WO2022223505A1

WO2022223505A1 - Control of n-terminal truncation by methionine supplementation

Info

Publication number: WO2022223505A1
Application number: PCT/EP2022/060221
Authority: WO
Inventors: Andreas KNEPPER; Stefan HUTWIMMER
Original assignee: Sandoz Ag
Priority date: 2021-04-19
Filing date: 2022-04-19
Publication date: 2022-10-27

Abstract

The present invention relates to controlling the amount of by-products resulting from N- terminal truncation of a polypeptide expressed in a host cell. In particular, the present invention relates to method of producing a recombinant polypeptide, in particular rhG-CSF or recombinant antibodies, with reduced impurities resulting from N-terminal truncation of said recombinant polypeptide.

Description

Control of N-terminal truncation bv methionine supplementation

The present invention relates to controlling the amount of by-products resulting from N- terminal truncation of a polypeptide expressed in a host cell. In particular, the present invention relates to method of producing a recombinant polypeptide, in particular recombinant human G- CSF (rhG-CSF) or recombinant antibodies, with reduced impurities resulting from N-terminal truncation of said recombinant polypeptide.

The biotechnological production of pharmaceutically relevant proteins brings about various challenges for the skilled person, inter alia due to the high regulatory standards for the purity of the pharmaceutically active protein. In order to achieve a level of purity complying with these standards, recombinantly produced proteins usually need to be purified after the initial isolation from the host cells. This process of removing impurities may involve several different steps. However, while each purification step improves the purity of the protein product, each purification step also brings about a loss in yield.

Impurities in a recombinant protein product may, inter alia, result from N-terminal truncation of the protein of interest. After isolation from the host cells the respective recombinant protein product is contaminated with fragments of the protein of interest, said fragments lacking one or more amino acids at the N-terminus. An example for a recombinant protein which is known to be prone to N-terminal truncation when recombinantly produced is recombinant human granulocyte colony stimulating factor. G-CSF is a polypeptide based hormone of mammals. It is a cytokine and stimulates inter alia the production of granulocytes. G-CSF also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. The natural human glycoprotein exists in two forms, a (more active) 174- and (less active) 177-amino-acid-long polypeptide.

The 174 amino acid long version of rhG-CSF has been used for several pharmaceutical applications. In oncology and hematology, rhG-CSF is used with certain cancer patients to accelerate recovery from neutropenia (i.e. abnormally low number of neutrophils) after chemotherapy. G-CSF is also used to increase the number of hematopoietic stem cells in the blood of the donor before collection for use in hematopoietic stem cell transplantation. Several other clinical applications are contemplated as well.

As mentioned previously, one problem arising in the biotechnological production of G-CSF is N-terminal truncation. G-CSF contains a non- structured, flexible N-terminal region of about 10 amino acids length which is prone to degradation. The amount of respective truncation by products must be reduced to meet the purity specifications for the pharmaceutical product. Said purification process in turn brings about a reduction in yield and concomitantly an increase in production costs. In WO 2017/067958 three commercially available G-CSF molecules were revealed to still contain such residual N-terminal truncation variants (see Fig. 1 of WO 2017/067958).

The prior art suggests for the biotechnological production of G-CSF to reduce impurities due to N-terminal truncation for example by way of using a specific type of coding sequence (WO 2017/067958) or by culturing host cells expressing the recombinant protein in a culture medium in presence of elevated levels of a nitrogen source such as (NH₄)₂S0₄, (NH₄)H₂P0₄, (NH₄)₂HP0₄, NH₄C1, (NH₄)₂Fe(S0₄)₂ 6 H₂0, CH₄N₂0, and NH₄N0₃ (WO 2017/067959). Other options are to change the secretory leader peptide (WO 2017/072310), by inhibiting the protein degradation pathway with a molecular inhibitor (WO 2017/118726) and more specifically by blocking the metalloproteinase in mammalian cell culture (WO 2008/135498). In the production of recombinant hG-CSF, truncation of the protein may also be reduced by mutating the signal peptide sequence in cell culture applications (WO 2010/037855). However, these different measures are not always desirable when producing G-CSF.

Impurities resulting from N-terminal truncation are not limited to G-CSF but can also be encountered with other proteins of interest. For example, and without being limited thereto, antibodies, such as single chain antibodies, may also be subject to N-terminal degradation when biotechnologically produced.

