 to 7 g/(10 ¹² cells), the cells referring to the expected total number of viable cells at the end of the production phase.

Embodiment 5.

The method according to any of the preceding embodiments, wherein the total amount of tryptophan provided in the method is from 2.5 to 7 g/(10 ¹² cells), for example from 2.5 to 3.5 g/(10 ¹² cells/l ), with cells referring to the expected total number of viable cells at the end of the production phase.

Embodiment 1a.

A method for producing a recombinant protein, including:

a) culturing host cells capable of producing the recombinant protein into the medium;

b) passing the culture through a production phase, in which the cells produce the recombinant protein, and during said production phase the culture is fed:

cysteine or cystine to a total amount of 10 to 30 wt.% of the total amount of the produced recombinant protein; and/or tryptophan to a total amount of 8 to 35 wt.% of the total amount of recombinant protein produced,

c) and optionally recovering the recombinant protein from the cell culture medium.

Embodiment 2a.

The method of Embodiment 1, wherein the culture is fed with cysteine or cystine to a total amount of 12 to 28 wt% of the total amount of recombinant protein produced, for example a total amount of 12 to 25 wt%, for example 12 to 20 wt%, of the total amount of recombinant protein produced.

Embodiment 3a.

The method of embodiment 1 or 2, wherein the culture is fed with tryptophan to a total amount of 8 to 30 wt% of the total amount of recombinant protein produced, for example a total amount of 8 to 25 wt%, for example 8 to 20 wt%, of the total amount of recombinant protein produced

Embodiment 4a.

The method according to any of the preceding embodiments, wherein the total amount of cysteine or cystine provided in the method is from 2.9 to 12 g/(10 ¹² cells), for example from 2.9 to 7 g/(10 ¹² cells), for example from 5.6 to 7 g/(10 ¹² cells), cells referring to the total number of viable cells at the end of the production phase.

Embodiment 5a.

The method according to any of the preceding embodiments, wherein the total amount of tryptophan provided in the method is from 2.5 to 7 g/(10 ¹² cells), for example from 2.5 to 3.5 g/(10 ¹² cells/l ), with cells referring to the total number of viable cells at the end of the production phase.

Embodiment 6.

The method according to any 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:

a) at the beginning of the production phase,

b) one or more times at any time during the production phase,

c) by continuous addition during the production phase or

d) in any combination of a), b) and c).

Embodiment 7.

The method according to any of the preceding embodiments, which is a batch process, such as a fed-batch process.

Embodiment 8.

The method according to any of the preceding embodiments, wherein cysteine or cystine and/or tryptophan is provided by daily addition during the production phase.

Embodiment 9.

The method of embodiment 8, wherein before adding cysteine or cystine the next day, the culture is depleted of cysteine or cystine, for example by reducing the amount

- 2 044551 added cysteine or cystine to a level of from 5.6 to 7 g/(10 ¹² cells), the cells referring to the expected total number of viable cells at the end of the production phase.

Embodiment 9a.

The method of embodiment 8, wherein before adding cysteine or cystine the next day, the culture is depleted of cysteine or cystine, for example, by reducing the amount of cysteine or cystine added to a level of from 5.6 to 7 g/(10 ¹² cells), and cells refer to the total number of viable cells at the end of the production phase.

Embodiment 10.

The method according to embodiment 8 or 9, in which at a late stage of production, i.e. when cells have already reached maximum viable cell density, the culture is depleted of tryptophan before adding tryptophan the next day.

Embodiment 11.

The method according to any of the preceding embodiments, wherein the concentration of cysteine or cystine in the cell culture medium does not exceed 0.9 g/L at any time during the production phase, preferably, wherein the concentration of cysteine or cystine in the cell culture medium does not exceeds 0.3 g/l at any time during the production phase.

Embodiment 12.

The 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 during the production phase, preferably, wherein the tryptophan concentration in the cell culture medium does not exceed 0.3 g/l at any time during the production phase.

Embodiment 13.

A method in which the production phase is carried out for at least 7 days, preferably at least 14 days.

Embodiment 14. The method according to any of the preceding embodiments, wherein at any time during the 2nd half of the production phase:

the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the expected amount of produced recombinant protein; and/or the amount of tryptophan in the culture is from 8 wt.% to 35% of the expected amount of recombinant protein produced.

Embodiment 15.

The method according to any of the preceding embodiments, wherein at any time during the production phase:

the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the expected amount of produced recombinant protein; and/or the amount of tryptophan in the culture is from 8 wt.% to 35% of the expected amount of recombinant protein produced.

Embodiment 14a.

The method according to any of the preceding embodiments, wherein at any time during the 2nd half of the production phase:

the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the amount of produced recombinant protein; and/or the amount of tryptophan in the culture is from 8 wt.% to 35% of the amount of recombinant protein produced.

Embodiment 15a.

The method according to any of the preceding embodiments, wherein at any time during the production phase:

the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the amount of produced recombinant protein; and/or the amount of tryptophan in the culture is from 8 wt.% to 35% of the amount of recombinant protein produced.

Embodiment 16. The method according to any of the preceding embodiments, wherein the host cells are mammalian cells, preferably CHO cells.

Embodiment 17.

The method of any of the preceding embodiments, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof.

Embodiment 18.

The method of embodiment 17, wherein the antibody or antigen binding fragment thereof is:

1) an antibody or its antigen-binding fragment, which:

a) contains CDR-H1 having the sequence defined in SEQ ID NO: 1; CDR-H2 having the sequence defined in SEQ ID NO: 2; CDR-H3, having the sequence

- 3 044551 defined in SEQ ID NO: 3; CDR-L1 having the sequence defined in SEQ ID NO: 4;

CDR-L2 having the sequence defined in SEQ ID NO: 5, and CDR-L3 having the sequence defined in SEQ ID NO: 6; or

b) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having the sequence defined in SEQ ID NO: 8; or

c) contains a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity with the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

d) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain having the sequence defined in SEQ ID NO: 11; or

e) contains a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity with 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

2) an antibody that contains 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; or

3) an antibody that contains a light chain having at least 80% identity or similarity, preferably 90% identity or similarity with 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.

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, 2000 L or more, 5000 L or more, 10000 l or more or 20,000 l or more.

Embodiment 20.

The method according to any of the preceding embodiments, comprising the step of isolating the recombinant protein from a cell culture medium and the additional step of purifying the recombinant protein.

Embodiment 21.

The method of embodiment 20, wherein the purification comprises Protein A chromatography.

Embodiment 22.

The method of embodiment 20 or 21, further comprising the step of preparing a purified recombinant protein composition.

Embodiment 23.

The method of embodiment 22, wherein the recombinant protein composition is formulated as a liquid composition containing one or more amino acids and a surfactant.

Embodiment 24.

The method of embodiment 23, wherein the composition contains histidine and/or proline.

Embodiment 25.

The method of embodiment 24, wherein the formulation contains histidine at a concentration of 5 to 100 mM, for example at a concentration of 10 to 50 mM, and/or proline at a concentration of 100 to 500 mM at a pH of 5 to 7.4, for example from 5 to 6.5, for example, from 5 to 6.

Embodiment 26.

The method of embodiment 25, wherein the formulation contains 30 mM histidine and 250 mM proline at a pH of 5.2 to 6.0, preferably at about 5.6.

Embodiment 27.

The method of any one of embodiments 23-26, 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%, for example from 0.01% to 0.1%, for example from 0.01% to 0.05%, for example 0.03%.

Embodiment 28.

The method according to any one of embodiments 23-27, wherein the recombinant protein is an antibody, wherein the antibody is present in the composition at a concentration of from 10 to 250 mg/ml, for example from 20 to 250 mg/ml, for example from 50 to 250 mg/ ml, for example from 120 to 160 mg/ml, for example about 140 mg/ml.

Embodiment 29.

A method according to any of the preceding embodiments, providing a reduction in the heterogeneity of the resulting recombinant proteins, wherein said reduction in heterogeneity includes a reduction in:

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a) charge heterogeneity, preferably acidic peak group (acidic isoform peaks) (APG);

and/or

b) oxidation of amino acids, isomerization, fragmentation, glycation of other covalent adducts, deamidation, cysteinylation; and/or

c) color or color intensity, for example between different batches of recombinant protein; and/or

d) high molecular weight species (HMWS); and/or

e) instability of the recombinant protein.

Embodiment 30.

A method for producing a recombinant protein, including:

a) culturing host cells capable of producing recombinant protein in the medium;

b) passing the culture through a production phase in which the cells produce the recombinant protein and the cell culture medium is fed with cysteine or cystine and/or tryptophan, the total amount of cysteine or cystine provided in the method being from 2.9 to 7 g/(10 ¹² cells), for example from 5.6 to 7 g/(10 ¹² cells), wherein the cells refer to the expected total number of viable cells at the end of the production phase, and/or the total amount of tryptophan provided in the method is from 2.5 up to 3.5 g/(10 ¹² cells), and the cells refer to the expected total number of viable cells at the end of the production phase,

c) optionally, isolating the recombinant protein from the cell culture medium.

Embodiment 30a.

A method for producing a recombinant protein, including:

a) culturing host cells capable of producing the recombinant protein into the medium;

b) passing the culture through a production phase in which the cells produce the recombinant protein and the cell culture medium is fed with cysteine or cystine and/or tryptophan, the total amount of cysteine or cystine provided in the method being from 2.9 to 7 g/(10 ¹² cells), for example from 5.6 to 7 g/(10 ¹² cells), where cells refer to the total number of viable cells at the end of the production phase, and/or the total amount of tryptophan provided in the method is from 2.5 to 3.5 g/(10 ¹² cells), cells referring to the total number of viable cells at the end of the production phase, c) optionally, isolating the recombinant protein from the cell culture medium.

Embodiment 31.

The method of embodiment 30, having one or more additional features listed in any of embodiments 2-29.

Embodiment 32.

A method for reducing the heterogeneity of a population of recombinant proteins in a batch obtained during the production phase of recombinant host cells, including limiting the total amount

a) cysteine or cystine and/or

b) tryptophan present in the cell culture medium during the recombinant protein production phase.

Embodiment 33.

The method of embodiment 32, having one or more additional features listed in any of embodiments 2-29.

Embodiment 34.

A recombinant protein preparation that can be or is produced by the method of any of the preceding embodiments.

Embodiment 35.

A pharmaceutical composition comprising an antibody, wherein the composition has one or more additional features listed in any of embodiments 23-28, wherein the antibody is preferably the antibody specified in embodiment 18.

Brief description of the figures

Fig. 1. Description of the calculations to measure the total amount of amino acids, cysteine or cystine and tryptophan added during the production phase carried out in the bioreactor, based on the weight percentage of the recombinant protein produced.

