WO2025036564A1 - Nucleic acid sequence, vector, screening method and method of producing recombinant antibodies - Google Patents

Abstract

The present invention relates to a nucleic acid sequence with an elevated expression rate having at least one expression cassette for expression of a protein, wherein the expression cassette has at least one promoter element, and at least one first transcription unit that encodes at least one peptide, wherein the promoter element consists of nucleic acid sequence SEQ ID NO: 1 or of a nucleic acid sequence having at least 70% homology with SEQ ID NO: 1. The invention further relates to a vector or an isolated nucleic acid comprising at least one nucleic acid sequence of the invention in simple or repetitive form. The present invention additionally relates to an amino acid sequence comprising SEQ ID NO: 2 or an amino acid sequence having at least 70% homology with SEQ ID NO: 2, and to a cell comprising a vector of the invention or an isolated nucleic acid of the invention, or a nucleic acid sequence of the invention, or an amino acid sequence of the invention, wherein the cell is a photosynthetically active cell, especially a unicellular plant or Viridiplantae, preferably a diatom. The invention also relates to a screening method for selection of cells having an elevated expression rate of a nucleic acid sequence of the invention, and to a method of producing recombinant antibodies using a vector of the invention or an isolated nucleic acid of the invention and/or a nucleic acid sequence of the invention.

Description

NUCLEIC ACID SEQUENCE, VECTOR, SCREENING METHODS AND METHODS FOR THE PRODUCTION OF RECOMBINANT ANTIBODIES

TECHNICAL FIELD

The present invention relates to a nucleic acid sequence with an increased expression rate for the production of recombinant proteins, as well as a vector or an isolated nucleic acid for the heterologous expression of proteins. Furthermore, the present invention relates to a corresponding amino acid sequence, a cell, as well as a screening method for selecting cells with an increased expression rate and a method for producing recombinant antibodies.

STATE OF THE ART

A problem with common immunoassays that use antibodies is that the antibodies are of animal origin. Antibodies are produced either in animals or in animal cell cultures. CHO (Chinese hamster oviduct cells) and HEK-293T cells are used in particular.

(human embryonic kidney cells) are used. This cell culture-based production is very cost-intensive, so existing capacities are mainly used to produce high-priced therapeutic antibodies. Antibodies used in diagnostics have much lower margins and are therefore still often produced in animals. In addition to the associated animal suffering, such antibodies produced in animals also have low reliability of binding properties, limited availability and the risk of contamination with human pathogens.

The alternatives for producing antibodies and other complex proteins are quite limited. Bacteria such as Escherichia coli cannot carry out the necessary post-translational modifications to the antibodies, and production rates are extremely low. The same applies to yeast or P/c/7/a-based production systems. Here, too, the antibodies can rarely be folded correctly by the host cells and production rates are similarly low to those in bacteria. Transgenic plants have been discussed as alternative producers for many years, but here too the very low production rates do not allow for commercial exploitation. The production of antibodies in Phaeodactylum tricomutum, a microalgae, has also already been described. For example, EP 2 671 950 A1 discloses the expression and secretion of recombinant, fully assembled protein complexes by microalgae. EP 2 660 323 A1 discloses the production of secreted therapeutic antibodies in the microalgae Phaeodactylum tricornutum. Even using these expression systems, it was not possible to achieve production rates that would allow for economic production.

In order to induce overexpression of a specific protein in Phaeodactylum tricornutum and thus significantly increase the productivity of a recombinant protein, WO 2021/002630 A1 describes the use of the promoter HASP1. Furthermore, a signal peptide HASP1-SP, an expression vector comprising HASP1 and HASP1-SP, a transformant into which the expression vector has been introduced into a host cell, and methods for producing, expressing and increasing the expression level of a protein of interest in a host cell are disclosed. With this method, too, the production rate can be increased even further.

TASK

The present invention is therefore based on the technical problem of providing a nucleic acid sequence, a vector, an amino acid sequence, a cell, as well as a screening and a production method which allow the expression level and thus the production rate of recombinant proteins to be further increased compared to the methods and genetic components known in the prior art.

SOLUTION

The object is achieved by a nucleic acid sequence having the features of claim 1, as well as a vector or an isolated nucleic acid, an amino acid sequence, a cell, a screening method and a method for producing recombinant antibodies having the features of the independent claims. Further advantageous embodiments can be found in the subclaims, the description and the embodiments. The object is achieved in particular by a nucleic acid sequence with an increased expression rate for the production of recombinant proteins, comprising at least one expression cassette for the expression of one or more peptides. According to the invention, the expression cassette has at least one promoter element and at least one first transcription unit which codes for a protein, wherein the promoter element consists of the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence with a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, further preferably at least 99% to SEQ ID NO: 1.

For the purposes of the invention, the term “homology” refers to the similarity between nucleotide sequences of DNA or RNA and/or between amino acid sequences of proteins.

Advantageously, SEQ ID NO:1 represents a promoter, hereinafter referred to as HASP1 ^mod , which has proven to be particularly suitable for regulated protein production. The HASP1 ^mod promoter is a promoter element derived from the natural HASP1 promoter, whereby a partial sequence of the natural HASP1 promoter has been duplicated.

SEQ ID NO:1 is as follows, where the underlined part represents the duplication:

5'-

CATACAGTGAATGTAACTTTCGAATTGACAGTATTAGTAGTCGTATTGACAGTGAGGCAC GCCCCTCAATGTGCGAGGTGGAAAATATACCAGCATGACAATGAATCTTGGAGATTCTT TTGCTGTCATCAAGATTCACCGCCAAATCTTCAGGAACCTATCACGTCCACAGGCGATG TTAATTCTTGAGTCGTCAAAACAAAGTCCTGTCCTACCTGTAGAAGTTGACAGCGAGCAA TTGTATGCAAACTTCTGACTTTGTTATAATAACATTAAAGGTAATTAAGTATCTTCAATTAG GCATTTTGTCACTGTCAGTCCGTTCCGACAATATAGGTAGATTTGGAATGAATCTTTTCT ATGCTCATACAGTGAATGTAACTTTCGAATTGACAGTATTAGTAGTCGTATTGACAGTGA GGCACGCCCCTCAATGTGCGAGGTGGAAAATATACCAGCATGACAATGAATCTTGGAGA TTCTTTTG CTGTCATCAAG ATTCACCG CCAAATCTTCAG G AACCTATCACG TCCACAG GC GATGTTAATTCTTGAGTCGTCAAAACAAAGTCCTGTCCTACCTGTAGAAGTTGACAGCGA GCAATTGTATGCAAACTTCTGACTTTGTTATAATAACATTAAAGGTAATTAAGTATCTTCA ATTAGGCATTTTGTCACTGTCAGTCCGTTCCGACAATATAGGTAGATTTGGAATGAATCT TTTCTATGCTGCTGCGAATCTTGTACACCTTTTGAGGCCGTAGATTCTGTCCGACGAAGC GATAATTATTGCAAAATACATGGACTCATTATTTTGATTCGATTTCTTTTTGGTATCCGAC TCGAAAAGATCCATCACGGCGAGC-3' The invention also encompasses nucleic acid sequences whose promoter element has individual HASP1 sequence sections repetitively, for example with respect to the start codon ATG between -100 and -1, between -200 to -101, between -300 to -201, between -400 to -301 and/or between -500 to -400. These sections can be combined in any constellation and copy number. This results in a new sequence that has less than 85% homology to the native and HASP1 promoter.

According to a preferred embodiment of the present invention, at least one transcription unit comprises a polynucleotide which encodes an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence with a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, further preferably at least 99% to SEQ ID NO: 2.

SEQ ID NO:2 represents an amino acid sequence of the hinge region of an antibody, which was derived from equine immunoglobulin sequences and has been found to be particularly protease-resistant. The use of this protease-resistant hinge region significantly reduces the proteolysis of the antibodies produced in different formats and from different species, both in vivo and in vitro.

The SEQ ID NO:2 is as follows:

VIKEPCCCPKCP

The object is further achieved by an amino acid comprising SEQ ID NO: 2 or an amino acid sequence having a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, further preferably at least 99% to SEQ ID NO: 2.

Furthermore, the object is achieved by a vector or an isolated nucleic acid which comprises a nucleic acid according to the invention in simple or repetitive form, and by a cell comprising a vector according to the invention or an isolated nucleic acid according to the invention or a nucleic acid sequence according to the invention or an amino acid sequence according to the invention.

According to the invention, the cell is a photosynthetically active cell, in particular a viridiplantae or a unicellular plant, preferably a diatom. The object is further achieved by a screening method for selecting cells with an increased expression rate of a nucleic acid sequence according to the invention. The screening method comprises the steps a) providing cells which have been transformed with a vector according to the invention or an isolated nucleic acid according to the invention and/or with a nucleic acid sequence according to the invention, b) isolating and transferring the cells into a culture medium, c) enriching the culture medium with a phosphate concentration in the range of 0.1 pM to 200 pM, preferably 0.5 pM to 200 pM, particularly preferably 1 pM to 200 pM, very particularly preferably 1 pM to 35 pM, and d) examining the cells for a desired expression rate.

Advantageously, the screening method according to the invention not only makes it possible to find producing cell lines, but also to identify the cell lines that produce the highest protein yields at the earliest possible stage of the process.

Furthermore, the object is achieved by a method for producing recombinant antibodies using a vector according to the invention or an isolated nucleic acid according to the invention or a nucleic acid sequence according to the invention, wherein the method comprises the following steps: a) providing cells with a nucleic acid sequence according to the invention, preferably determined by a screening method according to the invention, b) culturing the transformed cells in a culture medium, wherein the cultivation is initially carried out in a pre-culture and then in a main culture and wherein the culture conditions are adapted in such a way that the yield of heterologously produced proteins is maximized, and c) extracting the heterologously produced proteins.

It may be useful for the culture medium to contain phosphate in an amount of from 20 pM to 300 pM, preferably from 20 pM to 400 pM, particularly preferably from 20 pM to 500 pM. GENERAL BENEFITS

The invention describes a method and the components required for it, which for the first time enables efficient and vegan production of recombinant proteins in diatoms, for example in Phaeodactylum tricornutum. Vegan production is understood to mean production that does not use animals or animal cells or substances obtained from animals. The production method according to the invention is primarily aimed at the heterologous expression of antibodies of various formats, but can also be used for other proteins, preferably from animals and humans. In contrast to methods already described, the method according to the invention allows for the first time production quantities of at least 50 mg recombinant protein * L ^-1 culture. The quantities thus achieved are at least 20x higher than those described to date and have not yet been achieved by any stably transformed, plant or Sar-based production system. In addition, the production method according to the invention meets the requirements for completely vegan production, as long as the DNA sequences of the proteins to be produced do not come from animals, but for example from recombinant (human) antibody banks.