Given the loss in yield caused by truncation and the risk of failing regulatory requirements, there is thus a need in the art to establish further means for reducing said loss in yield. It was thus the object of the present invention to provide a means for reducing the amount of truncation by-products of biotechnologically produced proteins, for instance for reducing N-terminal truncation products of biotechno logically produced rhG-CSF or antibodies, such as single-chain antibodies.

This problem is solved by the subject-matter as set forth below and in the appended claims.

In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the scope of the invention to these specific examples.

Figure 1 illustrates the effect of methionine feeding in a rhG-CSF fermentation process conducted with different yeast autolysate lots from different suppliers. At least five (n = 5) different yeast autolysate lots from two (n = 2) different suppliers have been evaluated in six (n = 6) independent batches of a rhG-CSF fermentation process with or without methionine supplementation. The settings are explained in table 2. The 95 % confidence interval is given for the mean of each condition, respectively. The concentration for the fraction of truncated rhG-CSF protein is < 0.50 % (dashed line).

Figure 2 illustrates the effect of methionine feeding on the quality of a recombinant antibody at capture eluate (CAP.E) level. The reference without methionine feeding is depicted in a fine black line. Batches with methionine supplementation are detailed in Table 5 and represented here in black colour (dotted, dashed and straight line, respectively).

The inventors of the present invention have surprisingly found that production of a recombinant polypeptide can be achieved in a purer form if the culture medium of the host cells producing the recombinant polypeptide is supplemented with sufficient amounts of methionine. In doing so, the formation of by-products resulting from N-terminal truncation of the recombinant polypeptide is reduced, thereby increasing purity and yield of the recombinant protein.

Therefore, the present invention relates in a first aspect to the use of methionine for inhibiting formation of N-terminally truncated variants of a biotechnologically produced (e.g. recombinant) polypeptide by using methionine as a cell culture supplement for a host cell expressing the (e.g. recombinant) polypeptide (in a cell culture medium). In a second aspect, the present invention relates to a method of inhibiting formation of N- terminally truncated variants of a (e.g. recombinant) polypeptide in a cell culture process, wherein the method comprises the step of culturing a host cell expressing the (e.g. recombinant) polypeptide in presence of methionine. Without being limited thereto, such method will for example be particularly useful in a situation where a polypeptide is labelled for detection at the C-terminus, but biological activity and/or localisation is dependent on presence of an intact N- terminus. N-terminally truncated variants would in this case give rise to false positive signals.

In a third aspect, the present invention relates to a method of producing a (e.g. recombinant) polypeptide with reduced impurities resulting from N-terminally truncated variants of said (e.g. recombinant) polypeptide, the method comprising the steps of: a) culturing a host cell expressing the (e.g. recombinant) polypeptide in the presence of methionine, b) harvesting the (e.g. recombinant) polypeptide from the cell culture, c) optionally analysing the (e.g. recombinant) polypeptide for presence of contaminations with N-terminally truncated variants of the (e.g. recombinant) polypeptide, and d) optionally purifying said (e.g. recombinant) polypeptide.

As used herein, a “polypeptide” is understood to be a polymer of L-amino acid residues linked by peptide bonds in a specific sequence. The amino acid residues of a polypeptide may be modified by e.g. covalent attachments of various groups such as carbohydrates and phosphate. Other substances may be more loosely associated with the polypeptide, giving rise to conjugated polypeptides, which are also comprised by the term “polypeptide” as used herein. The term as used herein is intended to encompass also proteins. Thus, the term “polypeptide” also encompasses for example complexes of two or more amino acid polymer chains. The term also encompasses fusion proteins, fused peptides etc. The term "polypeptide" does encompass embodiments of polypeptides which exhibit optionally modifications typically used in the art, e.g. biotinylation, acetylation, pegylation, chemical changes of the amino-, SH- or carboxyl- groups (e.g. protecting groups) etc. The polypeptide may be a non-naturally occurring polypeptide. Furthermore, the term "polypeptide", as used herein, is not limited to a specific length of the amino acid polymer chain. However, typically a polypeptide according to the present invention will not exceed a length of 1500 amino acids. And usually (but not limited thereto) a polypeptide within the context of the present invention will be at least 150 amino acids long. The term “recombinant polypeptide”, as used herein, includes any heterologous polypeptide produced by a host cell (i.e. is a polypeptide not naturally occurring in the host cell). Typically, the recombinant protein will be encoded in the host cell by heterologous recombinant nucleic acid sequences. The polypeptide may for example be of mammalian origin, in particular of human origin, while the host cell is a prokaryotic cell, e.g. an E. coli cell. The polypeptide may also be an artificial polypeptide, i.e. does neither naturally exist in the host cell nor any other organism in nature. It is understood that in the context of the present invention the recombinant polypeptide is in particular any polypeptide prone to N-terminal truncation, if (over-)expressed in host cells such as E. coli. Truncated forms of the polypeptide will lack one or more consecutive amino acids at the N-terminus, e.g. 1 to 25, 1 to 20, 1 to 18 or 1 to 7 amino acids of the N-terminus.