Fig. 2. Effect of total added tryptophan and cysteine or cystine in wt% (g/g) of total mAb1 produced on b* value normalized to 40 mg/mL (a) and acid peak group (APG) variant ( b), respectively. Maximum concentrations of tryptophan and cysteine or cystine do not affect b* value (c) or APG% (d).

Fig. 3. Effect of the total amount of added tryptophan and cysteine or cystine in wt.% of the total mass of mAb1 produced (g/g) on the b* value normalized to 40 mg/ml (a) and on the variant

- 5 044551 acidic peak groups (APG) (b), respectively, and no correlation between the maximum concentrations of cysteine or cystine (c) and tryptophan (d) on the APG.

Fig. 4. Multiple linear regression model of the dependence of the acid peak group (APG) variant of the recombinant monoclonal antibody mAb1 on the added total amount of cysteine or cystine and tryptophan in wt.% of the total amount of mAb1 produced (g/g).

Fig. 5. Multiple linear regression model of the dependence of the variant group of the main peaks of the recombinant monoclonal antibody mAb1 on the total amount of added cysteine or cystine and tryptophan in wt.% of the total amount of recombinant mAb1 produced (g/g).

Fig. 6. Multiple linear regression model of the dependence of the high molecular weight species (HMWS) variant of the recombinant monoclonal antibody mAb1 on the total amount of added cysteine or cystine in wt.% of the total amount of recombinant mAb1 produced (g/g).

Fig. 7. Multiple linear regression model of the dependence of the variant normalized to 40 mg/ml b* value of the recombinant monoclonal antibody mAb1 on the total amount of added cysteine or cystine and tryptophan in wt.% of the total amount of recombinant mAb1 produced (g/g).

Fig. 8. Contour plots showing the effect of added total cysteine or cystine and tryptophan in wt% of total recombinant mAb1 produced, (g/g), on variants of (a) high molecular weight species (HMWS), (b) b* values, normalized to 40 mg/mL, (c) acid peak groups (APG) and (d) major peak groups. The square indicated by the black dotted line corresponds to the ideal added total amount of cysteine or cystine and tryptophan in wt% of the total amount of recombinant mAb1 produced (g/g) in terms of APG reduction, HMWS normalized to 40 mg/ml b* value and increasing the variant of the main peak group, where the added amount of cysteine or cystine corresponds to 12.06 and 28.03 wt.%, respectively, of the total amount of mAb1 produced (g/g), and the added amount of tryptophan ranges from 8.84 to 32 .06 wt.% of the total amount of mAb1 produced (g/g).

Fig. 9. Effect of added total cysteine or cystine and tryptophan in wt.% of the mass of the volumetric cell culture (CSV) on the total number of viable cells (IVCC), normalized to CSV. A multiple linear regression model of CSV-normalized cumulative IVCC versus added total cysteine or cystine and tryptophan as a wt% of CSV is shown in (a). A contour plot of the effect of added total cysteine or cystine and tryptophan in wt% of CSV weight on cumulative IVCC normalized to CSV is shown in (b).

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 the weight of CSV on the final titer of mAb1 measured by HPLC (mAb HPLC). A multiple linear regression model of the final HPLC titer of mAb1 versus added total cysteine or cystine and tryptophan in wt % of CSV is shown in (a). A contour plot of the effect of added total cysteine or cystine and tryptophan in wt.% of CSV weight on the final mAb titer determined by HPLC is shown in (b).

Fig. 11. A contour plot of the effect of total cysteine or cystine and tryptophan added to cell culture medium during the production phase per IVCC* 10 ^-12 at the end of the production phase on IVCC for 14 days is shown in (a). A contour plot showing the effect of the total amount of cysteine or cystine and tryptophan added to cell culture medium during the production phase, per IVCC* 10 ^-12 at the end of the production phase, on the final mAb titer determined by HPLC is shown in (b). . A description of the calculations for measuring the amount of amino acids, cysteine or cystine and tryptophan added during the production phase carried out in the bioreactor, based on IVCC* 10 ^-12 at the end of the production phase, is shown in (c).

Fig. 12. Contour plot of the effect of maximum concentrations of cysteine or cystine and tryptophan achieved in cell culture medium during the production phase on IVCC normalized to CSV.

Fig. 13. Total cysteine or added cysteine and Total cysteine or added cysteine on IVCC for the conditions described in table. 6.

Fig. 14. Effect of cysteine or cystine depletion on cell growth and mAb titer. Viable cell concentration (VCC) profile (a), mAb titers (b) and Cys concentrations before feed addition (c) are shown as a function of three experimental conditions: without cysteine depletion or cystine throughout the production phase [No depletion - (34, 35 g/L Cys in feed)] and two conditions with daily cysteine depletion or cystine depletion from day 6 to the end of production in a fed-batch mode and Cys concentration in feed of 17.17 g/L and 6.87 g/L .

Fig. 15. Effect of added total amount of cysteine or cystine and tryptophan in wt.% of the total amount of produced recombinant antibody, by weight, on (a) APG variant and b* value normalized to 40 mg/ml for mAb2; (b) APG variant for mAb3 and (c) APG, BPG variants

- 6 044551 (group of alkaline peaks (peaks isomorphic with base properties) and variants of the main group for mAb4.

Fig. 16. Effect of added total amount of cysteine or cystine and tryptophan in wt.% of the total amount of recombinant mAb1, mAb2, mAb3 and mAb4 on APG. (a) is a graph of the APG variant for mAb1, mAb2, mAb3 and mAb4 versus the added total amount of cysteine or cystine in wt.% of the total amount of recombinant mAb1, mAb2, mAb3 and mAb4 produced, by wt. (b) shows a multiple linear regression model of the dependence of the APG variant of the recombinant monoclonal antibodies mAb1, mAb2 and mAb3 and the recombinant polyspecific antibody mAb4 on the added total amount of cysteine or cystine and tryptophan in wt.% of the total amount (by weight) of the recombinant mAb produced.

Fig. 17. An equation was derived to predict the acid peak group (APG) level based on data obtained for the CHO DG44 cell line expressing the mAb1 antibody (Table 3).

Fig. 18. Comparison of the experimental acid peak group (APG) level (APG exp.) with the predicted APG level (APG prog.) for the CHO cell line DG44 expressing mAb1 antibodies. The data were obtained using the production (cultivation) method in a continuous mode using Alternating Tangential Flow (ATF) technology in 2-liter bioreactors. APG prediction is based on the equation shown in FIG. 17.

Detailed Description of the Invention

The invention is based on the discovery that limiting the total amount of cysteine or cystine and/or tryptophan used in the cell culture medium during the production phase of a method for producing a recombinant protein makes it possible to reduce the heterogeneity of the resulting recombinant proteins. Therefore, the present invention provides for the use of a limited amount of cysteine or cystine and/or tryptophan in the cell culture medium to reduce the heterogeneity of the antibody or antigen binding fragment expressed in the cell culture medium.

Reducing heterogeneity is advantageous with respect to:

a) charge, preferably acidic peak group (APG) heterogeneity; and/or

b) oxidation of amino acids, isomerization, fragmentation, glycation of other covalent adducts, deamidation, cysteinylation; and/or

c) color or color intensity (b* value normalized to 40 mg/ml); and/or

d) formation of high molecular weight species (HMWS); and/or

e) instability of the recombinant protein and/or

f) their combinations.

As used herein, the term heterogeneity refers to differences between individual molecules, eg, recombinant proteins, in a population of molecules produced by the same production method or in the same production lot. Heterogeneity may result from incomplete or heterogeneous modifications of recombinant proteins, for example due to post-translational modifications of the expressed protein. Such modifications may result from deamination reactions and/or oxidation reactions and/or covalent addition of small molecules, such as glycation reactions and/or isomerization reactions and/or fragmentation reactions and/or other reactions, and may also involve changes in the nature of glycation. The chemical-physical manifestation of such heterogeneity results in varying characteristics of the resulting recombinant protein preparations, which include, but are not limited to, charged variant profile, color or color intensity, and molecular weight.

The term production phase of the present invention includes that step of culturing cells in a recombinant protein production method in which the cells express (ie produce) the recombinant protein(s). The production phase begins with increasing the titer of the desired product and ends with the collection of cells, fluid or cell culture supernatant. Typically, at the beginning of the production phase, the cell culture is transferred to a bioreactor. Harvesting is a step in which cell culture fluid is removed, for example from a vessel to produce a recombinant protein, such as a recombinant antibody, which must be isolated and purified in subsequent steps. The term initial cell culture weight as used herein refers to the weight of the culture at the beginning of the production phase, typically the weight when introduced into the bioreactor.

In a first aspect, the invention relates to a method for producing a recombinant protein, comprising:

a) culturing host cells capable of producing the recombinant protein into the medium;

b) passing the culture through a production phase, in which the cells produce the recombinant protein, and during said production phase the culture is fed:

a) cysteine or cystine to a total amount of 10 to 30% by weight of the expected total amount of recombinant protein produced; and/or

b) tryptophan to a total amount of 8 to 35% by weight of the expected total amount of recombinant protein produced,

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c) and optionally recovering the recombinant protein from the cell culture medium.

As will become apparent from the description of the invention, the culture is fed with cysteine or cystine and/or tryptophan; such recharge can be performed using:

1. cysteine; or

2. cystine; or

3. cysteine and cystine; or

4. cysteine and tryptophan; or

5. cystine and tryptophan; or

6. cysteine, cystine and tryptophan; or

7. tryptophan.

When used herein, the expression total amount of cysteine or cystine or cysteine or cystine to total amount... refers to a) the total amount of cysteine only if the method does not use cystine, b) the total amount of cystine only if the method does not use cysteine, or c) to the total amount of cysteine+cystine if both compounds are used in the method. Cysteine and cystine in cell culture media are in a constant equilibrium in which two cysteine molecules are oxidized to a cystine molecule and reduced back to two cysteine molecules.

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 percentage by weight. Total refers to the total quantity determined at the end of the production phase, i.e. to the total amount of cysteine or cystine and/or tryptophan added during the production phase, and to the total amount of recombinant protein produced during the production phase, the total amount of recombinant protein produced being measured at the end of the production phase.

In fig. 1 shows how the total amount of cysteine or cystine and/or tryptophan is calculated based on the wt.% of the recombinant protein produced. The total amount of cysteine, cystine or tryptophan added is calculated as a function of the feed rate (or feed volume) and the concentration of cysteine, cystine or tryptophan in that feed, and the concentration of cysteine, cystine or tryptophan in the medium to which the feed is added, per volume added recharge (Fig. 1, A). The amount of recombinant protein produced is calculated as a function of the final volume of cell culture medium and the final titer of recombinant protein (Fig. 1 B). The ratio of these two calculated parameters represents the total amount of cysteine or cystine and/or tryptophan added per amount of recombinant protein produced (Fig. 1 C).