Another advantage over the production of polyclonal antisera is the consistent quality and reproducibility of the antibodies produced, since, for example, the animals producing the antibodies die after a certain time and antibodies from another animal have different properties, i.e. the quality of the antibodies produced using the conventional method varies greatly. In addition, antibodies and auxiliary proteins obtained from a diatom, unicellular plant or viridiplantae are precisely defined antibodies, i.e. antibodies whose amino acid sequence is predefined and consistent.

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of the invention, the recombinant antibodies are obtained by expression from the diatom (diatoms), the green algae (Chlorobionta) or a seed plant, whereby very high yields are advantageously achieved, for example for expression from seed plants yields of more than 20 mg of antibodies per liter of culture (in the case of single cells) or per 100 g of fresh weight. The recombinant antibodies are particularly preferably obtained from the diatom or the green alga, in particular in the diatom, such as Phaeodactylum tricornutum. nucleic acid sequence

The invention relates on the one hand to a nucleic acid sequence with an increased expression rate for the production of recombinant proteins, comprising at least one expression cassette for the expression of a protein, wherein the expression cassette has at least one promoter element and at least one first transcription unit which encodes at least one peptide, preferably an antibody, and wherein the promoter element consists of the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence with a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, further preferably at least 99% to SEQ ID NO: 1.

For the purposes of the invention, the term “homology” refers to the similarity between nucleotide sequences of DNA or RNA and/or between amino acid sequences of proteins.

An expression cassette in the sense of the invention consists of at least one regulatory promoter element, a 5'-UTR (untranslated region at the 5' end), a codon-determining sequence (CDS, which is translated into a peptide), a 3'UTR (untranslated region at the 3' end) and a terminator, which acts as a stop codon and causes the termination of translation. The CDS can contain various subelements, such as signal peptides, which direct antibodies or antigens to a specific cell compartment, tag sequences such as Strep-Tag or ßx His-Tag, which primarily serve to purify or detect the peptide, or fluorescent markers such as GFP (Green Fluorescent Protein) etc., which primarily serve to detect via their fluorescence.

The term "promoter" in the sense of the present invention refers to a polynucleotide sequence that is located upstream of a gene and regulates the transcription of a functional gene. The promoter forms a recognition and binding site for an RNA polymerase, which initiates the transcription of the gene.

The use of a promoter element derived from the HASP1 promoter (Highly Abundant Secreted Protein from P. tricorn ut um) has proven to be particularly advantageous and is referred to below as HASP1 ^mod . The nucleic acid sequence of HASP1 ^mod is listed under SEQ ID NO:1 and is as follows: 5'-

CATACAGTGAATGTAACTTTCGAATTGACAGTATTAGTAGTCGTATTGACAGTGAGGCAC GCCCCTCAATGTGCGAGGTGGAAAATATACCAGCATGACAATGAATCTTGGAGATTCTT TTGCTGTCATCAAGATTCACCGCCAAATCTTCAGGAACCTATCACGTCCACAGGCGATG TTAATTCTTGAGTCGTCAAAACAAAGTCCTGTCCTACCTGTAGAAGTTGACAGCGAGCAA TTGTATGCAAACTTCTGACTTTGTTATAATAACATTAAAGGTAATTAAGTATCTTCAATTAG GCATTTTGTCACTGTCAGTCCGTTCCGACAATATAGGTAGATTTGGAATGAATCTTTTCT ATGCTCATACAGTGAATGTAACTTTCGAATTGACAGTATTAGTAGTCGTATTGACAGTGA GGCACGCCCCTCAATGTGCGAGGTGGAAAATATACCAGCATGACAATGAATCTTGGAGA TTCTTTTG CTGTCATCAAG ATTCACCG CCAAATCTTCAG G AACCTATCACG TCCACAG GC GATGTTAATTCTTGAGTCGTCAAAACAAAGTCCTGTCCTACCTGTAGAAGTTGACAGCGA GCAATTGTATGCAAACTTCTGACTTTGTTATAATAACATTAAAGGTAATTAAGTATCTTCA ATTAGGCATTTTGTCACTGTCAGTCCGTTCCGACAATATAGGTAGATTTGGAATGAATCT

TTTCTATGCTGCTGCGAATCTTGTACACCTTTTGAGGCCGTAGATTCTGTCCGACGAAGC GATAATTATTGCAAAATACATGGACTCATTATTTTGATTCGATTTCTTTTTGGTATCCGAC TCGAAAAGATCCATCACGGCGAGC-3'

The invention also encompasses nucleic acid sequences whose promoter element has individual HASP1 sequence sections repetitively, for example with respect to the start codon ATG between -100 and -1, between -200 to -101, between -300 to -201, between -400 to -301 and/or between -500 to -400. These sections can be combined in any constellation and copy number. This results in a new sequence that has less than 85% homology to the native and HASP1 ^mod promoter.

According to one embodiment of the invention, the promoter element can consist of a nucleic acid sequence in which up to 30% of the bases differ, particularly in the proximal promoter regions. The latter can also originate from P. tricornutum or from other organisms, including those from humans or vertebrates. This modification is another reason for the high performance of the developed expression system.

According to a preferred embodiment, the expression cassette further comprises at least one second transcription unit which encodes a reporter protein. This second transcription unit is also referred to herein as a reporter gene. The reporter gene used is preferably a gene which does not normally occur in eukaryotic cells, preferably in photosynthetically active cells, in particular in unicellular plants or viridiplantae. The reporter gene primarily serves to indicate the activity of the promoter under which it was introduced and/or the expression of the first transcription unit with which it is functionally linked. For example, a reporter enzyme, such as a bioluminescent enzyme, a fluorescent reporter protein, such as GFP (green fluorescent protein), or a detectable antigen can be used as a reporter protein.

Reporters are proteins that directly or indirectly generate a detectable signal. This signal can, for example, B. be a fluorescence (e.g. tGFP (turbo green fluorescent protein), GFP (green fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), CFP (cyan fluorescent protein), DsRed (Discosoma sp. red fluorescent protein)), a luminescence (e.g. luciferase), a coloration by chromoproteins (e.g. meffBlue, aeBlue, cjBlue, amilGFP, fwYellow, amajLime, scOrange, amilCP gfasPurple, eforRed, asPink (asCP), meffRed, tsPurple and spisPink, as well as mCherry), a color reaction (e.g. ß-glucoronidase, ß-galatosidase) or the conversion of a substrate into a product, which changes the absorption spectrum of the respective solution.

In combination with a nutrient deficiency-triggered or a nutrient-triggered promoter, a reporter gene can be used to simplify optimization of culture conditions and/or to control cell vitality or nutrient availability in a cell culture. This enables in-time analysis of the cultured cells. The strength of the signal correlates with the availability or lack of the nutrient under investigation. It has been found that the HASP1 ^mod promoter (according to SEQ ID NO:1) is inhibited by phosphate and is triggered by phosphate deficiency. Here, "phosphate" refers to all phosphate salts and phosphate-containing organic compounds and all chemical forms of phosphorus.

Phosphate concentrations of over 360 pM are particularly important for the regulation of

HASP1 ^mod - promoter is suitable. Such high concentrations not only allow the expression rate of the HASP1 ^mod promoter to be inhibited very efficiently and over a longer period of time, but also allow a higher cell density to be achieved before a strong increase in expression occurs due to the consumption of phosphate. The HASP1 ^mod - promoter thus provides the ideal control to achieve high cell densities without expression of the controlled gene, before expression is massively increased due to the consumption of phosphate. On the other hand, its expression strength can be controlled very finely and the maximum possible expression rates are very high.

Preferably, the expression cassette further comprises at least one sequence region which encodes a selection marker, preferably a resistance gene. Resistance genes are genes that encode factors that make the cell resistant to certain substances, such as antibiotics and/or plant pathogens and/or heavy metals. Resistance genes are usually used as selection markers to detect whether a vector has been transformed into a cell.

Examples of preferred resistance genes are the zeocin resistance gene (zeocin is the commonly known trade name for phleomycin D1), the nourseothricin resistance gene, the blasticidin resistance gene and the chloramphenicol resistance gene.

It is particularly advantageous if the resistance gene is coupled to the second transcription unit, which encodes a reporter protein.

It can be provided that the resistance gene is linked to the reporter gene via genetic coupling in a polycistronic mRNA. The person skilled in the art knows that polycistronic mRNA is understood to mean an mRNA that is encoded by several consecutive genetic units on the nucleic acid sequence of the DNA and which therefore has several open reading frames (ORFs).

In a further advantageous embodiment, the second transcription unit is fused to the first transcription unit, with separation occurring during or immediately after translation, for example caused by P2 peptides (e.g. P2A, P2B, P2T) or IRES elements (internal ribosome entry site). Representatives of both P2 peptides and IRES elements have been shown to be active in P. tricornutum.

Another possibility for coupling the reporter protein is direct coupling at the protein level to the protein to be detected, preferably to the antibody to be detected.

According to a preferred embodiment, individual genetic elements, preferably the promoter element and/or the first transcription unit and/or a complete expression cassette, are repetitively present. The repetitive use, i.e. the repeated use, of genetic elements, in particular regulatory elements such as the promoter element, can increase the expression rate of the nucleic acid sequence according to the invention. In particular, the genetic elements and/or the expression cassette in question are used at least twice, preferably at least three times, preferably immediately one after the other, in the case of repetitive use.

The repetitive use of transcription units, in particular the first transcription unit, which codes for a recombinant protein to be produced, in particular an antibody, leads to a significant increase in the yield and the number of clones or the proportion of clones that show detectable production. This minimizes the process effort. Several copies of the genetic information or the transcription unit of the protein (or antibody) to be produced are cloned on a plasmid or vector. These transcription units preferably flank a resistance gene. The position effect is exploited, i.e. when integrated into the genome at a "favorable" locus, the so-called gene of interest (GOI) is present in multiple copies and ensures an increased amount of transcript, which ultimately increases the yield of recombinant protein.