A “host cell”, as used herein, includes any cell suitable for producing (e.g. recombinant) polypeptides. A “host cell” is preferably an isolated host cell. A “host cell” is also preferably not part of a human or animal organism. Host cells particularly contemplated by the present invention are bacterial cells such as E. coli and yeast cells, with the former being most preferred. In this context, it is also noted that herein the term “cell culture” is used to refer generally to all forms of single cell cultivation, be it now of procaryotic or eukaryotic origin. The term covers bacterial fermentation processes, yeast fermentation as well as mammalian cell culture procedures. Preferably, the cell culture process is a microbial cell culture process, e.g., a bacterial or yeast fermentation process, most preferably a bacterial fermentation process. The culture conditions will typically be aerobic, but may be - in particular for bacterial fermentation processes - also anaerobic.

“N-terminally truncated variants”, as used herein, are polypeptides which are fragments of the polypeptide of interest. To wit, they lack a sequence of one or more N-terminal amino acid residues present at the N-terminus of the polypeptide of interest. Preferably, such N-terminally truncated variant lacks up to 10 % of consecutive amino acid residues present on the N-terminus of the polypeptide of interest. It is further understood that variants with merely substitutions or internal deletions in the N-terminal sequence are not covered by the term “N-terminally truncated variants”, as these species do not reflect an N-terminal truncation and are not fragments of the polypeptide of interest. The term “methionine” as used herein, in particular in the context of supplementation of cell culture media, encompasses D-methionine, L-methionine and mixtures thereof (D,L methionine), unless specified otherwise.

The terms “single-chain antibody”, “single chain variable fragment” and “scFv” are used interchangeably herein.

The term "comprising", as used herein, shall not be construed as being limited to the meaning "consisting of" (i.e. excluding the presence of additional other matter). Rather, "comprising" implies that optionally additional matter, features or steps may be present. The term "comprising" encompasses as particularly envisioned embodiments falling within its scope "consisting of' (i.e. excluding the presence of additional other matter) and "comprising but not consisting of" (i.e. requiring the presence of additional other matter, features or steps), with the former being more preferred.

The use of the word “a” or “an”, when used herein, may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

In the following, several embodiments of the invention are illustrated. All of these are applicable in similar manner to the first, second as well as to the third aspect of the invention mentioned above.

In a particularly preferred embodiment of the present invention the host cells are cultured in the continuous presence of methionine throughout the production phase of the recombinant polypeptide. Continuous presence of methionine may be achieved by supplementing the culture medium once with sufficient amounts of methionine (e.g. already at the beginning of the protein production process). Sufficient amounts are amounts of methionine which are known to last essentially for the entire duration of the planned fermentation process, i.e. which are after addition not fully consumed by the host cells in the course of the cell culture process. It is evident that in this scenario longer cell culture periods will require higher amounts of methionine and vice versa. In an alternative and more preferred embodiment, the host cells are not only supplied with methionine once (e.g. at the beginning of the protein production process) but repeatedly, for example two, three, four, five times or more than five times. In this scenario the methionine may be added for example in regular intervals. A further and even more preferred possibility is to continuously feed methionine to the host cells (in particular if the polypeptide is recombinant G-CSF or an antibody, such as a single-chain antibody, and/or the host cell is an E. coli cell). In this scenario the methionine concentrations may be lower at the beginning of the protein production process, as the methionine pool is replenished in the course of the cell culture process. Irrespective if methionine is added in regular intervals or constantly fed to the host cells, the methionine levels are chosen in an amount sufficient to essentially ensure continuous presence of methionine throughout the cell culture process and to avoid preferably (temporary or permanent) full depletion of methionine from the culture medium. Preferably, the addition of methionine occurs in the stage of main stage fermentation, i.e. in the process stage where protein product formation occurs.