The host cells may first (in step a.) be grown in a cell culture medium, which may or may not include cysteine or cystine and tryptophan. If the cell culture medium already contains an initial amount of cysteine or cystine and/or tryptophan, then the total amount will include the specified initial amount.

In one embodiment of the method of the invention, the culture is fed with cysteine or cystine to a total amount of from 12.06 wt.% to 28.03 wt.% of the expected total amount of recombinant protein produced, for example a total amount of from 12 wt.% to 28 wt. %, for example from 12 to 25 wt.%, for example from 12 to 20 wt.%, of the expected total amount of recombinant protein produced.

In another embodiment of the method of the invention, the culture is fed with tryptophan to a total amount of from 8.84 to 32.06 wt.% of the expected total amount of recombinant protein produced, for example a total amount of from 8 wt.% to 30 wt.%, for example from 8 to 25% by weight, for example 8 to 20% by weight, of the expected total amount of recombinant protein produced.

Alternatively, the total amount of cysteine or cystine and/or tryptophan can be expressed as the total amount added in the process relative to the total number of viable cells at the end of the production phase. In one embodiment, the total amount of cysteine and/or cystine provided in the method is from 2.9 to 12 g/(10 ¹² cells), for example from 2.9 to 7 g/(10 ¹² cells), for example from 5.6 to 7 g/(10 ¹² cells), the cells referring to the expected total number of viable cells at the end of the production phase. In another embodiment, the total amount of tryptophan provided in the method is from 2.5 to 7 g/(10 ¹² cells), for example from 2.5 to 3.5 g/(10 ¹² cells), the number of cells being the expected the total number of viable cells at the end of the production phase.

It should be understood that one skilled in the art is aware of methods for measuring the amount of cysteine or cystine and/or tryptophan added and/or present in a cell culture at a certain phase, such as the production phase. For example, this can be done as described in the examples provided herein. Likewise, one skilled in the art is aware of methods for measuring the total amount of recombinant protein produced by a cell culture and, therefore, is able to apply the disclosure of the present invention to achieve the desired technical effect. For example, this can be done, as disclosed in the examples provided herein, for example, using a model ForteBio Octet analyzer (ForteBio, Inc., Menlo Park, Calif.) or high pressure liquid chromatography (HPLC) using cell culture supernatant samples, which are stored at -80°C until analysis.

To develop a method of the invention in which the amount of cysteine or cystine and/or tryptophan per expected total amount of recombinant protein produced is maintained within certain limits, it may be necessary to perform one or more initial experiments to determine the approximate levels of recombinant protein produced by specific host cells under certain cultivation conditions. When the approximate total levels of recombinant protein produced are known, the method of the invention can be designed in which the amount of cysteine or cystine and/or tryptophan per expected total amount of recombinant protein produced will be maintained within these limits.

To achieve this total amount of cysteine or cystine and/or tryptophan in the cell culture medium during the production phase, various strategies can be used. In one embodiment, the total amount can be achieved by adding cysteine or cystine and/or tryptophan immediately at the beginning of the production phase, for example only once, or the total amount can already be included in the cell culture medium used for production. In another embodiment, the total amount can be achieved by adding up the supplements, such as daily supplements or as a continuous addition, during the production phase. In another embodiment, the total amount can be achieved by a combination of the initial concentration of cysteine/cysteine and/or tryptophan in the cell culture fluid at the beginning of the production phase and additions.

Accordingly, 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:

a) at the beginning of the production phase,

b) one or more times at any time during the production phase,

c) by continuous addition during the production phase or

d) in any combination of a), b) and c).

In a preferred embodiment, cysteine or cystine and/or tryptophan is added at the beginning of the production phase and added as daily bolus supplements during the production phase. Preferably, the production phase lasts at least 7 days, more preferably more than 7 days, for example 10 days, more preferably 14 or more days.

Preferably, the concentration of cysteine or cystine in the cell culture medium does not exceed 0.9 g/L at any time during the production phase, preferably the concentration of cysteine or cystine in the cell culture medium does not exceed 0.3 g/L at any time time during the production phase.

Furthermore, preferably the concentration of tryptophan in the cell culture medium does not exceed 0.6 g/L at any time during the production phase, preferably the concentration of tryptophan in the cell culture medium does not exceed 0.3 g/L at any time during the production phase.

In one embodiment, cysteine or cystine is added daily to the cell culture medium, and by day 6 the maximum concentration of cysteine or cystine in the cell culture medium is increased to 0.3 g/L, and from day 7 to day 14 the maximum concentration of cysteine or cystine in the cell culture medium is increased to 0.9 g/l.

In some embodiments, the amount of cysteine or cystine and/or tryptophan is within specified ranges calculated for the entire production phase, not only at the end of the production phase, but also at any point in time at any point during the production phase, or even at any point in time during throughout the production phase. Thus, in one embodiment, at any time during the 2nd half of the production phase (for example, from day 7 to day 14 of the 14-day production phase):

the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the expected amount of produced recombinant protein; and/or the amount of tryptophan in the culture is from 8 wt.% to 35% of the expected amount of recombinant protein produced.

In another embodiment, at any point in time during the production phase:

the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the expected amount of produced recombinant protein; and/or the amount of tryptophan in the culture is from 8 wt.% to 35% of the expected amount of recombinant protein produced.

When cysteine or cystine is delivered to cells by daily supplementation, the culture can be ensured to be depleted of cysteine or cystine before the next daily supplement is added. In one

- 9 044551 of embodiments, the culture is depleted of cysteine or cystine before adding cysteine or cystine the next day, for example by reducing the addition of cysteine or cystine to a level of 5.6 to 7 g/(10 ¹² cells). In a second embodiment, during cell culture, the culture is depleted of tryptophan at a late stage of production, i.e. when cells have already reached maximum viable cell density, for example, depletion begins on day 8 or later during the 14-day production phase.

Without being limited to any theory, it is believed that, despite depletion of cysteine or cystine, cells in the production phase do not become depleted of cysteine or cystine, and presumably have an internal mechanism that allows cysteine or cystine obtained during supplementation to be stored as an inactive metabolite , which, under conditions of depletion, is capable of being converted into cysteine or cystine, which is supplied to the cell culture medium.

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

a) culturing host cells capable of producing the recombinant protein into the medium;

b) passing the culture through a production phase in which the cells produce the recombinant protein and the cell culture medium is fed with cysteine or cystine and/or tryptophan, the total amount of cysteine or cystine provided in the method being from 2.9 to 7 g/(10 ¹² cells), for example from 5.6 to 7 g/(10 ¹² cells), wherein the cells refer to the expected total number of viable cells at the end of the production phase, and/or the total amount of tryptophan provided in the method is from 2.5 up to 3.5 g/(10 ¹² cells), and the cells refer to the expected total number of viable cells at the end of the production phase,

c) optionally, isolating the recombinant protein from the cell culture medium.

In one embodiment of this method, the cell culture medium is fed with cysteine or cystine to a total amount of from 12 to 28 wt.% of the expected total amount of recombinant protein produced, for example a total amount of from 12 wt.% to 25 wt.%, for example from 12 up to 20 wt.% of the expected total amount of produced recombinant protein.

In another embodiment of this method, the cell culture medium is fed with tryptophan to a total amount of 8 to 30 wt% of the expected total amount of recombinant protein produced, for example a total of 8 wt% to 25 wt%, for example 8 to 20 wt% .%, of the expected total amount of recombinant protein produced.

In another embodiment of this 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:

a) at the beginning of the production phase,

b) one or more times at any time during the production phase,

c) by continuous addition during the production phase or

d) in any combination of a), b) and c).

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

In another embodiment of this method, the cell culture medium is depleted of cysteine or cystine before adding cysteine or cystine the next day, for example by reducing the addition of cysteine or cystine to a level of between 5.6 and 7 g/(10 ¹² cells).

In another embodiment of this method, the cell culture medium is depleted of tryptophan before it is added the next day late in production, i.e. when cells have already reached maximum viable cell density.

In another embodiment of this method, the concentration of cysteine or cystine in the cell culture does not exceed 0.9 g/l at any time during the production phase; preferably, the concentration of cysteine or cystine in the cell culture at any time during the production phase does not exceed 0.3 g/l.

In another embodiment of this method, the concentration of tryptophan in the cell culture does not exceed 0.6 g/l at any time during the production phase; it is preferable when the concentration of tryptophan in the cell culture at any time during the production phase does not exceed 0.3 g/l.

In another embodiment of this method, the production phase is carried out for at least 7 days, preferably at least 14 days.

In one embodiment of this method, at any time during the 2nd half of the production phase:

a) the amount of cysteine or cystine in the cell culture medium is from 10 to 30% by weight of the expected amount of recombinant protein produced; and/or

b) the amount of tryptophan in the cell culture medium is from 8 to 35 wt.% of the expected value;

- 10 044551 given amount of produced recombinant protein.

In another embodiment of this method, at any time during the production phase:

a) the amount of cysteine or cystine is from 10 to 30% by weight of the expected amount of recombinant protein produced; and/or

b) the amount of tryptophan is from 8 to 35% by weight of the expected amount of recombinant protein produced.

In another embodiment of this method, the host cells are mammalian cells, preferably CHO cells.

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

In another embodiment of this 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, 2000 L or more, 5000 L or more, 10,000 L or more, or 20,000 l or more.

In one embodiment, the method includes the step of isolating the recombinant protein from the cell culture medium and the additional step of purifying the recombinant protein.

In a further embodiment of this method, purification involves Protein A chromatography.

In a further embodiment, the method includes the additional step of obtaining a purified recombinant protein composition.

In one embodiment of this method, the recombinant protein formulation is prepared as a liquid formulation containing one or more amino acids and a surfactant.

In a further embodiment of this method, the composition contains histidine and/or proline.

In another embodiment of this method, the composition contains histidine at a concentration of from 5 mM to 100 mM, for example at a concentration of from 10 to 50 mM, and/or proline at a concentration from 100 to 500 mM at a pH of from 5 to 7.4, for example from 5 to 6.5, for example, from 5 to 6, for example, from 5.5 to 6.

In another further embodiment of this method, the formulation contains 30 mM histidine and 250 mM proline at a pH of 5.2 to 6.0, such as about 5.6.

In a further embodiment of this 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%, for example 0.01 % to 0.1%, for example 0.01% to 0.05%, for example 0.03%.

In another further embodiment of this method, the recombinant protein is an antibody, wherein the antibody is present in the composition at a concentration of from 10 to 250 mg/ml, for example from 20 to 250 mg/ml, for example from 50 to 250 mg/ml, for example from 120 to 160 mg/ml, for example about 140 mg/ml.

In another embodiment, the method reduces the heterogeneity of the recombinant proteins produced, wherein said reduction in heterogeneity includes reducing:

a) charge heterogeneity, preferably acidic peak group (APG); and/or

b) oxidation of amino acids, isomerization, fragmentation, glycation of other covalent adducts, deamidation, cysteinylation; and/or

c) color or color intensity, for example between different batches of recombinant protein; and/or

d) high molecular weight species (HMWS); and/or

e) instability of the recombinant protein.