Preferably, in the case of a repetitive presence of a transcription unit or in the case of the presence of several different transcription units, the resistance gene is arranged particularly "closely" coupled to the transcription units. In particular, "close" coupling means that the genetic connection between the transcription unit and the resistance gene is not severed by genetic recombination (via internal or externally supplied recombinases).

According to a preferred embodiment, the nucleic acid according to the invention is codon-optimized for use in a specific host organism, for example in diatoms, preferably in P. tricornutum

A sequence of a nucleic acid is codon-optimized in particular when the codons are optimally adapted to the frequency of the corresponding tRNAs, so that efficient translation is not inhibited by missing tRNAs loaded with the corresponding amino acid. Furthermore, it is considered an optimization that certain recognition sites of restriction enzymes, which can complicate the cloning process, have been eliminated. The invention therefore also encompasses a nucleic acid encoding an antibody, wherein the sequence of the nucleic acid is codon-optimized for expression in a diatom, unicellular plant or viridiplantae, in particular in a diatom.

The inventors have also discovered that the profile of the amino acid sequence from the original sequence can be taken into account during codon optimization of the sequence of the nucleic acid. This improves in particular the correct folding of the antibody compared to the native counterpart of the antibody, which leads to increased stability of the antibody and increased biological activity of the antibody expressed in the host organism (as defined herein).

In addition, a codon-optimized sequence of a nucleic acid has the advantage that the expression rate in the host organism is increased by at least a factor of 10, preferably at least a factor of 20, particularly preferably at least a factor of 30, very particularly preferably at least a factor of 40 compared to a non-codon-optimized sequence of a nucleic acid. For example, the production rate in the diatom can thereby be increased from the conventional maximum of 3 mg of antibodies per liter of culture to 160 mg of antibodies per liter of culture.

In addition, a sequence of a nucleic acid codon-optimized for expression in a host organism (as defined herein) has the advantage that the folding of the antibody is improved in accordance with the native counterpart of the antibody, which leads to increased stability of the antibody and increased biological activity of the antibody expressed in the host organism.

According to a preferred embodiment, the nucleic acid sequence according to the invention has at least one replicative viral unit, preferably a replicon from the weed dwarf virus (WDV). Such viral elements enable independent replication of the transformed DNA in the form of DNA or RNA. This increases the copy number of transformed DNA or RNA, which, for example, increases the amount of transcript (mRNA) and produces more protein. Furthermore, an increased copy number of the transcription unit to be introduced can result in this DNA being integrated into the genome several times, which increases the chance of integration at a "favorable" locus.

Furthermore, the nucleic acid sequence according to the invention preferably has at least one sequence region which is responsible for a retention signal, preferably for the amino acid sequence KDEL and/or HDEL. Equipping heterologously expressed proteins with a retention signal ensures that the expressed protein remains in the endoplasmic reticulum. Examples of such retention signals are the amino acid sequences KDEL and HDEL. Normally only the heavy chain is equipped with such a sequence. When antibodies are produced in the diatom, however, not only the heavy chain is equipped with such a signal, but also the light chain. This is intended to prevent the possible transport of the light chain into the Golgi apparatus and to promote the assembly of both molecules. In addition, keeping the expressed proteins in the endoplasmic reticulum ensures protection against proteases and successful glycosylation, which increases the yield and maintains functionality.

According to a preferred embodiment of the present invention, the first transcription unit comprises a polynucleotide which encodes an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence with a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, further preferably at least 99% to SEQ ID NO: 2.

SEQ ID NO:2 represents an amino acid sequence of the hinge region of an antibody, which was derived from equine immunoglobulin sequences and has been found to be particularly protease-resistant. The use of this protease-resistant hinge region significantly reduces the proteolysis of antibodies of different formats and from different species produced in P. tricornutum, both in vivo and in vitro.

The SEQ ID NO:2 is as follows:

VIKEPCCCPKCP

vector or isolated nucleic acid

A vector according to the invention or an isolated nucleic acid according to the invention for the heterologous expression of proteins, preferably of antibodies, has at least one nucleic acid sequence according to the invention in simple or repetitive form.

Reference is hereby expressly made to the description of the nucleic acid sequence according to the invention. The number of transcription cassettes in the vector or in the isolated nucleic acid is preferably more than 1 and particularly preferably more than 2. The vector according to the invention or the nucleic acid according to the invention thus has at least two, preferably at least three nucleic acid sequences according to the invention. The transcription cassettes regulated in this way preferably encode a protein, particularly preferably an antibody (in other words: several transcription cassettes all encode one and the same protein or one and the same antibody), the protein, preferably the antibody, having a sequence identity of at least 50%, particularly preferably at least 60%, very particularly preferably at least 70%, in particular at least 80% at the amino acid level.

A transcription cassette (as described herein) consists of at least one regulatory promoter element, a 5'-UTR, a codon-determining sequence (CDS, which is translated into a peptide), a 3'UTR and a terminator. The CDS can contain various subelements, such as signal peptides, which direct antibodies or antigens to a specific cell compartment, tag sequences, such as Strep-Tag or 6x His-Tag, which primarily serve to purify or detect the peptide, or fluorescent markers such as GFP (Green Fluorescent Protein) etc., which primarily serve to detect via their fluorescence.

Preferably, the vector according to the invention or the isolated nucleic acid according to the invention has at least two, preferably at least three, particularly preferably at least four expression cassettes in repetitive form.

The expression cassettes of a vector or of an isolated nucleic acid can be identical to one another or have an identity of at least 70%, preferably at least 80%, to one another. Furthermore, one or more second and/or further expression cassettes differing from a first expression cassette can be arranged in the vector according to the invention or the isolated nucleic acid according to the invention.

By repetitive use, i.e. repeated use, of one or more expression cassettes, an increase in the expression rate of the nucleic acid sequence according to the invention can be achieved. The number of producers with higher production quantities, the number of transgenic clones and the proportion of clones with a detectable signal also increase. The signal of the reporter protein is a direct measure of protein production/antibody products. In particular, the expression cassettes in question are used in repeated use at least in duplicate, preferably at least in triplicate, preferably immediately one after the other.

According to a preferred embodiment, the vector or nucleic acid according to the invention further comprises at least one sequence region which encodes a selection marker, preferably a resistance gene (as described herein).

amino acid sequence

The invention also relates to an amino acid sequence comprising SEQ ID NO: 2 or an amino acid sequence having a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, further preferably at least 99% to SEQ ID NO: 2.

The amino acid sequence according to the invention forms a particularly protease-resistant hinge region in a recombinant antibody. The resulting recombinant antibody is advantageously modified in such a way that it has increased stability against diatom-, human-, plant- or microalgae-specific proteases. This can increase the stability of the antibody in the host organism in which the antibody is expressed and thus also the expression rate.

The term "antibody" refers herein to an immunoglobulin (Ig) or a derivative of an immunoglobulin as produced by the acquired immune system of vertebrates. Examples of naturally occurring antibodies are antibodies of class M (IgM), G (IgG), A (IgA), and E (IgE), in particular from mammals such as humans, rabbits, mice, rats, camels, llamas, goats, and/or horses. Furthermore, artificial formats based on such proteins are also included, examples of which are scFvs (single chain variable fragments), or scFv-Fc (single chain variable fragments fused to a crystalline fragment).

A “native antibody” in the sense of the present invention is a natural antibody as is found in an individual as defined herein, in particular a vertebrate, particularly preferably a mammal, most preferably a primate, in particular a human. Preferably, the amino acid sequence according to the invention is a cysteine-rich amino acid sequence which contains at least 20 cysteines, preferably at least 15 cysteines, particularly preferably at least 12 cysteines. According to a particularly preferred embodiment, the amino acid sequence in the hinge region comprises or consists of (a) the sequence VIKEPCCCPKCP or (b) a sequence identity which differs from this amino acid sequence by a maximum of 30%, in particular by a maximum of 20%, particularly preferably by a maximum of 15%, or (c) an amino acid sequence which has only one amino acid exchange compared to the variant according to (a).

Also according to the invention is a nucleic acid sequence coding for an amino acid sequence comprising SEQ ID NO: 2 or for an amino acid sequence having a homology of at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, further preferably at least 99% to SEQ ID NO: 2.

cell

The invention further relates to a cell comprising a vector according to the invention or an isolated nucleic acid according to the invention (as described herein) or a nucleic acid sequence according to the invention (as described herein), wherein the cell is a photosynthetically active cell, in particular a unicellular plant or viridiplantae, preferably a diatom.

Advantageously, by using photosynthetically active cells, at least a large part or even the entire process for producing recombinant proteins can be carried out in non-animal organisms and thus meet the requirements of vegan production.

screening procedures

A screening method according to the invention for selecting cells with an increased expression rate of a nucleic acid sequence according to the invention (as described herein) comprises the steps: a) providing cells which have been transformed with a vector according to the invention or an isolated nucleic acid according to the invention and/or with a nucleic acid sequence according to the invention, b) isolating and transferring the cells into a culture medium, c) enriching the culture medium with a phosphate concentration in the range of 0.1 pM to 200 pM, preferably 0.5 pM to 200 pM, particularly preferably 1 pM to 200 pM, most particularly preferably 1 pM to 20 pM. d) examining the cells for a desired expression rate.

Common methods such as ballistic methods or electroporation are used to transform the cells with the genetic material according to the invention. While DNA-coated nanoparticles are shot at the cells in ballistic transformation, cells are briefly treated with an electric shock in the range of 2000 V to 2500 V in electroporation, which makes the cell membrane permeable to the DNA.

The cells that have taken up the transgene must then be identified and selected. This is typically done by selecting against a selection substance that interacts with a selection marker contained in the expression cassettes.

Resistance genes (as described herein) are preferably used as selection markers. Following transformation, the cells are transferred to a selection plate and/or to a selection medium, with the selection plate or the selection medium containing the respective selection substance in a sufficient concentration. The person skilled in the art knows the concentration in which the selection substance must be used.

In a preferred embodiment, Zeocin (phleomycin D1) and/or blasticidin are used as selective reagents.

Only cells in which a successful gene transfer has taken place have (in addition to the gene of interest) the respective resistance gene and thus have resistance to the selection substance, which enables them to survive on or in a medium containing the corresponding selection substance. Experts refer to this as positive selection.