Preferably, the methionine concentration in the fermentation culture medium (i.e. the supernatant) is kept essentially throughout the protein production process constant, e.g. at or above 200 mg/L, preferably at or above 380 mg/L, even more preferably at or above 500 mg/L. The methionine concentration in the fermentation culture medium may for example be kept constantly in the range of about 500 to about 3200 mg/L.

Preferably, the (recombinant) polypeptide is overexpressed in the host cell.

The type of expression is not limited by the present invention. The polypeptide may be constitutively expressed or expression may be inducible. Expression may include intracellular protein production, protein production in form of inclusion bodies, secretion of the produced protein into the periplasm, secretion of the protein of interest into the surrounding media etc.

The host cells used in the context of the present invention may be any type of host cell suitable for producing the (e.g. recombinant) polypeptide of interest. The host cell may be a mammalian cell, such as a CHO cell, but may also be a bacterial cell such as an E. coli cell. The host cell may also be a yeast cell. Preferably, and in particular in cases where the recombinant protein is rhG-CSF or an antibody, such as a single-chain antibody, the host cells are bacterial cells, such as E. coli cells. E. coli B or K12 strains are preferred. In order to enable production of the polypeptide of interest, e.g. G-CSF or an antibody, such as a single-chain antibody, a host cell may comprise a respective nucleic acid encoding the polypeptide of interest. Said nucleic acid may further comprise elements operably linked to the sequence encoding said polypeptide, which allow the transcription of the nucleic acid sequence and translation of the resulting mRNA into the encoded polypeptide in the given host cell. In particular, the nucleic acid may comprise a heterologous promoter. Said heterologous promoter may be operably linked to the nucleic acid sequence encoding the polypeptide, e.g. granulocyte colony stimulating factor (G- CSF) or an antibody, such as a single-chain antibody, thereby providing for transcription of said nucleic acid sequence. A "heterologous promotor” for the nucleic acid encoding the polypeptide of interest is a promoter, that is not found in direct association with the respective nucleic acid sequence encoding said polypeptide in nature, i.e. is in nature not operably linked with the respective nucleic acid sequence encoding said polypeptide.

The “culture medium” used for the fermentation process may be any kind of medium suitable for producing in the respective host cell a (recombinant) polypeptide, e.g. for producing a recombinant polypeptide in E. coli cells. The choice of the culture medium will depend on the choice of host cells for the production of the polypeptide of interest. A person skilled in the art of cell culture and fermentation is readily aware of a number of suitable media for the respective host cell. The culture medium may be a “complex” medium or a “synthetic” medium. A “complex” medium contains components of unknown composition and concentration (i.e. they are not precisely defined), e.g. media containing yeast extract or serum. Examples for a complex medium are Luria broth (LB) or terrific broth (TB). In contrast, a “synthetic medium” (also termed “chemically defined medium” in the art) is typically composed of defined components in defined concentrations (and will typically be based on a basal medium or a balanced salt solution, without being limited thereto). According to a preferred embodiment of the invention the culture medium will be a “synthetic” medium. Frequently, a synthetic medium will only comprise those components absolutely required for allowing production of a recombinant polypeptide in the host cell. A particularly preferred culture medium for E. coli cells will comprise components as disclosed in Riesenberg et al. (Appl Microbiol Biotechnol. 1990 Oct;34(l):77-82) and Korz et al. (Journal of Biotechnology 39 (1995) 59-65), incorporated herewith by reference.