In a further independent aspect, the invention relates to a method of reducing the heterogeneity of a population of recombinant proteins in a batch produced during the production phase of recombinant host cells, comprising limiting the total amount of:

a) cysteine or cystine and/or

b) tryptophan present in the cell culture medium during the recombinant protein production phase.

In one embodiment, the method includes:

a) culturing host cells capable of producing the recombinant protein into the medium;

b) passing the culture through a production phase, in which the cells produce the recombinant protein, and during said production phase the culture is fed:

cysteine or cystine to a total amount of from 10 wt.% to 30 wt.% of the expected total amount of recombinant protein produced; and/or tryptophan to a total amount of from 8 wt.% to 35 wt.% of the expected total amount of recombinant protein produced,

c) optionally, isolating the recombinant protein from the cell culture medium.

In one embodiment of the method, the culture is fed with cysteine or cystine to a total amount of from 12 to 28 wt.% of the expected total amount of recombinant protein produced, for example a total amount of from 12 wt.% to 25 wt.%, for example from 12 to 20 wt. %, of the expected total amount of recombinant protein produced.

In another embodiment of the method, the culture is fed with tryptophan to a total amount of 8 to 30 wt.% of the expected total amount of recombinant protein produced, for example a total amount of 8 to 25 wt.%, for example 8 to 20 wt.%, of the expected total amount of recombinant protein produced.

In another embodiment of the method, the total amount of cysteine or cystine provided in the method is from 2.9 to 12 g/(10 ¹² cells), for example from 2.9 to 7 g/(10 ¹² cells), for example from 5.6 up to 7 g/(10 ¹² cells), the cells referring to the expected total number of viable cells at the end of the production phase.

In another embodiment of the method, the total amount of tryptophan provided in the method is from 2.5 to 7 g/(10 ¹² cells), for example from 2.5 to 3.5 g/(10 ¹² cells/l), the cells being to the expected total number of viable cells at the end of the production phase.

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

a) at the beginning of the production phase,

b) one or more times at any time during the production phase,

c) by continuous addition during the production phase or

d) in any combination of a), b) and c).

In another embodiment, the method is a batch process, such as a fed-batch process.

In another embodiment of the method, cysteine or cystine and/or tryptophan is provided by daily addition during the production phase.

In another embodiment of the method, the cell culture medium is depleted of cysteine or cystine before adding cysteine or cystine the next day, for example by reducing the addition of cysteine or cystine to a level of 5.6 to 7 g/(10 ¹² cells).

In another embodiment of the method, the cell culture medium is depleted of tryptophan before it is added the next day at a late stage of production, i.e. when cells have already reached maximum viable cell density.

In another embodiment of the method, the concentration of cysteine or cystine in the cell culture medium does not exceed 0.9 g/l at any time during the production phase, preferably when the concentration of cysteine or cystine in the cell culture medium does not exceed 0.3 g/l at any time during the production phase.

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

In another embodiment of the method, the production phase is carried out for at least 7 days, preferably at least 14 days.

In another embodiment of the method, at any time during the 2nd half of the production phase:

the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the expected amount of produced recombinant protein; and/or the amount of tryptophan in the culture is from 8 wt.% to 35% of the expected amount of recombinant protein produced.

In another embodiment of the method, at any time during the production phase:

the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the expected amount of produced recombinant protein; and/or the amount of tryptophan in the culture is from 8 wt.% to 35% of the expected amount of recombinant protein produced.

In another embodiment of the method, the host cells are mammalian cells, preferably CHO cells.

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

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, 2000 L or more, 5000 L or more, 10,000 L or more, or 20,000 l or more.

In one embodiment, the method includes the step of isolating the recombinant protein from the cell culture medium and the additional step of purifying the recombinant protein.

In a further embodiment of the method, purification involves Protein A chromatography.

In a further embodiment, the method includes the additional step of obtaining a purified recombinant protein composition.

In one embodiment of this method, the recombinant protein formulation is prepared as a liquid formulation containing one or more amino acids and a surfactant.

- 12 044551

In a further embodiment of this method, the composition contains histidine and/or proline.

In another further embodiment of this method, the composition contains histidine at a concentration of 5 mM to 100 mM, for example at a concentration of 10 to 50 mM, and/or proline at a concentration of 100 to 500 mM at a pH of 5 to 7.4, for example from 5 to 6.5, for example from 5 to 6, for example from 5.5 to 6.

In another further embodiment of this method, the formulation contains 30 mM histidine and 250 mM proline at a pH of 5.2 to 6.0, such as about 5.6.

In a further embodiment of this method, the surfactant is polysorbate 80, preferably at a concentration of from 0.001% to 0.1% w/v, for example from 0.005% to 0.1%, for example from 0.01% to 0 .1%, for example 0.01% to 0.05%, for example 0.03%.

In yet another further embodiment of this method, the recombinant protein is an antibody, wherein the antibody is present in the composition at a concentration of from 10 to 250 mg/ml, for example from 20 to 250 mg/ml, for example from 50 to 250 mg/ml, for example from 120 up to 160 mg/ml, for example about 140 mg/ml.

In another embodiment, the method reduces the heterogeneity of the recombinant proteins produced, wherein said reduction in heterogeneity includes reducing:

a) charge heterogeneity, preferably acidic peak group (APG); and/or

b) oxidation of amino acids, isomerization, fragmentation, glycation of other covalent adducts, deamidation, cysteinylation; and/or

c) color or color intensity, for example between different batches of recombinant protein; and/or

d) high molecular weight species (HMWS); and/or

e) instability of the recombinant protein.

In a further aspect, the invention relates to a recombinant protein preparation that can be or is produced by the method of the invention. In one embodiment, the drug is a bulk drug. In other embodiments, for example, when the method includes the additional steps of producing a product in the form of a protein drug formulation, the resulting drug is a protein drug, for example, a drug suitable for administration to a patient.

The recombinant proteins, preferably antibodies or antigen-binding fragments thereof, in said preparation thus obtained have reduced heterogeneity relative to the same recombinant proteins obtained in the same manner, but in which during the production phase the total amount of cysteine or cystine and/or tryptophan is not limited, as described in the present invention.

In another further aspect, the invention relates to a pharmaceutical composition containing an antibody, wherein the composition is a liquid composition with one or more amino acids and a surfactant.

In one embodiment, the pharmaceutical composition contains histidine and/or proline.

In a further embodiment, the pharmaceutical composition contains histidine and/or proline.

In a further embodiment, the pharmaceutical composition contains histidine at a concentration of 5 mM to 100 mM, for example at a concentration of 10 to 50 mM, and/or proline at a concentration of 100 to 500 mM at a pH of 5 to 7.4, for example 5 to 6.5, for example, from 5 to 6, for example, from 5.5 to 6.

In a further embodiment, the pharmaceutical composition contains 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.

In a further embodiment of the pharmaceutical composition, the surfactant is polysorbate 80, preferably at a concentration of from 0.001% to 0.1% w/v, for example from 0.005% to 0.1%, for example from 0.01% to 0.1%, for example 0.01% to 0.05%, for example 0.03%.

In a further embodiment of the pharmaceutical composition, the antibody is present in the composition at a concentration of 10 to 250 mg/ml, such as 20 to 250 mg/ml, such as 50 to 250 mg/ml, such as 120 to 160 mg/ml, such as about 140 mg/ml.

In a further embodiment of the pharmaceutical composition, the antibody is:

1) an antibody or its antigen-binding fragment, which

a) contains CDR-H1 having the sequence defined in SEQ ID NO: 1; CDR-H2 having the sequence defined in SEQ ID NO: 2; CDR-H3 having the sequence defined in SEQ ID NO: 3; CDR-L1 having the sequence defined in SEQ ID NO: 4; CDR-L2 having the sequence defined in SEQ ID NO: 5, and CDR-L3 having the sequence defined in SEQ ID NO: 6; or

b) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having the sequence defined in SEQ ID NO: 8; or

- 13 044551

c) contains a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity, with the sequence defined in

SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity, with the sequence defined in

SEQ ID NO: 8;

d) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain having the sequence defined in SEQ ID NO: 11; or

e) contains a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity with 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

2) an antibody that contains 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; or

3) an antibody that contains a light chain having at least 80% identity or similarity, preferably 90% identity or similarity with 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.

Host cells and culture conditions.

The recombinant protein, antibody, or antigen binding fragment thereof can preferably be produced by culturing mammalian host cells, most preferably Chinese hamster ovary (CHO) cells.

The term cell culture or grammatical variations thereof includes, without limitation, a plurality of host cells, preferably mammalian host cells, suitably engineered and/or manipulated to express (i.e., produce) one or more recombinant proteins that support or grown in cell culture medium for a specified period of time, such as the production phase.

Mammalian cells, and in particular CHO cells, can be cultured in any medium that is capable of supporting their growth and expression of the recombinant protein; preferably the medium is a medium that does not contain animal products such as animal serum and peptone. Various cell culture media are available to those skilled in the art, each media containing various combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose, or equivalent energy source, which are present in appropriate concentrations necessary for cell growth and production. squirrel. Suitable media are described, for example, in WO 98/08934 and US 2006/0148074 (both documents are incorporated herein in their entirety). Other suitable commercially available media that can be used in the present invention or that can be modified to meet cysteine/cysteine and/or tryptophan requirements include AmpliCHO CD Medium, Dynamis™ Medium, EX-CELL Advanced CHO Fed Batch System, CD Medium FortiCHO™, OptiCHO™ CP Medium, Minimum Essential Medium (MEM), BalanCD® CHO Growth A Medium, ActiPro™ Medium, DMEM (Dulbecco's Modified Eagle's Medium) and RPMI-1640 Medium.

The cell culture may be in any suitable container, such as a shake flask or bioreactor, which can be operated in a fed-batch or dry mode depending on the scale of production required. These bioreactors can be stirred tank reactors or airlift reactors. Various large-scale bioreactors are available with capacities ranging from 1000 to 50,000 L, preferably from 5000 to 20,000 L or up to 100,000 L. Alternatively, smaller volume bioreactors, such as 2 to 100 L, can also be used to produce the antibody or antibody fragment.

In a preferred embodiment of the present invention, regardless of where any previous phase (ie, growth phase) occurs, the production phase is carried out in a bioreactor or any other suspension culture container such as a shake flask or centrifuge tube. The production phase is preferably carried out in a fed-batch mode, but alternatively any other mode such as batch, continuous or chemostat mode can be used. In the case of continuous or chemostat mode, the ratios of the total amount of cysteine or cysteine and/or tryptophan used are calculated according to the continuous flow rate depending on the rate of removal of the produced recombinant protein from the production vessel.