The selection is preferably carried out on a solid nutrient medium, preferably on an agar plate, in particular on an agar plate with an agar concentration of less than 1.5%, preferably less than 1%. Advantageously, the growth time of the cells on agar plates with such a reduced agar concentration is reduced, compared to a standard agar concentration of 1.5%. According to a preferred embodiment, the selection is carried out on a solid medium or solid nutrient medium which contains 0.5% to 1% agar, preferably 0.6% to 1% agar, particularly preferably 0.7% to 1% agar, very particularly preferably 0.8% to 1% agar. Such a solid medium is advantageously also used for the maintenance of the strains of the cells used, both transformed and non-transformed cells.

A central requirement is not only to find producing cell lines, but also to identify the cell lines that produce the highest protein yields as early as possible in the selection process. To achieve this, according to a preferred embodiment, reporter proteins (as described herein) are used. These can be present either as a fusion protein or dicistronic (e.g. linked by means of an IRES element). The massively parallel fluorescence-based detection by the reporter proteins correlates clearly with the amount of recombinant proteins that are later produced by this cell line. However, when using nutrient or nutrient deficiency-triggered promoters, such as the modified HASP1 promoter HASP1 ^mod described here, it is not possible to establish a correlation between reporter signal and protein production directly after selection. In order to establish a meaningful correlation, the clones must be transferred from the selection plate or from the selection medium to a liquid medium (e.g. into a multiwell plate). The clones must then be measured over a specific period of time, ideally at a specific time, in order to achieve a meaningful correlation. The time of the evaluation depends on the concentration of the relevant nutrient (e.g. phosphate in the case of HASP1 ^mod ). The concentration must be set so that the cells do not exceed a critical value and the time until evaluation is as short as possible and at the same time as close to reality as possible. For example, the intensity of the fluorescence of the reporter proteins can be correlated with the amount of antibody formed. However, as shown below, this correlation is not trivial and depends on many factors (e.g. the number of cells). The selection process described here is therefore designed so that the correlation between fluorescence and antibody production can be established very reliably with minimal effort.

The preselected cells are separated according to step (b) and transferred to a culture medium. A modified f/2 medium can be used as a culture medium, for example. Preferably, the transfer takes place at least one multiwell plate of at least 6 wells, preferably at least 12 wells, particularly preferably at least 24 wells, very particularly preferably at least 48 wells, further preferably at least 96 wells, in particular at least 384 wells. The resulting clones are separated.

The use of multiwell plates represents a high-throughput method that significantly reduces the effort and costs required for selecting, for example, clones with high expression levels, as this enables simultaneous measurement of up to 384 independent clones.

The medium contains a phosphate concentration of 0.1 pM to 50 pM, preferably 0.25 pM to 35 pM, particularly preferably 0.5 pM to 30 pM, most preferably 0.75 pM to 25 pM, in particular 0.1 pM to 20 pM, furthermore in particular 1 pM to 20 pM, according to step (c) advantageously leads to the phosphate deficiency-triggered modified promoter HASP1 ^mod described herein reacting with increased activity due to the low phosphate concentration of the medium and the protein production thus triggered can be evaluated at an earlier point in time than at higher phosphate concentrations. The resulting increased production of the reporter protein can be easily read out.

Here, “phosphate” refers to all phosphate salts and phosphate-containing organic compounds and all chemical forms of phosphorus.

When evaluating reporter signals, the clones can be analyzed directly after the cells have been isolated and transferred to a culture medium, but the number of cells is low at this point, which increases the measurement inaccuracy. To address this problem, the cells are cultured in the culture medium, preferably in a multiwell plate, to an optimal cell density. It must be taken into account, especially when working with nutrient deficiency-triggered promoters, that the initial nutrient concentration has a significant influence on the expected cell density during the course of the cultivation and thus also on the time of evaluation. This is influenced by several factors: In order to obtain reliable reporter signals, the cell density must not be too high, because above a certain cell density the signal strength of the reporter is no longer proportional to the number of cells. Although a qualitative comparison might be possible under certain circumstances, it is not permissible for a quantitative statement.

Advantageously, the cells are examined for an expression rate of the

Nucleic acid sequence at several times during cell cultivation. In particular, Using the phosphate deficiency-triggered HASP1 ^mod promoter described here in combination with a fluorescent reporter protein, there is no longer a linear relationship between the number of cells and the signal intensity once a critical cell density has been exceeded. The phosphate deficiency must therefore occur before the cell density exceeds the critical value, otherwise a comparison between the clones is not possible. When using nutrient deficiency-triggered promoters, it must also be ensured that only clones that show a similar number of cells are compared with each other and global peaks of the individual signal time series must be used to evaluate production.

The expert knows how to determine the number of cells. A suitable method is to determine the optical density (OD), for example by means of absorption measurement, where a light beam is directed into the medium and the light loss is measured with a detector. Up to a certain cell density, the optical density shows an essentially linear increase.

For the photometric measurement (both for determining the cell density and for examining the cells for an expression rate of the nucleic acid sequence, in particular by determining the activity of the reporter protein, for example via fluorescence measurements), the multiwell plate is preferably filled with the culture medium to a volume of 30% to 70%, preferably 45% to 55% of the total volume of each individual well. The measurement inaccuracy is highest in this range, with 50% filling volume being suitable both for culturing the cells in the multiwell format and for a more error-tolerant measurement (e.g. in the case of volume deviations due to evaporation during cultivation or due to pipetting errors). Therefore, when transferring the preselected cells according to step (b), the wells of the multiwell plate are preferably filled with the culture medium to a volume of 30% to 70%, preferably 45% to 55% of the total volume of each individual well. A microplate reader can be used to read the parameters.

To determine the number of cells in a Phaeodactylum tricornutum culture, different wavelengths can be used, which are preferably between 600 and 800 nm. The measurement is particularly preferably carried out at a wavelength of 600 to 750 nm.

In both fluorescence measurements and optical density measurements, the number of cells must not exceed a certain number in order to establish a linear relationship. to obtain reliable, linear measurement results. The range in which the cell number must lie depends on the cell size and type and is preferably determined empirically, e.g. using dilution series. For diatoms, the cell number at which the examination according to step (d) is preferably carried out is 0.1 million cells * mL ^-1 to 120 million cells * mL ^-1 , preferably 0.25 million cells * mL ^-1 to at least 100 million cells * mL ^-1 , particularly preferably 0.5 million cells * mL ^-1 to 100 million cells * mL ^{-1 ,} most particularly preferably 1 million cells * mL ^-1 to 90 million cells * mL ^-1 , further preferably 2 million cells * mL ^-1 to 80 million cells * mL ^-1 , even more preferably 3 million cells * mL ^-1 to 70 million cells * mL ¹ , in particular 4 million cells * mL ^-1 to 60 million cells * mL ^-1 .

It is advantageous to take into account the morphology of the respective cells when determining the cell number, particularly with regard to a possible influence on the measurement results of the method used. For example, when determining the cell number of Phaeodactylum, the occurrence of two different cell shapes (fusiform cells and oval cells) can lead to an incorrect correlation between cell number and optical density.

Preferably, an immunobiochemical and/or photometric method, preferably a high-throughput method, is carried out to examine the cells for an expression rate of the nucleic acid sequence according to step (d). Examples of immunobiochemical methods that can be used are ELISA, SDS-PAGE or dot blot. Examples of photometric methods that can be used are the photometric determination of cell density and/or chlorophyll content and/or fluorescent proteins and/or chromoproteins and/or color reactions and/or luminescence. All of the methods that can be used can also be used in addition to and/or on the basis of one another or as an alternative to one another.

According to a preferred embodiment, the examination according to step (d) is carried out after a period of 1 to 21 days, preferably from 1.5 to 21 days, particularly preferably from 2 to 14 days after the implementation of step (c). It has been shown that the optimal cell density for carrying out the examination according to step (d) can be expected after this period.

Preferably, in the screening method according to the invention, at least 5, preferably at least 10, particularly preferably at least 30, very particularly preferably at least 60, further preferably at least 96 different clones are examined in order to identify cells with an increased, preferably with the highest expression rate of a nucleic acid sequence according to the invention.

Following the screening method according to the invention, additional investigations of the cells that express the most strongly, such as ELISA or SDS-PAGE, can be carried out to determine whether and which antibodies are produced, in which quality and with which binding properties.

The disruption of cells in a multiwell format (especially 2 - 384 well) is preferably carried out using a buffer (e.g. PBS, HEPES, Tris-HCl, CHES, CAPSO, AMP, CAPS, CABS) containing detergents or by means of a temperature increased compared to the physiological state or a combination of detergents and a temperature increased compared to the physiological state.

The temperature is preferably more than 40 °C, particularly preferably more than 50 °C and most preferably at least 60 °C. Incubation times of between 0.5 h and 72 h are preferably used.

Suitable detergents are known to the person skilled in the art. Examples of these are Tween20, Triton X 100, sodium dodecyl sulfate (SDS), digitonin or cetyltrimethylammonium bromide (CTAB). The respective detergent is used in a final concentration preferably between 0.1% and 10%, particularly preferably between 0.2% and 10%, very particularly preferably between 0.3% and 10%, further preferably between 0.4% and 10%, in particular between 0.5% and 10%.

Centrifugation after cell disruption leads to better quality results when analyzing the purification, e.g. by ELISA or SDS-PAGE.

The digested material can be used for screening in the ELISA method using multiwell plates, but also - after adding so-called SDS-PAGE loading buffer and heating to 80 °C to 100 °C - directly in SDS-PAGE. Such methods are well known to those skilled in the art. However, the sedimentation of the solids and pigments described here leads to better quality results in ELISA and SDS-PAGE and plays a central role in the analysis of the purification steps. manufacturing process

The method according to the invention for producing recombinant antibodies using a vector according to the invention and/or an isolated nucleic acid according to the invention and/or a nucleic acid sequence according to the invention comprises the following steps: a) providing cells with a nucleic acid sequence according to the invention, preferably determined by a screening method according to the invention, b) culturing the transformed cells in a culture medium, wherein the cultivation is initially carried out in a pre-culture and then in a main culture and wherein the culture conditions are adapted in such a way that the yield of heterologously produced proteins is maximized, and c) extracting the heterologously produced proteins.

The provision of the cells according to step (a) may also comprise transforming the cells (as described herein) by suitable methods such as ballistic transformation or electroporation.

When culturing the transformed cells according to step (b), the focus is on optimizing high yields of heterologously produced proteins, especially in closed cultivation systems.