A particularly preferred polypeptide in the context of the present invention is granulocyte colony stimulating factor (G-CSF). As used herein, the term “granulocyte colony stimulating factor”, or G-CSF, encompasses all forms of G-CSF known or conceivable for a person skilled in the art. The G-CSF may, for example, be of any mammalian origin, such as human G-CSF (hG-CSF), which is particularly preferred, mouse G-CSF (mG-CSF), or bovine G-CSF (bG- CSF). The term encompasses all allelic variants. The term encompasses recombinant G-CSF (i.e. with or without L-methionine at the N-terminus), for instance recombinant human G-CSF (SEQ ID NO: 1), as well as natural G-CSF (i.e. without L-methionine at the N-terminus), for instance natural human G-CSF (SEQ ID NO: 2). The term “G-CSF” does also encompass mutated versions of naturally occurring G-CSF. Preferably, such mutated versions of G-CSF do still comprise the flexible N-terminal region of about 10 amino acids length of G-CSF. Most preferably, such mutated versions do still exhibit the biological activity of G-CSF. Preferably, such mutated versions of naturally occurring G-CSF are at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 or more than 160 amino acids long. The nucleic acid encoding the granulocyte colony stimulating factor (G-CSF) may also encode a G-CSF precursor exhibiting a signal peptide at the N-terminus which is then posttranslationally proteolytically cleaved, yielding the actual protein product. The term G-CSF also encompasses fusion proteins comprising G-CSF on the one hand and one or more further fusion partners on the other hand. Particularly preferred are fusion proteins, in which G-CSF forms the N-terminal part of the fusion protein. Examples for potential fusion partners are, without being limited thereto, conventionally used tags, such as His-tags, or detectable markers such as GFP. The eventually produced G-CSF may be glycosylated, may be pegylated, may be both (i.e. is glycosylated and pegylated) or may be none of it (i.e. is neither glycosylated nor pegylated). Typical truncation by-products which are formed when (over-)expressing (e.g. recombinant) G-CSF lack up to 7 N-terminal amino acids in comparison to the full length protein. In particular, the truncated G- CSF forms whose formation can be inhibited by using the present invention are “group II truncations”. “Group II truncation products of G-CSF” obligatorily lack all amino acids N- terminal of the first leucine residue occurring on the N-terminal end of G-CSF and lack 0 to 4 further amino acid residues (and not more) of the N-terminal amino acid residues of G-CSF. With respect to natural human G-CSF (SEQ ID NO: 2), said “group II truncations” thus may lack the N-terminal residues TP (SEQ ID NO: 3), TPL (SEQ ID NO: 4), TPLG (SEQ ID NO: 5), TPLGP (SEQ ID NO: 6), or TPLGPA (SEQ ID NO: 7) of SEQ ID NO: 2. The group II truncation products of G-CSF according to SEQ ID NO: 2 are thus truncated by two, three, four, five and/or six amino acids at the N-terminus. With respect to recombinant human G-CSF (SEQ ID NO: 1) this means that said “group II truncations” are truncated versions of recombinant G-CSF lacking the N-terminal sequence motifs MTP (SEQ ID NO: 8), MTPL (SEQ ID NO: 9), MTPLG (SEQ ID NO: 10), MTPLGP (SEQ ID NO: 11), or MTPLGPA (SEQ ID NO: 12) of SEQ ID NO: 1. The Group II truncation products of G-CSF according to SEQ ID NO: 1 are thus truncated by three, four, five, six and/or seven amino acids at the N-terminus. The table below illustrates “group II truncation products of G-CSF” (i.e. N-terminally truncated variants of G-CSF) with respect to the truncated (i.e. missing) residues (left side) as well as for the resulting N-terminal sequence of the truncated by-product (right side). “(M)” is intended to reflect that the N-terminal L-methionine may be present (in recombinant G-CSF) or absent (in native G-CSF). The amount of these by-products can be reduced when using methionine as supplement to the cell culture.

Table 1: missing residues N-terminus of fragment

The concept of “group II truncation products of G-CSF” has been illustrated above for SEQ ID NO: 1 and SEQ ID NO: 2. However, a person skilled in the art will readily be capable of applying said concepts to G-CSF of other origin (e.g. other mammalian origin such as bovine or murine G-CSF, G-CSF with point mutations in said stretch etc.,) as well, where the individual amino acid sequence at the N-terminus of said G-CSF may be individually distinct from the human sequence.

Other particularly preferred polypeptides in the context of the present invention are antibodies, such as single-chain antibodies. The eventually produced antibody may be glycosylated, may be pegylated, may be both (i.e. is glycosylated and pegylated) or may be none of it (i.e. is neither glycosylated nor pegylated). Typical truncation by-products which are formed when (over-) expressing an antibody, such as a single-chain antibody, may lack for example 1 to 18 N- terminal amino acids in comparison to the full length protein. Specific truncated by-products of a single-chain antibody, which have been shown by the inventors to be reducible by the present invention, lack 2, 4, or 18 amino acids of the N-terminus. While the concept of the present invention is illustrated in the examples on basis of G-CSF and a single-chain antibody (i.e. deriving from very different classes of proteins), the present invention is not limited to these proteins.