In one embodiment, the method includes the step of isolating the recombinant protein from the cell culture medium and the additional step of purifying the recombinant protein.

In a further embodiment, purification includes Protein A chromatography.

In a further embodiment, the method includes the additional step of preparing a purified recombinant protein composition.

- 14 044551

In one embodiment, the recombinant protein formulation is formulated as a liquid formulation containing one or more amino acids and a surfactant.

In a further embodiment, the composition contains histidine and/or proline.

In another further embodiment, the composition contains histidine at a concentration of 5 mM to 100 mM, for example at a concentration of 10 to 50 mM, and/or proline at a concentration of 100 to 500 mM at a pH of 5 to 7.4, for example 5 to 6.5, for example from 5 to 6, for example from 5.5 to 6.

In another further embodiment, the composition contains 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.

In a further embodiment, the surfactant is polysorbate 80, preferably at a concentration of 0.001% to 0.1% w/v, such as 0.005% to 0.1%, such as 0.01% to 0.1 %, for example from 0.01% to 0.05%, for example 0.03%.

In another further embodiment, the recombinant protein is an antibody, wherein the antibody is present in the formulation at a concentration of 10 to 250 mg/ml, such as 20 to 250 mg/ml, such as 50 to 250 mg/ml, such as 120 to 160 mg /ml, for example about 140 mg/ml.

The recombinant protein, such as an antibody or an antigen-binding fragment thereof, is typically found in the supernatant of a mammalian cell culture, typically a CHO cell culture. In the case of CHO host cells, the antibody or antigen-binding fragment thereof is secreted into the supernatant, and said supernatant can be collected by methods known in the art, typically by centrifugation.

Recombinant proteins that can be produced by the method of the invention.

The method of the invention can be used to produce any type of recombinant polypeptide or protein, including, for example, peptides or larger proteins whose tertiary structure is significant, as well as, for example, glycoproteins and multimeric proteins. However, preferably the recombinant protein produced by the method of the invention is an antibody or an antigen-binding fragment thereof. As used herein, the term antibody or antibodies includes, for example, both monoclonal and polyclonal antibodies, as well as both monospecific and polyspecific antibodies, such as bispecific antibodies.

The term antibody or antibodies includes antibodies of any species, particularly mammalian species, typically containing two heavy chains and two light chains, human antibodies of any isotype, including IgA1, IgA2, IgD, IgG1, IgG2a, IgG2b, IgG3 IgG4 IgE and IgM, and modified ones thereof variants, and antibodies from non-human primates, such as chimpanzee, baboon, rhesus or cynomolgus monkey, antibodies from rodents, such as mouse, rat or rabbit; goat or horse antibodies and derivatives thereof, or antibodies from birds, such as chicken species, or fish, such as shark species. The term antibody or antibodies also refers to chimeric antibodies in which the first portion of at least one antibody heavy and/or light chain sequence belongs to a first species and the second portion of the antibody heavy and/or light chain sequence belongs to a second species. Chimeric antibodies of interest herein include primatized antibodies containing antigen binding variable domain sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences . Humanized antibodies are chimeric antibodies that contain sequence derived from non-human antibodies. In general, humanized antibodies are human antibodies (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region or complementarity determining region (CDR) of a non-human species (donor antibody), such as mouse, rat, rabbit, chicken or non-human primate having the required specificity, affinity and activity. In most cases, the remnants of the human (recipient) antibody are outside the CDR; those. in the framework region (FR), are further replaced by corresponding non-human remains. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. These modifications are performed to further improve the characteristics of the antibodies. Humanization reduces the immunogenicity of non-human antibodies in humans, thereby facilitating the use of antibodies for the treatment of human diseases. Humanized antibodies and several different technologies for their production are well known in the art. The term antibody or antibodies also refers to human antibodies that can be generated as an alternative to humanization. For example, it is possible to produce transgenic animals (eg, mice) that are capable of producing the full repertoire of human antibodies after immunization without producing endogenous murine antibodies. For example, it has been reported that homozygous deletion of the antibody heavy chain junction (JH) region gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of a pool of human germline immunoglobulin genes into such mutant mouse germlines will result in the production of human antibodies with specificity for a particular antigen when a transgenic animal carrying the germline immunoglobulin genes is immunized with said antigen

- 15,044,551 people. Technologies for producing such transgenic animals and technologies for isolating and obtaining human antibodies from such transgenic animals are known in the art. Alternatively, in a transgenic animal, such as a mouse, only the immunoglobulin genes encoding the murine antibody variable regions are replaced with the corresponding human immunoglobulin variable region gene sequences. The mouse germline immunoglobulin genes encoding the antibody constant regions remain unchanged. Thus, the effector functions of antibodies in the immune system of the transgenic mouse and, consequently, the development of B cells are largely unaffected, which may lead to an improved antibody response to antigen challenge in vivo. Once genes encoding a particular antibody of interest are isolated from such transgenic animals, the genes encoding the constant regions can be replaced with human constant region genes, thereby producing a fully human antibody. As used herein, the term antibody or antibodies also refers to an aglycosylated antibody.

As used herein, the term antigen-binding fragment or grammatical variations thereof refer to an antibody fragment. The antibody fragment contains at least one immunoglobulin heavy or light chain domain, as known in the art, and binds to one or more antigens. Examples of antibody fragments of the invention include Fab, Fab', F(ab') ₂ and Fv and scFv fragments; a also diabodies, triantbodies, tetrabodies, minibodies, domain antibodies (dAbs) such as sdAb, VHH or camelid antibodies (e.g. those derived from camels or llamas such as Nanobodies™) and VNAR fragments, single chain antibodies, bispecific, trispecific , tetraspecific or polyspecific antibodies formed from antibody fragments or antibodies, including, without limitation, Fab-Fv or Fab-Fv-Fv constructs. The antibody fragments defined above are known in the art.

In a particularly preferred embodiment, the antibody or antigen binding fragment thereof produced by the methods of the invention is (Table 1):

1) an antibody or its antigen-binding fragment, which

a) contains CDR-H1 having the sequence defined in SEQ ID NO: 1; CDR-H2 having the sequence defined in SEQ ID NO: 2; CDR-H3 having the sequence defined in SEQ ID NO: 3; CDR-L1 having the sequence defined in SEQ ID NO: 4; CDR-L2 having the sequence defined in SEQ ID NO: 5, and CDR-L3 having the sequence defined in SEQ ID NO: 6; or

b) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having the sequence defined in SEQ ID NO: 8; or

c) contains a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity with the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;d. contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain having the sequence defined in SEQ ID NO: 11; or

e) contains a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity with 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

2) an antibody that contains 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; or

3) an antibody that contains a light chain having at least 80% identity or similarity, preferably 90% identity or similarity with 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.

In the present description, complementarity determining regions (CDRs) are defined in accordance with the definition according to Kabat. The Kabat designation is a standard for numbering residues in an antibody and is commonly used to identify CDR regions (Kabat et al., (1991), 5th edition, NIH Publication No. 91-3242).

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Region and op SEQ ID Amino acid sequence CDR-H1 GFTFSNYGMV SEQ GO NO: 1 CDR-H2 YIDSDGDNTYYRDSVKG SEQ GO NO:2 CDR-H3 GIVRPFLY SEQ ID NO:3 CDR-L1 KSSQSLVGASGKTYLY SEQ ID NO:4 CDR-L2 LVSTLDS SEQ ID NO:5 CDR-L3 LQGTHFPHT SEQ ID NO:6 Variable DIQMTQSPSS LSASVGDRVT ITCKSSQSLV GASGKTYLYW lung area LFQKPGKAPK RLIYLVSTLD SGIPSRFSGS GSGTEFTLTI chains SSLQPEDFAT YYCLQGTHFP HTFGQGTKLE IK SEQ GO NO:7 Variable EVPLVESGGG LVQPGGSLRL SCAVSGFTFS NYGMVWVRQA region PGKGLEWVAY IDSDGDNTYY RDSVKGRFTI SRDNAKSSLY heavy chain LQMNSLRAED TAVYYCTTGIVRPFLYWGQG TLVTVS SEQ GO NO:8 Light chain DIQMTQSPSS LSASVGDRVT ITCKSSQSLV GASGKTYLYW SEQ GO NO:9 LFQKPGKAPK RLIYLVSTLD SGIPSRFSGS GSGTEFTLTI SSLQPEDFAT YYCLQGTHFP HTFGQGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC Heavy chain EVPLVESGGG LVQPGGSLRL SCAVSGFTFS NYGMVWVRQA SEQIDNO: 10 PGKGLEWVAY IDSDGDNTYY RDSVKGRFTI SRDNAKSSLY LQMNSLRAED TAVYYCTTGI VRPFLYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK Heavy chain EVPLVESGGG LVQPGGSLRL SCAVSGFTFS NYGMVWVRQA

- 17 044551

Fab SEQ GO NO: 11 PGKGLEWVAY IDSDGDNTYY RDSVKGRFTI SRDNAKSSLY LQMNSLRAED TAVYYCTTGI VRPFLYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC

The recombinant protein or preferred antibody or antigen binding fragment thereof can typically be produced by host cells containing a vector encoding the nucleotide sequence of the protein or antibody.

The antibodies or antigen binding fragment thereof may contain only a heavy or light chain protein, in which case only the sequence encoding the heavy chain or light chain protein is used to transfect cells. To produce products containing both heavy and light chains, cells can be transfected with two vectors: a first vector encoding a light chain protein and a second vector encoding a heavy chain protein. Alternatively, a single vector may be used that includes sequences encoding light chain and heavy chain proteins.

In a preferred embodiment, the invention relates to a method for producing an antibody or antigen binding fragment thereof, comprising:

a) culturing CHO cells capable of producing an antibody or an antigen-binding fragment into the medium;

b) passing the culture through a production phase in which the cells produce the antibody or antigen-binding fragment thereof, wherein during said production phase the culture is fed with cysteine or cystine to a total amount of from 10 to 30% by weight of the expected total amount of antibody or antigen-binding fragment thereof produced;

c) and, optionally, isolating the antibody or antigen-binding fragment thereof from the cell culture medium, wherein the total amount of cysteine or cystine provided in the method is from 2.9 to 12 g/(10 ¹² cells), for example from 2.9 up to 7 g/(10 ¹² cells), for example from 5.6 to 7 g/(10 ¹² cells), wherein the cells refer to the expected total number of viable cells at the end of the production phase, and wherein cysteine or cystine is provided by daily addition during production phase, and wherein the concentration of cysteine or cystine in the cell culture medium does not exceed 0.9 g/l at any time during the production phase, preferably when the concentration of cysteine or cystine in the cell culture medium does not exceed 0.3 g/ l at any time during the production phase, and wherein said antibody or antigen-binding fragment thereof is preferably

a) contains CDR-H1 having the sequence defined in SEQ ID NO: 1; CDR-H2 having the sequence defined in SEQ ID NO: 2; CDR-H3 having the sequence defined in SEQ ID NO: 3; CDR-L1 having the sequence defined in SEQ ID NO: 4; CDR-L2 having the sequence defined in SEQ ID NO: 5, and CDR-L3 having the sequence defined in SEQ ID NO: 6; or

b) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having the sequence defined in SEQ ID NO: 8.