Cultivation can be carried out in batch format, i.e. under the static conditions of a constant culture medium, or in fed-batch format, i.e. with regular addition of fresh medium, whereby the expert knows which culture can achieve the highest yield.

It is pointed out at this point that there are different cultivation methods, such as batch, fed-batch and the continuous cultivation method, and that the invention relates to all cultivation methods, but in particular to batch cultivation, in which all nutrient concentrations are only adjusted at the beginning of the cultivation.

In fed-batch cultivation, nutrients are added during the cultivation process. It is crucial that the multiple addition of phosphate in a fed-batch cultivation accelerates the induction of the promoter (described here) and thus reduces the cultivation time. In addition, phosphate (regardless of the chemical form, in in which the phosphate is present), especially at the end of the cultivation, in small quantities to avoid complete exhaustion of the phosphate and thus optimize the yield.

The invention is not limited to the culture sequence (pre-culture -> main culture). The cultivation can also be carried out without prior pre-culture. According to the invention, the cells are brought to a certain cell density during the pre-culture. These cells are then transferred to the main culture. The desired product is obtained from the main culture. The vitality and division rate of the cells in the pre-culture is crucial for the increase in biomass in the main culture; the more vital and actively dividing the cells in the pre-culture, the higher the increase in biomass. Higher biomass leads to a higher yield in the main culture.

Preferably, the cells are therefore transferred from the pre-culture to the main culture at a certain division rate and, accordingly, the division rate of the cells is determined to determine the optimal time for transferring the pre-culture to the main culture.

According to a preferred embodiment, the pre-culture according to step (b) is carried out without external gassing of the culture medium.

In this embodiment, the pre-culture is transferred to the main culture in the range of a certain division rate, preferably after a period of at least 4 days, particularly preferably at least 7 days, very particularly preferably at least 12 days. The period refers to the start of the culture. The specialist personnel knows how the division rate can be determined.

According to a further preferred embodiment, the pre-culture according to step (b) is carried out with external gassing of the culture medium.

In this further embodiment, the pre-culture is transferred to the main culture in the range of a certain division rate, preferably after a period of at least 4 days, particularly preferably of at least 7 days, very particularly preferably of at least 12 days. The period refers to the start of the culture.

In particular, the optimal time to transfer the pre-culture into the main culture depends on the vitality of the cells. The vitality in turn depends on the nutrient composition of the culture medium. The lower the The more nutrients are available, the faster the optimal time for transferring the pre-culture to the main culture is exceeded. The following phosphate concentrations are particularly relevant for the pre-culture: 37 pM - 5000 M phosphate; particularly preferred 40 pM - 5000 pM; most particularly preferred 50 pM - 5000 pM.

In Phaeodactylum tricornutum, an alternative to the division rate can be the morphology of the cells, in particular the ratio between duplicate and single cells, or the amount of chlorophyll per cell or another measure representing the number of cells, such as the optical density (OD).

Phaeodactylum cells can take on different morphotypes, a fusiform and an oval shape. Both morphological types are present in every culture, whereby the ratio between the proportions can vary between > 0 and < 1. A morphology change can be achieved by the following method:

1. Spreading the cells on an agar plate and selecting colonies,

2. Subsequently, a “limiting dilution” is carried out, in which individual cells become the starting point for new colonies, all of which belong to the same morphotype.

Alternatively, the enrichment or switch to a particular morphology can be promoted or induced by physical or chemical conditions.

It has been shown that a change in morphology can significantly increase transformation efficiency. Fusiform cells achieve a higher transformation efficiency than oval cells. Furthermore, the change in morphology to the optimal cell shape can significantly increase the yield of recombinantly produced proteins. Another reason in favor of a change in morphology is that this process can significantly reduce the "clumping" of cells after the change in morphology. "Clumped" cells disrupt the cultivation process, the overgrowth process and the extraction of heterologous proteins.

For the optimal time of transfer of the pre-culture into the main culture in Phaeodactylum tricornutum, the ratio of single cells to at least double cell chains is between 0.1 and 10, preferably between 0.1 and 4, particularly preferably between 0.1 and 3.

Furthermore, the division rate can be used to determine the optimal time for transferring the pre-culture into the main culture of Phaeodactylum tricornutum. the optimal extrapolated or actual weekly division rate is log ₂ > 0.1, preferably log ₂ > 0.2, particularly preferably log ₂ > 0.3, most preferably log ₂ > 0.5, further preferably between log ₂ >= 0.5 and 20, further particularly preferably log ₂ >= 0.5 and 10, further particularly preferably log ₂ >= 0.5 and 5.

Advantageously, a culture medium is used which is adapted to the genetic elements according to the invention, in particular the nucleic acid sequence according to the invention or the vector according to the invention or the isolated nucleic acid according to the invention.

In particular, the culture medium comprises phosphate in an amount of 20 pM to 300 pM, preferably from 20 pM to 400 pM, particularly preferably from 20 pM to 500 pM.

Phosphate not only plays a central role in the activation of the HASP1 ^mod promoter (as defined herein), but is also crucial for achieving highest cell densities. It has been shown that pro:

8 -50 million cells*!.' ¹ : 20 pM - 500 pM phosphate,

Preferably 12 -50 million cells*!.' ¹ : 20 pM - 400 pM phosphate, particularly preferably 15 -50 million cells*!.' ¹ : 20 pM - 300 pM phosphate, are required in the medium. In this way, the amount of phosphate required to achieve the desired biomass and avoid premature phosphate depletion can be calculated.

It should be noted that phosphate concentrations of over 360 pM are particularly suitable for the regulation of the HASP1 ^mod promoter described here. Such high concentrations not only inhibit the expression rate of the HASP1 ^mod promoter very efficiently and over a longer period of time, but also allow a higher cell density to be achieved before a strong increase in expression occurs due to the consumption of phosphate. The H AS P1 ^mod promoter thus provides the ideal control to achieve high cell densities without expression of the controlled gene before expression is massively increased by the degradation of phosphate. On the other hand, its expression strength can be controlled very finely and the maximum possible expression rates are very high.

According to a preferred embodiment, the culture medium comprises glycerol as carbon source in an amount of more than 50 mM, preferably more than 60 mM, particularly preferably more than 70 mM, very particularly preferably more than 80 mM, further preferably more than 90 mM, in particular more than 100 mM. According to a further preferred embodiment, the initial culture medium comprises glucose as carbon source in an amount of more than 0.5 g/L, preferably more than 1 g/L, particularly preferably more than 2 g/L, very particularly preferably more than 5 g/L, further preferably more than 10 g/L, in particular more than 20 g/L.

During the entire further cultivation, glucose is administered at 0.5 g/L, preferably more than 1 g/L, particularly preferably more than 2 g/L, very particularly preferably more than 5 g/L, further preferably more than 10 g/L, in particular more than 20 g/L every 2 to 14 days; preferably every 2 to 10 days, particularly preferably every 3 to 7 days. The administration can contain only glucose but also other nutrients such as phosphate, any nitrogen source, particularly preferably NO3 salts. Either the entire medium can be replaced or the nutrients can be added directly to the culture (continuous culture or fed-batch).

Alternatively, acetate, sucrose, fructose, starch or an equivalent carbon source may be present in the culture medium as a carbon source.

A carbon source is necessary because the light penetration of the medium is continuously reduced by the increasing cell density.

Despite the carbon source, growth is limited by the light supply: It has been found that light-related cell growth decreases significantly at a cell density between 30 million cells*ml ^-1 million and 400 million cells*ml ^-1 . Thus, at a cell density between 35 million cells*ml ^-1 and 350 million cells*ml' ¹ , preferably at a cell density between 40 million cells*ml ^-1 and 300 million cells*ml ^{_ 1} , particularly preferably at a cell density between 45 million cells*ml ^-1 and 400 million cells*ml ^-1 and especially preferably at a cell density between 55 million cells*ml ^-1 and 200 million cells*ml ^-1 , either light-independent cell growth or continuous dilution of the cell culture or a process which allows light to penetrate the medium is required. Glycerol and/or glucose have proven to be particularly advantageous in the amounts stated here for the production of high biomass with simultaneous high production of heterologous protein.

Furthermore, the culture medium preferably comprises nitrogen, preferably in the form of NO ₃ and/or NH ₄ , in an amount of more than 10 mM, preferably more than 15 mM, particularly preferably more than 20 mM, very particularly preferably more than 30 mM, further preferably more than 40 mM, in particular more than 50 mM. In this case, a sufficient availability of a nitrogen source, particularly in the form of NO ₃ and/or NH ₄ , is advantageously crucial for optimal protein production. If the nitrogen concentration is below a critical value, the cell density itself may remain unaffected, but the protein yield drops dramatically. In order to avoid losses in yield, the amount of nitrogen to achieve a certain cell density must be within a certain range. Within the scope of the present invention, NH ₄ and/or NO ₃ can be present in any chemical form and bond.

Nitrogen is added either initially and/or gradually to achieve the specified values. Nitrogen is preferably added initially and gradually.

Preferably, the initial N0 ₃ concentration of the culture medium is between 5 mM and 250 mM, preferably between 10 mM and 250 mM, particularly preferably between 20 mM and 250 mM, especially preferably between 50 mM and 250 mM.

Preferably, the successive NO ₃ addition to the culture medium is between 5 mM and 250 mM, preferably between 10 mM and 250 mM, particularly preferably between 20 mM and 250 mM, especially preferably between 50 mM and 250 mM.

If NH ₄ is used as a nitrogen source, continuous monitoring of the pH value and continuous buffering of the culture medium are preferred, since otherwise a drop in the pH value leads to a strong reduction in the vitality of the cell culture.

According to a further advantageous embodiment, the culture medium has an initial amount of micronutrients/trace elements and vitamins of 2 to 400 times, preferably 3 to 400 times, particularly preferably 4 to 400 times, very particularly preferably 5 to 400 times above the respective “normal” concentration.

"Normal" concentrations for trace elements and vitamins have been described for the cultivation of diatoms and are known to those skilled in the art. They can vary from publication to publication. The reference values for "normal" concentrations within the scope of the present invention refer to the f/2 medium and are shown below: Table 1: Composition of the media: trace elements contained.

Table 2: Trace elements added to the medium in increased concentrations.

The typical concentrations of the f/2 medium (Guillard, 1975) are listed here.