The present invention according to the first, second or third aspect contemplates the (preferably continuous) presence of methionine in the culture medium during the production phase of the recombinant protein. Methionine may be present in pure form, as D-methionine or L- methionine or in a combination of L-methionine with D-methionine (including racemic mixtures of methionine, “D, L-methionine”). Methionine may also be provided in the form of nutrition supplements like in Alimet® supplement (Novus International, Inc. Missouri, USA). Preferably, L-methionine or D, L-methionine is used.

The method according to the third aspect of the invention foresees harvesting of the (recombinant) polypeptide. A person skilled in the art is readily aware of means to harvest the polypeptide of interest from a cell culture. If the polypeptide of interest is produced intracellularly, harvesting will typically involve pelleting of the host cell, removal of the supernatant and lysis of the host cell in a suitable buffer. If the polypeptide of interest is already secreted into the medium, harvesting may simply involve obtaining the supernatant. Harvesting, as used herein, encompasses obtaining a small sample from the fermentation process, e.g. for further analysis, but refers preferably to the final phase of the fermentation process where production of the polypeptide of interest is essentially stopped and all protein produced so far isolated.

Furthermore, the method according to the third aspect of the invention also contemplates to optionally analyze the polypeptide harvested from the cell culture for presence, type and/or amount of contamination with N-terminally truncated variants of the respective (recombinant) polypeptide. Such analysis is routine for the skilled person and may involve analytic methods such as analytical chromatography (e.g., size exclusion chromatography), mass spectrometry, N-terminal sequencing (e.g., EDMAN sequencing) etc. If the polypeptide is G-CSF, the analysis preferably involves checking for presence, type and/or amount of contaminations selected from those as set out in table 1, right column.

Finally, the polypeptide of interest may, depending on the ultimate purpose, require still further purification. For example, further purification will most often be required if regulatory standards must be met. Various protein purifications techniques are known to the person skilled in the art. Such purification may for example involve various chromatography types like cation exchange chromatography.

In a further (fourth) aspect, the present invention relates to a composition comprising a polypeptide of interest, such as G-CSF or an antibody, such as a single-chain antibody, said composition being obtainable or obtained by a method according to the third aspect of the present invention.

In particular, the present invention relates to a composition comprising an antibody, such as a single-chain antibody, said composition further comprising N-terminally truncated variants of an antibody, such as a single-chain antibody in an amount of 0.9 % (as measured by area of all peaks in a chromatogram) or less, in particular 0.8 % or less, or even 0.7 % or less. The composition may for example comprise up to 0.9 %, up to 0.8, or only up to 0.7% N-terminally truncated variants of an antibody, such as a single-chain antibody. It is particularly preferred if the composition comprises N-terminally truncated variants of an antibody, such as a single chain antibody, in the range of 0 to 0.9 %, e.g. in the range of 0.5 to 0.7 %. The N-terminally truncated variants of a single-chain antibodyare preferably as defined above for the first, second and third aspect of the present invention. It is understood that wherever herein percentages of impurities resulting from truncation products are mentioned, that these percentages are given vis-a-vis the total content of the respective polypeptide (non-truncated polypeptide + truncated polypeptides). The composition according to the present invention comprising a single-chain antibody may be, for example, a cell lysate, in particular a cell lysate of E. coli. However, the composition according to the present invention is most preferably a pharmaceutical composition comprising a single-chain antibody and a pharmaceutically acceptable carrier, diluent and/or excipient.

While the present invention particularly contemplates culturing the host cell in presence of methionine in order to avoid formation of N-terminally truncated variants, there may be technical applications where it is desirable to increase the proportion of such by-products. Therefore, the present invention relates in further aspects also to methods seeking the opposite effect, namely an increase in N-terminally truncated variants of the (e.g. recombinant) polypeptide of interest. To be more precise, the present invention relates in a fifth aspect to a method of promoting formation of N-terminally truncated variants of a (e.g. recombinant) polypeptide in a cell culture process, wherein the method comprises the step of culturing a host cell expressing the polypeptide in a culture medium with low levels of or even in absence of methionine. Furthermore, the present invention also relates in a sixth aspect to a method of producing a (e.g. recombinant) polypeptide with impurities resulting from N-terminal truncation of said polypeptide, the method comprising the steps of: a) culturing a host cell expressing the polypeptide in a culture medium lacking methionine or exhibiting only low levels of methionine (e.g. below 0.15 mg/L), b) harvesting the polypeptide from the cell culture, c) optionally analysing the polypeptide for presence of impurities with N-terminally truncated variants of the polypeptide, and d) optionally purifying said polypeptide.