In a further preferred embodiment, the invention relates to a method for producing an antibody or antigen binding fragment thereof, comprising:

a) culturing CHO cells capable of producing an antibody or an antigen-binding fragment into the medium;

b) passing the culture through a production phase in which the cells produce the antibody or an antigen-binding fragment thereof, wherein during said production phase the cell culture medium is fed with:

cysteine or cystine to a total amount of from 10 to 30 wt.% of the expected total amount of produced antibody or antigen-binding fragment; and/or tryptophan to a total amount of 8 to 35% by weight of the expected total amount of antibody or antigen-binding fragment thereof produced,

c) and, optionally, isolating the antibody or antigen binding fragment thereof from the cell culture medium, wherein the total amount of cysteine or cystine provided in the method is from 2.9 to 12 g/(10 ¹² cells), for example from 2.9 up to 7 g/(10 ¹² cells), for example from 5.6 to 7 g/(10 ¹² cells), cells being relative to the expected total number of viable cells at the end of the production phase, and

- 18 044551 wherein the total amount of tryptophan provided in the method is from 2.5 to 7 g/(10 ¹² cells), for example from 2.5 to 3.5 g/(10 ¹² cells), wherein the number of cells refers to the expected total number of viable cells at the end of the production phase, and wherein cysteine or cystine and/or tryptophan is provided by daily addition during the production phase, and wherein the concentration of cysteine or cystine in the cell culture medium does not exceed 0.9 g/l at any time point in time during the production phase, preferably when the concentration of cysteine or cystine in the cell culture medium does not exceed 0.3 g/l at any time during the production phase, and the concentration of tryptophan in the cell culture does not exceed 0.6 g/l medium at any time during the production phase, preferably when the concentration of tryptophan in the cell culture medium does not exceed 0.3 g/l at any time during the production phase, and wherein said antibody or antigen-binding fragment thereof is preferably

a) contains CDR-H1 having the sequence defined in SEQ ID NO: 1; CDR-H2 having the sequence defined in SEQ ID NO: 2; CDR-H3 having the sequence defined in SEQ ID NO: 3; CDR-L1 having the sequence defined in SEQ ID NO: 4; CDR-L2 having the sequence defined in SEQ ID NO: 5, and CDR-L3 having the sequence defined in SEQ ID NO: 6; or

b) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having the sequence defined in SEQ ID NO: 8.

The invention is further described below by way of examples with reference to the embodiments shown in the accompanying drawings.

Examples

Abbreviations:

mAb: monoclonal antibody;

MFCS: Multi-Fermentation Control System;

Cys: cysteine or cystine;

Tgr: tryptophan

Materials and methods.

Cell line, cell culture and experimental procedure.

The cell line CHO-DG44 was used. Cells were cultured in chemically defined inoculation medium without animal components containing cystine (0.05 g/L) and tryptophan (0.2 g/L) under standard operating conditions (pH 7, temperature 36.8°C ) in a glass 2 L stirred bioreactor with feed towers (C-DCUII, Sartorius Stedim Biotech) controlled by a multifermentation control system (Sartorius Stedim Biotech). Four different producer cell lines were used, each of which produced a monoclonal antibody (mAb), designated mAb1, mAb2, mAb3 and mAb4, respectively. mAb1 is an anti-FcRn 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.

The production process was carried out in an experimental mode with periodic feeding for 14 days. During this phase, monoclonal antibodies were secreted into the medium. Samples were collected daily for determination of viable cell density (VCD), viability, off-line pH, pCO2, osmolality, glucose-lactate concentration, amino acid concentration, and mAb concentration (which were stored at -80°C). If necessary, antifoam was added manually each day to prevent foam formation. 72 hours after inoculation, the process of continuous feeding with nutrients began at a given rate. The continuous nutrient feeding medium did not contain cysteine/cystine or tryptophan. During this time, cysteine/cystine and tryptophan were added daily for 10 days as a feed bolus in the amounts shown in the examples below. The amount of cysteine/cystine and tryptophan indicated in the examples represents the total amount of bolus supplements that began to be administered 72 hours after inoculation, taking into account the initial amount of these amino acids already present in the inoculation medium. The culture was fed with a glucose bolus when the glucose concentration fell below 6 g/L (starting on day 6), and the glucose concentration was measured daily. Before adding feed, samples were taken for amino acid analysis. Post-feed concentrations were calculated based on the feed composition and the measured nutrient concentrations before the addition of the feed.

Analytical methods.

Cell counts were performed using a VI-CELL® XR automated cell counting device (Beckman-Coulter, Inc., Brea, CA), which operated on a trypan blue exclusion basis.

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

To determine osmolality, we used a model 2020 freezing point osmometer (Advanced

- 19 044551

Instruments, Inc., Norwood, MA). Offline gas and pH measurements were performed using a BioProfile pHOx® blood gas analyzer (Nova Biomedical Corporation,

Waltham, MA).

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

Product titer analysis was performed using a ForteBio Octet model analyzer (ForteBio, Inc., Menlo Park, Calif.) or high pressure liquid chromatography (HPLC) using cell culture supernatant samples that were stored at -80°C prior to analysis.

Amino acids were analyzed by reverse-phase ultraperformance liquid chromatography (UPLC) (Waters AccQTagultra method) after ultrafiltration using Amicon Ultra-0.5 mL centrifugal filters (Merck Millipore, Billerica, MA).

Protein A purification (AKTA Xpress system) was used to purify mAbs from cell culture supernatant samples. The relative percentages of major, acidic (APG for acid peak group) and alkaline (BPG for alkaline peak group) isoforms of purified mAb were determined by capillary electrophoresis with visualization of the results (ProteinSimple iCE3). High molecular weight species (HMWS), monomeric and low molecular weight species (LMWS) levels of purified mAb were determined by size exclusion chromatography (SE-UPLC).

The color intensities of concentrated mAb1 and mAb2 formulations were measured in concentrated Protein A eluates using a transmission mode spectrophotometer (UltrascanPro) and compared according to the Internationale de L'eclairage (SP.) scale. Numerical results were normalized to a concentration of 40 mg/ml.

Purified mAb1 was analyzed by electrospray ionization mass spectrometry (ESI-MS). Peptide mapping was performed to identify post-translational modifications of the antibodies. Statistical analysis was performed using SAS JMP 11 software.

Example 1.

CHO cells producing mAb1 were inoculated into two-liter bioreactors at a seeding density of 0.35x10 ⁶ cells/ml. Eight experimental conditions were tested in the fed-batch process as described in Materials and Methods, with different maximum concentrations of cysteine or cystine and tryptophan achieved during cell culture and different total amounts of cysteine or cystine (Cys) and tryptophan (Trp). , expressed as wt.% of the total mass of mAb1 produced (Table 2a). The first goal was to evaluate the effect of cysteine or cystine and tryptophan on the heterogeneity of recombinant mAb1. The second objective was to determine whether this effect was due to the high concentrations of cysteine or cystine and/or tryptophan achieved during cell culture and/or the total amount added, expressed as % wt of the total mAb produced.

Table 2a

Bioreactor or ID Max. Cys concentration Total Cys/mAbl wt% (g/g) Max. Tgr concentration Total Trp/mAbl wt% (g/g) 1 0.05 g/l 17.51 0.20 g/l 11.79 2 0.05 g/l 13.35 0.20 g/l 10.34 3 0.30 g/l 15.72 0.30 g/l 10.58 4 0.05 g/l 18.94 0.20 g/l 11.27 5 0.12 g/l 16.18 0.20 g/l 9.87 6 0.05 g/l 13.30 0.20 g/l 8.96 7 0.05 g/l 20.79 0.20 g/l 12.37 8 0.05 g/l 20.840612 0.20 g/l 8.5684363

Charged variants of the recombinant protein and color intensity were measured as described in Materials and Methods. Data were analyzed by one-way Anova statistical analysis for a linear model, and p values <0.05 were accepted as statistically significant.

As can be seen from FIG. 2b, there is a correlation between the increased level of the charged acidic peak group variant of mAb1 (APG%) and the increase in the total amount of added cysteine or cystine per total weight percent (g/g) of mAb1 produced.

Regarding the color intensity of mAb1, there is a correlation between the increased color intensity of mAb1 (b* value normalized to 40 mg/ml) and the increased total amount of added tryptophan based on the total weight percentage (g/g) of mAb1 produced (Fig. 2a).

However, when the data were analyzed against the maximum concentration of tryptophan, cysteine, or cystine, it was found to have no effect on color or APG (Figures 2c and 2d).

To confirm that it is the total amount of cysteine or cystine and tryptophan per total mass of mAb1 produced that influences mAb1 heterogeneity, rather than the maximum concentration of cysteine or cystine and/or tryptophan achieved during the fed-batch production phase, we tested 8 experimental conditions with different bolus

- 20 044551 supplementation with cysteine or cystine and tryptophan on day 3 to achieve high concentrations of these 2 amino acids (Table 2b). The feeding strategy was adapted to obtain the same amount of added cysteine or cystine and tryptophan in wt% (g/g) of the total mass of mAb1 produced. As seen from fed-batch conditions, there is a correlation between the increased level of mAb1's charged variant acidic peak group (APG%) and the increase in added total cysteine or cystine per total mAb1 wt% (g/g) produced (Fig. 3b) and between the increased color intensity of mAb1 (b* value normalized to 40 mg/ml) and the increased added total amount of tryptophan based on the total amount of mAb1 produced in wt.% (g/g) (Fig. 3a). However, there is no correlation between the charged APG variant and the maximum concentrations (g/L) of cysteine or cystine and tryptophan (Fig. 3c and 3d, respectively). These results confirm that the charged APG variant and color intensity are influenced by the total amount of cysteine and tryptophan added during cell culture based on the wt% (g/g) of the total mass of mAb1 produced. However, the maximum concentration of cysteine or cystine and tryptophan does not have any effect on the charged version of APG and color intensity.

Table 2b

Ш bioreactor Maximum Cys concentration (g/l) Maximum concentration Tgr (g/l) Total Cys/mAbl wt% (g/g) Total Trp/mAbl wt% (g/g) 9 0.06 0.20 13.85 9.34 10 0.06 0.60 17.31 11.71 eleven 0.90 0.60 16.00 10.79 12 0.48 0.38 13.60 9.17 13 0.90 0.38 13.12 8.88 14 0.90 0.20 13.10 8.84 15 0.06 0.20 14.66 9.88 16 0.48 0.38 13.85 9.37

Conclusions.