Examples of vitamins that are included in the culture medium at the indicated increased concentration are biotin (used as standard in a “normal” concentration of 2 pM), cyanocobalamin (used as standard in a “normal” concentration of 0.37 pM) and thiamine (used as standard in a “normal” concentration of 297 pM).

The culture medium preferably has a pH of 6 to 9.5, preferably 6.5 to 9, particularly preferably 7 to 8.5. The culture medium is preferably buffered using Tris buffer, although other buffer-active substances known to the person skilled in the art may also be used. which have equivalent properties in the pH range mentioned, for example HEPES or MOPS buffers.

In particular, the culture medium contains more than 2.5 mM Tris-HCl, pH 8, preferably more than 5 mM Tris-HCl, pH 8, particularly preferably more than 7.5 mM Tris-HCl, pH 8, very particularly preferably more than 10 mM Tris-HCl, pH 8.

According to a preferred embodiment, the culture medium has an oxygen saturation in the range of 40% to 100%, preferably in the range of 50% to 100%, particularly preferably in the range of 60% to 100%, very particularly preferably in the range of 70% to 100%, in particular in the range of 80% to 100%.

Particularly at higher cell densities, the carbon source (for example in the form of glycerol, acetate, glucose, sucrose, fructose, starch) of the culture medium has a strong influence on the increase in biomass. When light availability is low, it is no longer CO2 but O2 that drives cell growth. Oxygen can be added to the culture medium either from the air or as concentrated _O2 or as a mixture containing _O2 . The decisive factor here is the saturation of the culture medium with O2.

In heterotrophic or mixotrophic conditions, cultivation is carried out with air or O ₂ or a mixture containing O _2. This type of aeration ensures that the culture medium remains supplied with oxygen.

When aerating with air, the initial aeration is at least 1 L * min ^-1 * L ^-1 culture medium, preferably at least 2 L * min ^-1 * L ^-1 culture medium, particularly preferably at least 3 L * min ^-1 * L ^-1 culture medium, very particularly preferably at least 4 L * min ^-1 * L ^-1 culture medium. The aeration with air is preferably gradually increased to at least 8 L * min ^-1 * L ^-1 culture medium.

When aerating with oxygen, the initial aeration is at least 0.1 L * min ^-1 * L ^-1 culture medium, preferably at least 0.2 L * min ^-1 * L ^-1 culture medium, particularly preferably at least 0.3 L * min ^-1 * L ^-1 culture medium, very particularly preferably at least 0.4 L * min ^-1 * L ^-1 culture medium. The aeration with O ₂ and/or air is preferably gradually increased to at least 8 L * min ^-1 * L ^-1 culture medium. For photoautotrophic cultivation, a gas mixture containing air or another gas mixture with CO ₂ is preferably used to aerate the culture medium. In this application, the initial aeration when aerating with air is also at least 1 L * min ^-1 * L ^-1 culture medium, preferably at least 2 L * min ^-1 * L ^-1 culture medium, particularly preferably at least 3 L * min ^-1 * L ^-1 culture medium, very particularly particularly preferably at least 4 L * min ^-1 * L ^-1 culture medium. Preferably, the aeration with air is gradually increased to at least 8 L * min ^-1 * L ^-1 culture medium.

When aerating with a gas mixture containing CO ₂ , the CO ₂ content of the initial aeration is at least 0.1 L * min ^-1 * L ^-1 culture medium, preferably at least 0.2 L * min ^-1 * L ^-1 culture medium, particularly preferably at least 0.3 L * min ^-1 * L ^-1 culture medium, very particularly preferably at least 0.4 L * min ^-1 * L ^-1 culture medium. The aeration with CO ₂ is preferably gradually increased to at least 8 L * min ^-1 * L ^-1 culture medium.

Increased ventilation leads to a significantly faster growth of biomass.

Another essential parameter for optimized cultivation in terms of biomass and heterologous protein production is the type (regarding the spectrum used) and intensity of the light exposure.

According to a further preferred embodiment, the culture medium is exposed during the cultivation according to step (b) to an illumination with an illumination intensity (light intensity) of 20 - 400 pmol *nr ² * s ^-1 , preferably of 40 - 400 pmol *nr ² * s ^-1 , particularly preferably of 60 - 400 pmol *nr ² * s ^-1 , very particularly preferably of 80 - 400 pmol *nr ² * s ^-1 , in particular of 100 - 400 pmol *nr ² * s ^-1 , measured in the interior of the culture medium, preferably in the middle of the culture medium.

The measurement of the light intensity is preferably carried out either with a PAR sensor or an ePAR sensor (Extended Photosynthetically Active Radiation), both of which allow the measurement of the total photon flux intensity, whereby the measuring range of the ePAR sensors (400 nm to 750 nm) in the spectrum exceeds that of PAR sensors (400 nm to 700 nm). Alternatively, the measurement of the light intensity can be carried out with another suitable sensor that detects light intensity and spectrum.

Preferably, the sensor used for measuring light is aligned at an angle of 90° to the light source. The sensor is located at a distance relative to the vessel wall closest to the light source of 0.3 to 0.5 times the diameter of the cultivation vessel.

It can further be provided that a successive increase in the exposure intensity by 20 - 400 pmol *nr ² * s ^-1 , preferably from 40 - 400 pmol *nr ² * s ^-1 , particularly preferably from 60 - 400 pmol *rrr ² * s ^-1 , most preferably 80 - 400 pmol *m ^-2 * s ^-1 , in particular 100 - 400 pmol *m ^-2 * s ^-1 , measured (as described herein) in the interior of the culture medium, preferably in the middle of the culture medium.

Following the cultivation of the transformed cells, the heterologously produced proteins are extracted according to the invention.

In addition to the heterologously produced protein or proteins, also referred to herein as protein of interest, the disrupted cells can contain a large number of low molecular weight substances that can represent a disruptive factor for the subsequent processes. These substances include, for example, pigments, i.e. colorants that are visible to the naked eye. It has been found that after cooling the disrupted cells, in particular the disrupted diatom cells, the pigments can be separated, which can be done using common separation methods such as centrifugation or filtration. For this purpose, the following temperatures are set in the separation methods: preferably a maximum of 4 °C, particularly preferably a maximum of 2 °C and very particularly preferably a maximum of 0 °C. Furthermore, the pH value of the disruption buffer is preferably between pH 2 - 12 and particularly preferably between pH 4 - 10.

The extraction of the heterologously produced proteins is also carried out by methods known to the person skilled in the art.

EXAMPLES OF IMPLEMENTATION

The present invention is explained in more detail with reference to the following figures and embodiments, without limiting the invention to these.

This shows

Fig. 1: A schematic overview of the relationship between individual invention objects;

Fig. 2A: the repetition of two promoters;

Fig. 2B: a vector according to the invention with repetitively inserted expression cassettes;

Fig. 3: a diagram showing the formation of fluorescence as a function of the copy number of the promoter used in a nucleic acid sequence according to the invention or a vector according to the invention;

Fig. 4A: a diagram showing the distribution of signal intensities depending on the number of expression cassettes in a vector according to the invention;

Fig. 4B: a diagram of the number of transgenic colonies and the proportion of fluorescent clones as a function of the number of expression cassettes in a vector according to the invention;

Fig. 5A: a diagram showing the distribution of signal intensities depending on the type of vector modification with respect to a replicon;

Fig. 5B: Evidence of the formation of a replicon in Phaeos;

Fig. 6A: the result of three gel electrophoreses comparing the proteolytic degradation depending on the hinge region used;

Fig. 6B: the amino acid sequence of the hinge region according to the invention;

Fig. 7: a diagram showing the dependence of the HASP1 ^mod promoter on phosphate consumption or repression of the HASP1 ^mod promoter by regular phosphate addition;

Fig. 8A: a diagram showing the dependence between cell density and measured GFP signal;

Fig. 8B: a graph of the responses of seven independent clones to phosphate deficiency compared to a graph of the same clones under phosphate addition; Fig. 9: a diagram of the phosphate deficiency-dependent activation of the HASP1 ^mod promoter according to the invention;

Fig. 10: a diagram showing the shortening of the harvest cycle by phosphate addition;

Fig. 11: an embodiment of a vector according to the invention;

Fig. 12: a graph comparing the tGFP signal intensities of independent clones at three different time points in 96-well plates with the reference in 100 ml flasks.;

Fig. 13A: Microscopic image of fusiform cells of P. tricornutunr,

Fig. 13B: Microscopic image of oval cells of P. tricornutunr,

Fig. 13C/D: Diagrams of the correlation of optical density and cell number in fusiform cells (C) and oval cells (D) of P. tricornutunr,

Fig. 13E: a diagram showing the correlation between expected and measured optical density as a function of cell density;

Fig. 14: a diagram showing the correlation between ELISA signals and GFP signals following a screening method according to the invention;

Fig. 15A: a microscopic image of P. tricornutum-ZeWerr,

Fig. 15B: a diagram showing the proportion of different cell types depending on the culture age;

Fig. 15C: a representation of viability and division rate of cells of a preculture of P. tricornutum at different times during the cultivation;

Fig. 15D: a diagram showing the growth of the main culture depending on the age of the previous culture;

Fig. 16A: a diagram showing the relationship between protein production, culture age and amount of nitrogen source;

Fig. 16B: a diagram showing the relationship between cell density, culture age and amount of nitrogen source;

Fig. 16C: a diagram showing the relationship between protein production, culture age and amount of urea source;

Fig. 16D: a diagram showing the relationship between cell density, culture age and amount of urea source;

Fig. 17: a diagram showing the dependence of cell growth on phosphate concentration; Fig. 18: a diagram showing the dependence of absolute biomass on the available concentration of trace elements and vitamins;

Fig. 19: a diagram showing the dependence of the increase in cell number on ventilation;

Fig. 20: the result of gel electrophoresis after digestion, separation of pigments and purification of antibodies in IgG format from P. tricornutunr,

Fig. 21 : Correlation between fluorescence and expression rate in 10 independent clones; and

Fig. 22: A diagram comparing the binding affinity of two diatom antibodies (formats) with those of the same sequence from human cell culture (Expi ^293F ).

Figure 1 shows a schematic overview of the relationship between individual invention subjects. All of the sub-steps shown (individually or in combination) lead to an improvement/optimization of the heterologous production of proteins, in particular antibodies, in particular in the diatom Phaeodactylum tricornutum.