To the extent applicable, embodiments set forth above for the first, second and third aspect of the invention apply likewise to the fifth and sixth aspect of the invention. For example, the host cell is preferably a bacterial cell, such as an E. coli cell. The low levels of methionine are preferably low levels of L- methionine.

Examples

In the following, specific examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1 : Analysis of the abundance of N-terminallv truncated G-CSF variants in presence and absence of methionine supplementation

• Cultivation and expression was conducted in the presence and absence of methionine supplementation (either in complex or in synthetic medium)

• Broth was taken from the G-CSF fermenter operated with different media (complex and synthetic) and with and without methionine supplementation at dedicated times

• Samples (broth) were stored either in the fridge or in the freezer until analysis

• If samples had been frozen they were thawed before mechanical disruption of the cells

• Inclusion bodies were isolated from the homogenate

• Isolated inclusion bodies were then mixed with refold buffer in order to enable correct folding

• Correctly folded G-CSF was analyzed by anion exchange chromatography for product variants

The settings of the study is explained in Table 2.

Table 2: settings of the study to evaluate the effect of methionine feeding in the rhG-CSF fermentation process

At least five (n = 5) different yeast autolysate lots from two (n = 2) different supplier have been evaluated in six (n = 6) independent batches. As a result, methionine feeding significantly reduced the amount of truncated recombinant human G-CSF (rh-GCSF) protein below the concentration of 0.50 % in complex medium (see Fig. 1). This effect occurs during expression in complex medium regardless of the yeast autolysate lot used. It becomes even more pronounced if the duration of expression is extended from 8 hours to 12 hours.

In synthetic media, similar results were obtained, as demonstrated in table 4 below. The table summarizes the effect of methionine supplementation on the formation of rhG-CSF in chemically-defined medium (CDM). Three batches were conducted with methionine feeding and all three batches resulted in almost identical truncation values, which are 2- to 3-fold lower compared to the reference batch without methionine supplementation. The amount of truncated rhG-CSF protein is significantly reduced by methionine supplementation and equal to or slightly higher than 0.5 %. Thus, methionine feeding is demonstrated to also reduce the amount of truncated rhG-CSF proteins in synthetic medium. The formation of truncated rhG-CSF proteins is not related to the duration of the protein expression. The beneficial effect of methionine dosing is even stronger at extended duration of expression.

Table 4: Effect of methionine dosing on the formation of rhG-CSF in chemically-defined medium (CDM).

Cultivation procedure Methionine dosing Results yes no Sum high truncations (%)

8h of expression 12h of expression

CDM standard x 1.4 1.8

CDM standard x 0.6 0.6

CDM standard x 0.6 0.5

CDM: chemically-defined medium, synthetic medium Example 2: Analysis of the abundance of N-terminallv truncated scFv antibody fragment variants in presence and absence of methionine supplementation

• Cultivation and expression were conducted in the presence and absence of methionine supplementation

• Broth was taken from a scFv antibody fragment fermenter at dedicated times

• Inclusion bodies were isolated from the homogenate

• Isolated inclusion bodies were then mixed with refold buffer in order to enable correct folding of the scFv antibody fragment

• Correctly folded scFv antibody fragment was analyzed by anion exchange chromatography for product variants

• Peak assignment had been performed before by fraction collection, concentration of fractions and subsequent identification by MS

• The area of all peaks of a chromatogram was set to 100 %; all concentrations (in %) are given in area of certain species related to area of all peaks of the chromatogram

The effect of methionine feeding on impurities including truncated variants of a scFv antibody fragment under various conditions of cultivation was assessed. Three different settings were compared with and without methionine feeding (see Table 5). The results are summarized in Table 6. In the reference cultivations without methionine feeding (settings A to C) the fractions of impurities including truncated scFv antibody fragment variants were very prominent (at least 1.3 to 5.9 % of all antibody molecules contain truncated product variants), whereas under the same conditions but with methionine feeding (settings D to F) the fractions of impurities including truncated scFv antibody fragment variants were significantly reduced (0.3 to 0.7 %). Thus, methionine feeding was demonstrated to significantly reduce the amount of truncated scFv antibody fragment in synthetic medium. The formation of truncated scFv antibody fragment was not apparently related to the duration of the protein expression. The beneficial effect of methionine dosing was even much stronger at extended duration of expression. Table 5: Conditions of cultivation in order to assess the effect of methionine feeding on impurities including truncated variants of a scFv antibody fragment