The total amount of Cys and Trp added in wt.% of the total amount of recombinant mAb1 produced (g/g) influences the charged variant of mAb1 and color intensity. In contrast, the maximum concentration of cysteine or cystine and tryptophan in the cell culture medium did not affect the quality of mAb1.

Example 2.

To further study the effect of added total cysteine or cystine and tryptophan in wt% of total mAb produced (g/g) during cell culture, 48 experimental conditions (Table 3) were used in 2 L bioreactors as described in Materials and Methods. . The charged mAb1 variant, aggregates (HMWS), color intensity, titer, and viable cell growth were analyzed.

- 21 044551

As shown in FIG. 4, the total amount of cysteine or cystine and tryptophan added during the production phase in wt.% of the total amount of mAb1 produced (g/g) affects the charged variant of the acidic group (APG%). The saturation effect is approximately 50 wt.% of the total amount of cysteine or cystine added in the 14-day production phase, based on the total amount of mAb1 produced (g/g). The effects of cysteine or cystine and tryptophan are cumulative without any interaction. Decreasing the percentage of total cysteine or cystine and tryptophan added during the 14-day production phase as a percentage by weight of the total amount of mAb1 produced (g/g) results in a decrease in the percentage of acidic peak pro

- 22 044551 inducible mAbl.

In fig. 5 shows the effect of the total amount of cysteine or cystine and tryptophan (added during 14 days of cell culture) in wt.% of the total mass of mAb1 produced on the group of main peaks. As can be seen from APG%, the effect of cysteine or cystine and tryptophan is cumulative without any interaction. Decreasing the total amount of cysteine or cystine and tryptophan added during the 14-day production phase as a percentage by weight of the total weight of mAb1 produced results in an increase in the percentage of the main peak of recombinant mAb produced.

In fig. 6 shows the effect of the total amount of cysteine or cystine added during the 14-day production phase in wt.% of the total amount of mAb1 produced (g/g), on high molecular weight species (HMWS). The saturation effect is approximately 50% of the total amount of cysteine or cystine added during the 14-day production phase in wt.% of the total amount of mAb1 produced (g/g). Reducing the total amount of cysteine or cystine results in a decrease in the percentage of HMWS in the recombinant mAb produced. There was no observed effect of the total amount of added Trp on HMWS.

The results shown in FIG. 7 demonstrate the effect of the total amount of cysteine or cystine and tryptophan added during the 14-day production phase in wt.% of the total amount of mAb1 produced (g/g) on color intensity (b* value normalized to 40 mg/ml) recombinant mAb1. Decreasing the total amount of cysteine or cystine and tryptophan added during the 14-day production phase results in a decrease in color intensity of the resulting recombinant mAb1. The effect of the total amount of cysteine or cystine and tryptophan added during the 14-day production phase in wt.% of the total amount of mAb1 produced (g/g) is observed.

In fig. 8 shows contour plots demonstrating the optimal ranges for the percentage of total cysteine or cystine and tryptophan added during the 14-day production phase as % wt of total mAb1 produced (g/g) to achieve the lowest APG, HMWS values. , color intensity and highest values for a group of main peaks; the added total amount of cysteine or cystine and tryptophan is for cysteine or cystine from 12.06 to 28.03 wt.% of the total amount of mAb-1 produced (g/g), and for tryptophan from 8.84 to 32.06 wt. % of total mAb1 (g/g).

The cumulative total number of viable cells (IVCC) during the 14-day production phase was calculated and normalized to cell culture volume (CSV). The results shown in Fig. 9 show the effect on IVCC of the percentage of total cysteine or cystine and tryptophan added to the cell culture medium based on the initial mass of CSV. There are optimal ranges for the percentage of total cysteine or cystine and tryptophan added to the cell culture medium based on the initial mass of CSV, which for cysteine or cystine range from 0.08% to 0.24% and for tryptophan range from 0. 07% to 0.15%. There was no synergistic effect and only a cumulative effect was observed (Figs. 9a and 9b).

In fig. 10a and 10b show the effect on the mAb1 titer of the percentage of total cysteine or cystine and tryptophan added to the cell culture medium based on the initial weight of CSV. There is an optimal range, expressed as a percentage of the total amount of cysteine or cystine and tryptophan added to the cell culture medium based on the weight of CSV, which is for cysteine or cystine from 0.08% to 0.24% and for tryptophan from 0.07% up to 0.15% wt.% by weight of CSV. There is no interaction effect.

The contour plots shown in Fig. 11a and 11b show the optimal ranges for the total amount of cysteine or cystine and tryptophan added to the cell culture medium per IVCC* 10 ^-12 at the end of the production phase, which is for Cys from 2.9 to 12 g and for Trp from 2 .5 to 7 years

Example 3.

The recombinant monoclonal antibody was characterized using 3 experimental conditions in a fed-batch mode as described in Materials and Methods, with the addition of varying total amounts of cysteine or cystine and tryptophan in wt % of the total amount of recombinant mAb1 produced (Table 4).

Table 4

Ш bioreactor Total Cys/mAbl wt% (g/g) Total Trp/mAbl wt% (g/g) 65 11.72 7.21 66 22.16 11.89 67 94.20 46.72

- 23 044551

Mass spectroscopic analysis showed a shift in the mass of the most intense peak observed in the mass spectrum under nondenaturing and denaturing conditions and for glycosylated mAb1 as a result of increasing the concentration of cysteine or cystine and tryptophan. These observations lead to the conclusion that the modifications are not due to changes in glycosylation patterns. Analysis of light chain, heavy chain and half-mer (one heavy chain plus one light chain) mass spectra after manual deconvolution suggests the possibility of glycation of mAb1 at high total cysteine or cystine and tryptophan added during the 14-day production phase in wt.% of total mass of mAb produced. It is likely that a higher content of adducts can be observed, i.e. attachment of small molecules to mAb1. The adduction of cysteine to the light chain increases as the total amount of cysteine or cystine and tryptophan added increases. In table Figure 5 summarizes the peptide mapping characteristics of mAb1 for the three experimental conditions tested. The results indicate that increasing the total amount of cysteine or cystine and tryptophan added during the 14-day production phase, as a percentage by weight of the total mAb produced, results in increased oxidation of methionine in heavy chain threonine 19 and deamidation of heavy chain threonine 33. Additionally, the APG% and BPG% mAb1 variants increase sharply with increasing total cysteine or cystine and tryptophan added in wt% of total mAb produced during the 14-day production phase, while the main peak increases with decreasing total cysteine or cystine and tryptophan added in wt.% of the total mass of mAb produced during the 14-day production phase.

___________________________Table 5

Ш bioreactor

Characteristics of mAb 1 65 66 67 Oxidation of methionine NS TO 19 10.60% 15.20% 19.70% Deamination NS T023 2.60% 2.10% 2.20% Deamination NS TOZZ 4.80% 6.60% 7.00% APG (%) 39.30% 49.10% 82.90% BPG (%) 6.10% 7.30% 2.40% Main (%) 54.70% 43.70% 14.80%

Example 4.

To determine the growth inhibitory concentration of cysteine or cystine and tryptophan in the mAb1-expressing CHO cell line DG44, various bolus supplements of cysteine or cystine and tryptophan were tested on day 3 and administered to achieve high concentrations of these amino acids (Table 2b). To obtain the same amount of added cysteine or cystine and tryptophan in wt.% of the total mass of mAb1 produced, a feeding strategy was adapted. In fig. Figure 12 shows that high concentrations of cysteine or cystine and tryptophan from 0.3 g/L to 0.9 g/L and 0.6 g/L, respectively, significantly reduce cell growth (cumulative IVCC during the 14-day production phase, normalized to CSV).

Example 5.

It has been hypothesized that depletion of cysteine or cystine may affect the growth and productivity of the mAb1-expressing CHO cell line. Nine experimental conditions were analyzed in 2 L bioreactors (Table 6a): three control conditions without cysteine or cystine depletion throughout the production phase, two experimental conditions with daily depletion starting from day 6 until the end of the fed-batch production process, in which the cysteine or cystine concentration was 6.87 g/L, and four experimental conditions with cysteine or cystine depletion on day 6 and a fed cysteine or cystine concentration of 17.17 g/L. The wasting was cyclical due to daily supplementation of cysteine or cystine. The recharge 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 is shown in FIG. 13. Cys concentrations before feed addition are shown in FIG. 14s. As shown in FIG. 14a, depletion of cysteine or cystine on day 6 does not affect cell growth if the concentration of cysteine or cystine in the feed is approximately 17.17 g/L. Without being limited to any theory, it has been proposed that cysteine or cystine-related metabolites accumulate and are stored in cells and become available when cysteine or cystine is depleted. However, depletion of cysteine or cysteine affects the efficiency of mAb1 production by the cell line (Fig. 14b).

- 24 044551

Table 6a

Ш bioreactor Cys concentration in added feed (g/l) Total amount of Cys added per IVCC on day 14 (g/10 ¹² cells) 68 34.35 12.24 69 34.35 13.10 70 34.35 9.51 71 6.87 4.32 72 6.87 4.29 73 17.17 4.69 74 17.17 5.07 75 17.17 4.59 76 17.17 4.24

Table 6b

Cultivation Day Added makeup amount (%CSV/24h) 0 0 1 0 2 0 3 0.2 4 0.28 5 0.36 6 0.36 7 0.68 8 0.68 9 0.84 10 0.84 eleven 0.72 12 0.72 13 0 14 0

Example 6.

The effect of reducing heterogeneity due to controlling the total amount of cysteine or cystine and tryptophan added in wt.% of the total amount of recombinant mAb during the production phase is not exclusive to mAb1; In experiments with added total cysteine or cystine and tryptophan, three other CHO cell lines were tested, also producing recombinant antibodies (Table 7). As shown in FIG. 15a, increases in APG charged variant levels and color intensity correlate with increases in total cysteine or cystine and tryptophan added in wt% of total mAb2. Similar results were obtained when analyzing the charged APG variant of mAb3 (15b). Finally, the increase in the level of charged variants APG and BPG and the decrease in the main peak correlated with an increase in the total amount of added cysteine or cystine and tryptophan in wt.% of the total amount of mAb4 (15c). These results confirm the results obtained for mAb1.

- 25 044551

Table 7

Bioreactor or ID mAb Total Cys/mAb % (g/g) Total Trp/mAb% (g/g) 77 mAb2 24.02 14.89 78 mAb2 53.01 29.09 79 mAb2 33.35 21.81 80 mAb2 58.67 32.26 81 mAb3 15.97 17.25 82 mAb3 10.92 16.26 83 mAb4 81.15 57.12 84 mAb4 170.18 62.51 85 mAb4 151.22 55.55 86 mAb4 85.45 60.14

Example 7.