As already explained in detail, a sequence optimization (1) of a nucleic acid sequence according to the invention is first carried out. This can include, for example, codon optimization and/or the use of particularly protease-resistant genetic elements (as described herein), such as hinge regions derived from equine IgGs (immunoglobulin G).

The nucleic acid sequence according to the invention is then introduced into a vector according to the invention (or an isolated nucleic acid according to the invention) (2), whereby individual genetic elements and/or complete expression cassettes are used repetitively. Furthermore, special inducible promoters are used, in particular a promoter element from the nucleic acid sequence of SEQ ID NO: 1.

By using specific signal sequences (as described herein), the heterologously produced proteins are expressed in the endoplasmic reticulum of the cells, which leads to protection of the proteins from proteases and successful glycosylation, thereby increasing the yield and maintaining the functionality of the proteins.

In a next step, the transformation (3) of the vector according to the invention (or the nucleic acid according to the invention) into target cells takes place, preferably into photosynthetically active cells, in particular cells of a unicellular plant or viridiplantae, preferably cells of Phaeodactylum tricornutum. The transformation can take place ballistically or by means of electroporation in a suitable medium. This is followed by a screening method (4) according to the invention for selecting cells with an increased expression rate of a nucleic acid sequence according to the invention. In this case, a screening can be carried out using reporter genes for high-performance producers, i.e. for cells with an increased expression rate. Furthermore, the correlation between the expression of the reporter genes and the expression strength of the proteins to be produced can be determined. Multiwell plates are preferably used both for the screening method and for culture monitoring.

Following the screening method (4) according to the invention, a method according to the invention for producing recombinant proteins, preferably recombinant antibodies, (5) on the basis of the cells with an increased expression rate determined in the screening method (4) can be carried out.

According to the invention, recombinant proteins are produced in a culture medium that is tailored to the genetic elements used, in particular to the promoters used. The cultivation conditions, such as minimum light intensities and ventilation quantities, are also adapted (as defined herein).

Both in relation to the nucleic acid sequence according to the invention and in relation to the vector according to the invention (6) or the isolated nucleic acid according to the invention, the use of repetitive genetic elements leads to an enormous increase in the expression rates in P. tricornutum. Figure 2A shows the repetition of two HASP1 promoters. Figure 2B shows an example of an embodiment of a vector according to the invention (6) in which an expression cassette (9) was used repetitively, here in the form of a triple cassette for the production of antibodies in the scFv-Fc format. In particular, this repetitive use of the expression cassette (9) leads to a significant increase in the yield and the number of clones or the proportion of clones that show detectable production, thereby minimizing the overall process effort. In the embodiment shown, the expression cassette (9) has a promoter element (9.1), a first transcription unit (9.2) which codes for a protein to be produced recombinantly, and a second transcription unit (9.3) which codes for the reporter gene tGFP, wherein the individual genetic elements (9.1, 9.2, 9.3) in Figure 2B are shown as an example on one expression cassette (9), but are also present in the other expression cassettes (9).

Figure 3 shows a diagram of the formation of fluorescence as a function of the

Copy number of the promoter used (9.1) in a

nucleic acid sequence or a vector according to the invention (6) or a isolated nucleic acid according to the invention. In particular, Figure 3 shows the distribution of the medians of the GFP signals (fluorescence signals of the green fluorescent protein normalized using ODeoo) depending on the promoter used (9.1), whereby the HASP1 ^mod promoter was used either in a single version (left) or in a double version (right). It can be seen that the repetitive (here double) use of the HASP1 ^m ° ^d promoter leads to an amplification of the measured GFP signals.

Figure 4A shows a diagram of the distribution of the signal intensities depending on the number of expression cassettes (9) in a vector according to the invention (6). It can be seen that an increased number of expression cassettes (9) in the vector (6) leads to an increase in the fluorescence signal (measured as a normalized tGFP signal, note Iog2 scaling), which is due to the increased expression of the reporter gene. Figure 4B shows a diagram of the number of transgenic colonies and the proportion of fluorescent clones depending on the number of expression cassettes (9) in a vector according to the invention (6) (this is an Iog2 scale). It is clear that an increased number of expression cassettes (9) increases both the number of transgenic clones and the proportion of clones with a detectable fluorescence signal, with the fluorescent signal being a direct measure of protein production/antibody production.

Figure 5A illustrates the increase in the yield of heterologously produced protein depending on the use of replicative viral units. This is a diagram showing the distribution of signal intensities depending on the type of vector modification in relation to a replicon, whereby in the example shown, the replicon from the weed dwarf virus (WDV) was used. The use of the replicon (on the right in the diagram) leads to a significant increase in the fluorescence signal (measured as a normalized tGFP signal, note Iog2 scaling) compared to vectors not modified in this way (on the left in the diagram) and thus to an increase in the yield of heterologously produced proteins. This is based on the fact that the replicon enables independent division of the transformed DNA. This increases the number of copies of transformed DNA, which, for example, increases the amount of transcript (mRNA) and produces more protein. Furthermore, an increased copy number of the transcription unit to be introduced (9.2) can result in this DNA being integrated multiple times into the genome, which increases the chance of integration at a “favorable” locus.

Figure 5B shows a schematic representation of the repModi vector (vector modified with so-called replicon) and putative replicon and the analysis of some clones from the repModi approach. Top of the figure) Schematic representation of the repModi vector and of the putative replicon with the corresponding primer position for detection and sequencing. (Bottom left in the figure) PCR analysis of the control and the clones D6, E2, E12, F8 for antibodies and replicon formation. (Bottom right in the figure) Sequencing result of the replicon amplicons of the clones E12 and F8. Marker (M): GeneRuler 1 kb Plus. The primer pair R1/R2 was used to detect a replicon (480 bp) formation and the primer pair AK1/AK2 was used to detect the antibody sequence (390 bp). Black arrows in the figure bottom left point to the respective amplicons at the correct height.

The repModi vector (in Fig. 5B repModi) was equipped with genetic elements from the weed dwarf virus (WDV). The replicon forming units (2x LIR, RepA and SIR) flank the antibody cassette and are involved in the recombination of the vector. After the recombination event, the vector consists of a LIR element, the RepA, the SIR element and the antibody cassette, the backbone and a LIR element are removed. Such a unit is able to replicate independently in the host organism.

To check whether this recombination event had occurred, appropriate primers were synthesized. The primer pair R1/R2 was designed to detect the recombination event (480 bp) and the primer pair AK1/AK2 a small part of the antibody cassette (390 bp). In the case of recombination, the distance between primers R1 and R2 is reduced from 3720 bp to 480 bp.

The results of the PCR are shown in Figure 5B, bottom left. A clone was used as a control that contains the same antibody sequence as the repModi approaches, but had been cloned into the noModi vector. With the primer pair AK1/AK2, a band at approx. 400 bp was detected in all clones. With the primer pair R1/R2, a band of approx. 500 bp was detected in all tested clones D6, E2, E12 and F8, except for the control. The amplicons of the clones E12 and F8 were then sequenced. The sequencing revealed a base sequence as would be expected in a recombination (Figure 5B, bottom right). The PCR result and the sequencing strongly indicate the formation of a replicon.

Figure 6A shows the result of different gel electrophoresis to compare the proteolytic degradation depending on the hinge region used.

The lanes labelled with M always show the same size marker. Different IgGs are applied in the different lanes: Lane 1: equine lgG6, lane 2: equine scFv-Fc, lane 3: murine lgG1 and lane 4: human lgG1. The filled arrows indicate the non-proteolytically cleaved heavy chain (HC), the hatched arrows indicate the light chains (LC). This band is missing in lane 2 because in the scFv-Fc format the LC is covalently bound to the other regions of the scFv-Fc and is therefore not separated in SDS-PAGE. Therefore, the upper band in lane 2 has also traveled less far than those in all other lanes. The open arrows indicate the cleavage products that occur in murine and human IgGs. It can be seen that the use of protease-resistant elements, here a protease-resistant hinge region, significantly reduces the proteolysis of antibodies of different formats and from different species produced in P. tricornutum, both in vivo and in vitro.

Figure 6B shows the amino acid sequence of the equine hinge region from eqlgG6 according to the invention. The described effect of reducing proteolysis by the hinge region also applies to genetic elements in which this amino acid sequence has been changed by up to 30%.

Figure 7 shows a diagram of the dependence of the HASP1 ^mod promoter on phosphate consumption (dark gray curve) and repression of the HASP1 ^mod promoter by regular phosphate addition (light gray curve). The GFP signal is a direct measure of promoter activity and protein production. Several independent clones showed this phosphate dependence. One of the clones is shown here as a representative. It can be seen that the HASP1 ^mod promoter is inhibited by phosphate and triggered by phosphate deficiency. "Phosphate" here means all phosphate salts and phosphate-containing organic compounds and all chemical forms of phosphate. It should be noted that phosphate concentrations of over 360 pM are particularly suitable for regulating the promoter. Such high concentrations not only allow the expression rate of the HASP1 ^mod promoter to be inhibited very efficiently and over a longer period of time, but also allow a higher cell density to be achieved before a strong increase in expression occurs due to the consumption of phosphate. The HASP1 ^mod promoter thus provides the ideal control to achieve high cell densities without expression of the controlled gene, before expression is massively increased by the degradation of phosphate. On the other hand, its expression strength can be controlled very finely and the maximum possible expression rates are very high.

Figure 8A shows a diagram of the relationship between cell density and measured GFP signal in three different cell cultures (dark gray, medium gray, light gray). The dashed line shows the expected GFP signal (assuming unrestricted linear behavior) and the solid line shows the measured GFP signal. It can be seen that the measured signal deviates from the expected signal. The reason for this is the increasing cell density. The relationship between cell density and signal strength is shown in a certain range linear. If signals are measured above a critical cell density, an appropriate correction must be made. In particular, this deviation means that when using a nutrient deficiency-dependent promoter (e.g. HASP1 ^mod , alkaline phosphatase) or a nutrient-triggered promoter (e.g. nitrate reductase), the nutrient concentration of the cell culture must be carefully chosen for studies at the molecular level (e.g. testing of additional regulatory sequences as well as for cultivation studies). The deficiency must arise before the cell density exceeds the critical value, otherwise a comparison between the clones is not possible. When using nutrient deficiency-triggered promoters, it must also be ensured that only clones that show a similar number of cells are compared and global peaks of the individual signal time series must be used to evaluate production. If the critical cell density is exceeded, there is no linear relationship between the number of cells and the signal intensity. Since the underlying effects depend predominantly on the cell density, the measurement can be corrected using a standard.