No. pH (-) Temperature (°C) Exponential increase of Methionine feeding glucose feeding (h) _{Yes No}

A 7.2 25 13.0 x

B 6.9 28 12.0 x

C 6.9 28 13.5 x

D 7.2 25 13.0 x

E 6.9 28 12.0 x

F 6.9 28 14.0 x

Table 6: Effect of methionine feeding on impurities including truncated scFv variants under various conditions of cultivation

No. Impurities including truncated variants (%)

6h of expression lOh of expression 12h of expression 15h of expression

A 1.8 n.d. 5.6 5.5

B 2.0 n.d. 5.3 n.d.

C 1.3 n.d. 5.4 5.9

D 0.3 n.d. 0.4 0.3

E 0.4 0.7 n.d. n.d.

F 0.4 n.d. 0.5 0.3

The lever of the effect of methionine feeding on the quality of the antibody molecule becomes even more clear when looking at Figure 2. The four batches were harvested and purified to the capture eluate (CAP.E) level. The chromatography method separates the impurities including truncated antibody variants from the correct/complete antibody molecules and elutes them ahead of the product peak. This fraction is referred to with the term ‘pre-peaks’. In the reference cultivation without methionine feeding (grey color, setting B in Table 5) the pre-peaks are very prominent, whereas in all three batches with methionine feeding the pre-peaks are significantly reduced (settings D, E and F in Table 5; dotted, dashed and straight black lines, respectively). From this presentation, the strong effect of methionine feeding on the reduction of the amount of truncated antibody proteins in synthetic medium is readily evident.

Claims

1. Use of methionine for inhibiting formation of N-terminally truncated variants of a recombinant polypeptide by using methionine as a cell culture supplement for a host cell expressing the recombinant polypeptide.

2. A method of inhibiting formation of N-terminally truncated variants of a recombinant polypeptide in a cell culture process, wherein the method comprises the step of culturing a host cell expressing the recombinant polypeptide in a culture medium in presence of methionine.

3. A method of producing a recombinant polypeptide with reduced impurities resulting from N-terminally truncated variants of said recombinant polypeptide, the method comprising the steps of: a) culturing a host cell expressing the recombinant polypeptide in a culture medium in presence of methionine, b) harvesting the recombinant polypeptide from the cell culture, and c) analysing the recombinant polypeptide for presence of contaminations with N- terminally truncated variants of the recombinant polypeptide, and d) optionally purifying said recombinant polypeptide.

4. The use according to claim 1 or the method according to claim 2 or 3, wherein the host cell is cultured in continuous presence of methionine.

5. The use according to claim 1 or 4 or the method according to claim 2 to 4, wherein the methionine concentration is kept essentially constantly at or above or 200 mg/L, preferably at or above 380 mg/L, even more preferably at or above 500 mg/L.

6. The use according to any one of claims 1, 4 and 5, or the method according to any one of claims claim 2 to 5, wherein the recombinant polypeptide is overexpressed in said host cell.

7. The use according to any one of claims 1, and 4 to 6, or the method according to any one of claims claim 2 to 6, wherein the medium is a synthetic medium or wherein the medium is a complex medium.

8. The use according to any one of claims 1, and 4 to 7, or the method according to any one of claims claim 2 to 7, wherein the host cell is a bacterial cell such as an E. coli cell.

9. The use according to any one of claims 1, and 4 to 8, or the method according to any one of claims claim 2 to 8, wherein the recombinant polypeptide is human granulocyte-colony stimulating factor (hG-CSF), in particular hG-CSF according to SEQ ID NO: 1, or wherein the recombinant polypeptide is an antibody, in particular a single-chain antibody.

10. The use according to any one of claims 1, and 4 to 9, or the method according to any one of claims claim 2 to 9, wherein methionine is provided as D,L methionine or L- methionine.

11. The use according to any one of claims 1, and 4 to 10, or the method according to any one of claims claim 2 to 10, wherein the N-terminally truncated variants lack up to 10% amino acids at the N-terminus as compared to the recombinant polypeptide.

12. The use according to any one of claims 1, and 4 to 11, or the method according to any one of claims claim 2 to 11, wherein methionine is constantly feed to the cell culture.