The effect of the total amount of cysteine or cystine and tryptophan added as % wt of the total weight of the recombinant mAb produced during the 14-day production was analyzed from data obtained for the four different monoclonal antibodies tested herein (Tables 3 and 7). As shown in FIG. 16 (a and b), for all four antibodies analyzed, an increase in the level of the charged APG variant correlates with an increase in the total amount of added cysteine or cystine and tryptophan in wt.% of the total mass of recombinant antibodies produced. The results confirm that the relationship between the added total amount of cysteine or cystine and tryptophan in wt.% of the total weight of the produced recombinant antibody and the heterogeneity of the antibody is not limited to a specific antibody, but applies to any antibody.

Example 8.

Liquid pharmaceutical formulations.

Pharmaceutical formulations of mAbl monoclonal antibody were prepared in a fed-batch mode, i.e. in a 2000 L stainless steel bioreactor under standard operating conditions described in Materials and Methods, with the addition of varying total amounts of cysteine or cystine and tryptophan as indicated in Table. 9. Antibody sample buffer was replaced with diafiltration buffer (33 mM His and 250 mM Pro, pH 5.6) at least 7 times (7 dialysis volumes) followed by ultrafiltration through a 30 kDa molecular weight cutoff (MWCO) membrane. Once the antibody concentration was reached (140 mg/ml ±14 mg/ml), polysorbate 80 was added at the required concentration (0.03% w/v based on final concentration). Antibody concentration was measured using UV A280.

In table 8 shows the charged variants of mAbl in the composition. In the two production processes defined in Table. 8 and 9 as higher addition of Cys, the total amount of added cysteine or cystine in wt.% of the total amount of recombinant mAbl produced was greater compared to the other three production processes defined in table. 8 and 9 as lower addition of Cys. The increase in the level of charged APG variants correlates with an increase in the total amount of added cysteine or cystine in wt.% of the total amount of recombinant mAb 1 produced.

Claims (28)

Table 8 Higher amount of Cys added (Try #1) Higher amount of Cys added (Try #2) Lower amount of Cys added (Try #1) Lower amount of Cys added (Try #2) Lower amount added th Cys (experiment #3) iCE %APG 50.4% 45.5% 38.2% 36.4% 41.3% iCE % main peak 40.6% 44.7% 54.6% 54.9% 48.7% iCE %BPG 9.0% 9.8% 7.2% 8.7% 9.9% Higher amount of added Cys (run #1) Higher amount of added Cys (run #2) More low amount of Cys added (run #1) Lower amount of Cys added (run #2) lower amount of Cys added (run #3) Total Cys/mAbl wt.% (g/g) 15.66 14, 10 12.20 12.36 12.77 Total Trp/mAbl 8.89 8.00 8.13 8.22 8.50 wt.% (g/g) Example 9: Model for predicting the level of acidic species peak group ( APG) was developed based on data (Table 3) obtained for the CHO DG44 cell line expressing gpAA antibodies (Fig. 17). APG was expressed as a function of the total amount of cysteine or cystine and tryptophan added as % wt of the total weight of the recombinant mAb produced during 14 days of production using the Michaelis-Menten kinetic model. To apply this model to a continuous production process, each day during the continuous production process was defined as a new production batch. Therefore, the ratios of the total amount of cysteine or cysteine and/or tryptophan used were calculated according to the continuous flow rate depending on the rate of removal of the produced recombinant protein from the production vessel. The continuous production process was carried out in 2-liter bioreactors using Alternating Tangential Flow (ATF) technology. As shown in FIG. 18, the prediction of the charged variant of APG agrees well with the experimental data. The results support the ability to approximate the relationship between the added total amount of cysteine or cystine and tryptophan in wt.% of the total mass of the produced recombinant antibody and the heterogeneity of the antibody to other production modes, such as production in a continuous, batch or chemostat mode. CLAIM 1) an antibody or its antigen-binding fragment, which a) contains CDR-H1 having the sequence defined in SEQ ID NO: 1; CDR-H2 having the sequence defined in SEQ ID NO: 2; CDR-H3 having the sequence defined in SEQ ID NO: 3; CDR-L1 having the sequence defined in SEQ ID NO: 4; CDR-L2 having the sequence defined in SEQ ID NO: 5, and CDR-L3 having the sequence defined in SEQ ID NO: 6; or b) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having the sequence defined in SEQ ID NO: 8; or c) contains a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity with the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity , preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8; d) contains a light chain variable region having the sequence defined in SEQ ID NO: 7, and a heavy chain having the sequence defined in SEQ ID NO: 11; or e) contains a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity with 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 1. A method for producing a recombinant protein, including: a) culturing host cells capable of producing the recombinant protein in a medium that does not contain animal products; b) passing the culture through a production phase in which the cells produce the recombinant protein, the culture being fed during said production phase -27044551 cysteine or cystine to a total amount of from 10 to 30 wt.% of the expected total amount of recombinant protein produced; and tryptophan to a total amount of from 8 to 35 wt.% of the expected total amount of recombinant protein produced, c) and isolating the recombinant protein from the cell culture medium, wherein the recombinant protein is an antibody or an antigen binding fragment thereof, wherein the host cells are CHO cells, and performing one or more initial experiments to determine the expected total amount of recombinant protein produced . 2) an antibody that contains 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; or 2. The method of claim 1, wherein the culture is fed with cysteine or cystine to a total amount of from 12 to 28 wt.% of the expected total amount of recombinant protein produced, for example a total amount of from 12 to 25 wt.%, for example from 12 to 20 wt. .% of the expected total amount of recombinant protein produced. 3) an antibody that contains 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. 3. The method according to claim 1 or 2, wherein the culture is fed with tryptophan to a total amount of from 8 to 30 wt.% of the expected total amount of recombinant protein produced, for example a total amount of from 8 to 25 wt.%, for example from 8 to 20 wt. .% of the expected total amount of recombinant protein produced. 4. The method according to any of the preceding claims, wherein the total amount of cysteine or cystine provided in the method is from 2.9 to 12 g/(10 ¹² cells), for example from 2.9 to 7 g/(10 ¹² cells) , for example, 5.6 to 7 g/(10 ¹² cells), wherein the cells are relative to the expected total viable cell count at the end of the production phase, and wherein one or more initial experiments are performed to determine the expected total viable cell count at the end of the production phase . 5. The method according to any one of the preceding claims, wherein the total amount of tryptophan provided in the method is from 2.5 to 7 g/(10 ¹² cells), for example from 2.5 to 3.5 g/(10 ¹² cells) , where cells refer to the expected total number of viable cells at the end of the production phase. 6. The method as claimed in any one of the preceding claims, wherein the total amount of cysteine or cystine and tryptophan in the culture is achieved by adding cysteine or cystine and tryptophan to the cell culture medium: a) at the beginning of the production phase, b) one or more times at any time during the production phase, c) by continuous addition during the production phase or d) in any combination of a), b) and c). 7. The method according to any of the preceding claims, which is a batch process, such as a fed-batch process. 8. The method according to any of the preceding claims, wherein cysteine or cystine and tryptophan are provided by daily addition during the production phase. 9. The method of claim 8, wherein the culture is depleted of cysteine or cystine before adding cysteine or cystine the next day, for example by reducing the addition of cysteine or cystine to a level of 5.6 to 7 g/(10 ¹² cells), wherein the cells refer to the expected total number of viable cells at the end of the production phase. 10. The method according to claim 8 or 9, in which at a late stage of production, i.e. when cells have already reached maximum viable cell density, the culture is depleted of tryptophan before adding tryptophan the next day. 11. The method according to any of the preceding claims, wherein the concentration of cysteine or cystine in the cell culture medium does not exceed 0.9 g/l at any time during the production phase, preferably wherein the concentration of cysteine or cystine in the cell culture medium does not exceeds 0.3 g/l at any time during the production phase. 12. The method according to any of the preceding claims, wherein the tryptophan concentration in the cell culture medium does not exceed 0.6 g/l at any time during the production phase, preferably wherein the tryptophan concentration in the cell culture medium does not exceed 0.3 g/l at any time during the production phase. 13. Method according to any of the preceding claims, wherein the production phase is carried out for at least 7 days, preferably at least 14 days. 14. The method according to any of the preceding claims, wherein at any time during the second half of the production phase: the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the expected amount of produced recombinant protein; and the amount of tryptophan in the culture ranges from 8 wt.% to 35% of the expected amount of recombinant protein produced. 15. The method according to any one of the preceding paragraphs, in which at any point in time during - 28 044551 production phases: the amount of cysteine or cystine in the culture is from 10 wt.% to 30% of the expected amount of produced recombinant protein; and the amount of tryptophan in the culture ranges from 8 wt.% to 35% of the expected amount of recombinant protein produced. 16. The method according to any of the preceding claims, wherein the host cells are mammalian cells, preferably CHO cells. 17. The method according to any of the preceding claims, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof. 18. The method according to claim 17, in which the antibody or antigen-binding fragment thereof is: 19. The method according to any of the preceding claims, 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, 2000 L or more, 5000 L or more, 10,000 L or more or 20,000 L or more. 20. The method according to any one of the preceding claims, including the step of isolating the recombinant protein from the cell culture medium and the additional step of purifying the recombinant protein. 21. The method of claim 20, wherein the purification includes Protein A chromatography. 22. The method of claim 20 or 21, wherein the method further includes the step of preparing a purified recombinant protein composition, and wherein the recombinant protein composition is prepared in the form of a liquid composition containing one or more amino acids and a surfactant. 23. The method according to claim 22, in which the liquid composition contains histidine and/or proline. 24. The method according to claim 23, in which the liquid composition contains histidine at a concentration of 5 to 100 mM, for example at a concentration of 10 to 50 mM, and/or proline at a concentration of 100 to 500 mM at a pH of 5 to 7.4 , for example from 5 to 6.5, for example from 5 to 6. 25. The method according to claim 24, in which the liquid composition contains histidine at a concentration of 30 mm and proline at a concentration of 250 mm at a pH of 5.2 to 6.0. 26. The method according to any one of claims 22 to 25, wherein the surfactant is polysorbate 80, preferably in a concentration of 0.001% to 0.1% w/v, for example 0.005% to 0.1%, for example 0.01% to 0.1%, for example 0.01% to 0.05%, for example 0.03%. 27. The method according to any one of claims 22-26, in which the recombinant protein is an antibody, wherein the antibody is present in the composition at a concentration of from 10 to 250 mg/ml, for example from 20 to 250 mg/ml, for example from 50 to 250 mg/ml, for example 120 to 160 mg/ml, for example 140 mg/ml. 28. A method according to any of the preceding claims, providing a reduction in the heterogeneity of the resulting recombinant proteins, wherein said reduction in heterogeneity includes a reduction in: a) charge heterogeneity, preferably acidic peak group (APG); and/or b) oxidation of amino acids, isomerization, fragmentation, glycation of other covalent adducts, deamidation, cysteinylation; and/or -