Figure 8B shows a graph of the responses of seven independent clones to phosphate starvation (left) compared to a graph of the same clones with phosphate added (right). The wild type is shown as a graph marked with in both graphs. Without the addition of phosphate (left graph), the tGFP signals of all transgenic clones increased. With the addition of phosphate (right graph), the tGFP signals decreased to a low level.

A diagram of the phosphate deficiency-dependent activation of the H AS P1 ^mod promoter according to the invention is shown in Figure 9. The strength of the GFP signal is illustrated as a function of the culture age using four different cultures. The four cultures were cultivated in culture media with different phosphate concentrations, with 36 pM representing the lowest and 360 pM the highest phosphate concentration. Here too, the GFP signal is a direct measure of the activation of the promoter and protein production. As the phosphate concentration increases, the peak of the GFP signal shifts, since the phosphate deficiency occurs at a later time or at a higher cell density. While in a medium with the lowest amount of phosphate the peak occurs after about 16 days (36 pM), this peak occurs at higher concentrations on about day 24 (72 pM), day 29 (140 pM) or the peak is not reached (360 pM). When this peak is reached depends on the initial phosphate concentration, the growth rate of the cells and the absolute number of cells. Although the peak of the culture with the highest phosphate concentration is not reached, the yield is still highest here because the cell density is many times higher than the cell density due to the increased phosphate availability. other crops. The amount of phosphate can be used to precisely control both the yield and the “harvest time”.

Figure 10 shows a diagram of the shortening of the harvest cycle by adding phosphate when using a phosphate deficiency-triggered promoter. Compared to the control, peak production is reached earlier if the amount of phosphate in the medium is kept at a constant "low" level or if phosphate is added at certain intervals. This effect occurs because a lower phosphate content (than the maximum desired phosphate content) in the culture medium causes the cells to "expect" a phosphate deficiency at an earlier point in time (than when the maximum desired amount of phosphate was initially added), and therefore all phosphate-dependent promoters are regulated accordingly (such as HASP1 ^mod ). By adding phosphate again, the "expected" deficiency does not occur (or not to the "expected" extent), but the activation of the promoter caused by the "expectation" is restored. B. HASP1 ^mod promoter, however, continues to have an effect for some time, whereby the protein of interest linked to the HASP1 ^mod promoter (e.g. an antibody) is already produced. The above-mentioned process can thus be continued by leaving the cells "in the belief" that a phosphate deficiency is occurring. Whether and when this deficiency occurs depends on the desired duration of cultivation and is monitored by the person responsible. In the example shown, phosphate is added in the form of NaH ₂ PO ₄ at certain intervals.

Figure 11 shows an example of an embodiment of a vector (6) according to the invention for the transformation of P. tricornutum. Only elements that are important for P. tricornutum are shown. This construct leads to the production of an antibody in the so-called scFv-Fc format in the transformed diatom (the individual elements of which are: a variable heavy chain VH (9.21), which is linked to a variable light chain VL (9.22) by means of a linker. This is located in front of the constant regions, here a human intermediate sequence, the hinge region (9.23) and an equine constant region (CH2 and CH3) (9.24). This region is linked via a proteolytically cleavable flag tag to a reporter gene (9.3), here turboGFP, which in turn is fused to a Strep tag. Another genetic construct under the control of a constitutive fcpB promoter is a selection marker (9.5), here the Zeocin resistance gene. It becomes clear that genetic elements of different origins (here antibodies in scFv format with synthetic, human and equine origins) can be combined with one another. and still form functional proteins.

Figure 12 illustrates a comparison of the tGFP signal intensities of independent clones at three different time points in 96-well plates with the reference in 100 ml flasks. In order to establish an evaluation of the tGFP signals in a multiwell plate, it was checked at which time the tGFP signals in the multiwell plate were closest to the "final/actual" tGFP signal. For this purpose, the tGFP signals of 29 independent clones were measured at several time points in a 96-well plate and then the tGFP signals of the same cultures in 100 ml flasks were determined. In previous experiments, SDS-PAGE showed that the tGFP signals in 100 ml flasks correlate with the actual amount of protein. Figure 12 shows the development of the tGFP signals in the 96-well plates at 4 different time points after inoculation of the culture (day 1, day 4, day 6, day 8). The corresponding R ² values are given in the legend. The R ² values are 0.1; 0.86; 0.93 and 0.93 for the corresponding time points 1, 4, 6 and 8. There was no correlation between the measured tGFP signal in the 96-well plate and the maximum achievable tGFP signal on day 1 after inoculation. On day 4, the R ² value rose to 0.86 and increased to 0.93 on day 6. From day 6 to day 8, there was no change in the correlation measurable by the R ² value.

Figures 13A and 13B show microscopic images of different cell types in the P. tricornutum culture, with Figure 13A showing fusiform cells and Figure 13B showing oval cells. Both morphological types are present in each culture, but the ratio between the proportions can vary between > 0 and < 1. When determining the cell number of Phaeodactylum using optical density, the morphology of the cells must be taken into account to avoid erroneous correlation. The appropriate factor depends on the device.

Figures 13C and 13D show diagrams for the correlation of the optical density (OD) and the cell number in fusiform cells (C) and oval cells (D) of P. tricornutum. From the diagram shown in Figure 13E for the correlation between expected and measured optical density as a function of cell density, it is clear that the cells must not exceed a certain cell number in order to obtain a linear relationship or reliable, linear measurement results. The range in which the cell number must lie depends on the cell size, type and measuring instrument and must therefore be determined empirically, e.g. via dilution series. Different wavelengths can be used to determine the cell number in a Phaeodactylum tricornutum culture, which are preferably 600 - 800 nm. The measurement is particularly preferably carried out at a wavelength of 600-750 nm.

Figure 14 shows a diagram of the correlation between ELISA signals and GFP signals following a screening method according to the invention. GFP signals here represent the amount of protein. This correlation with an R ² value of 0.92 shows that the high-throughput method used can be used to lyse the cells in the ELISA procedure.

Figure 15A shows a microscopic image of P. tricomutum cells, where the cells are present either as a single cell (11.1), as a cell chain of two cells (11.2) or as multiple cell chains (11.3). The optimal time for transferring the pre-culture to the main culture (in the context of the production process according to the invention) is a time at which the ratio of single cells to double cell chains is between

0, 3 and 10.

A diagram of the proportion of different cell forms as a function of culture age is shown in Figure 15B. “1x” denotes the proportion of single cells (11.1), “2x” the proportion of cell chains made up of two cells (11.2) and “>2x” the proportion of multiple cell chains (11.3).

Figure 15C shows a representation of vitality and division rate of cells from a preculture of

P. tricornutum at different times during the cultivation of the pre-culture. Finally, Figure 15D shows a diagram of the growth of the main culture depending on the age of the pre-culture. Figures 15B, 15C and 15D make it clear that the time at which the pre-culture should be transferred to the main culture plays an important role. There is a connection between vitality, division rate and/or the ratio between single cells and cell chains made up of two or more cells in the pre-culture and the subsequent growth rate of the main culture. The proportion of living cells must be high and a certain division activity of the pre-culture must not be undercut in order to enable the optimal increase in biomass. Higher biomass leads to a higher yield in the main culture. To determine the optimal time, either the proportion of living cells (Fig. 15C), the ratio between duplicate and single cells can be used, or the division rate must be above a certain threshold value (Fig. 15A and 15B).

Figure 16A shows a diagram of the relationship between protein production, culture age and amount of nitrogen source and Figure 16B shows a diagram of the relationship between cell density, culture age and amount of nitrogen source. The information on the nitrogen sources is given in mM. While the cell density is only minimally influenced by the nitrogen concentration (Fig. 16B), the amount of protein produced depends very strongly on the amount of nitrogen source (Fig. 16A). Here too, the GFP signal is a direct measure of the amount of heterologously expressed protein. Sufficient availability of a nitrogen source is crucial for optimal Protein production (especially NO ₃ and NH ₄ ). If this is below a critical value, the cell density itself may remain unaffected, but the protein yield drops dramatically. To avoid losses in yield, the amount of nitrogen to achieve a certain cell density must be within a certain range (e.g. NH ₄ or NO ₃ in any chemical form and bond).

Figure 16C shows a diagram showing the relationship between protein production, culture age and amount of urea source, while Figure 16D shows a diagram showing the relationship between cell density, culture age and amount of urea source. Here too, the information on the urea sources is given in mM. Similar to Figures 16A and 16B, the amount of nitrogen also influences protein production. However, as can be seen in Figure 16D, urea is harmful to the culture above a certain concentration, as the culture dies after a certain time at a concentration of 10 mM urea. The initial concentration of urea is therefore limited to >10 mM.

Figure 17 shows a diagram showing the dependence of cell growth on phosphate concentration. A certain amount of phosphate is required to achieve a certain maximum cell density. The different phosphate concentrations are given in pM. This clearly shows the fine balance between activation of the HASP1 ^mod promoter and achieving a high cell density (compare with Figure 9).

Figure 18 shows a diagram showing the dependence of absolute biomass on the available concentration of trace elements and vitamins. In the control, vitamins and trace elements are used at a concentration as described in the f/2 medium (“standard medium”). In the four subsequent samples, vitamins (V) and trace elements (S) were used in an x-fold amount compared to the standard medium: 5 times the amount, 40 times the amount, 80 times the amount and 160 times the amount. It can be seen that even an extreme increase in concentration does not lead to a significant reduction in biomass. Especially in a batch culture, it is advantageous to enrich the medium with as many nutrients as possible in order to avoid a possible deficiency.

A diagram showing the dependence of the increase in cell number on the aeration of the culture medium is shown in Figure 19. Increased aeration with 3 L/min (upper line) showed a significantly faster increase in biomass compared to the control (aeration with 1 L/min, lower line). This shows that a sufficient supply of gases such as CO2 and O2 must be ensured. Above all, the flow of light through the medium decreases significantly at low cell densities, which impairs photosynthesis. comes to a standstill. From this point on, the cell increasingly relies on the uptake of external, organic C sources such as glycerol or glucose and thus requires more and more O ₂ .

Figure 20 (A and B) shows the result of gel electrophoresis after digestion and purification of antibodies in IgG format from P. tricornutum. Figure 20A shows the Coomassie-stained SDS-PAGE, Figure 20B the corresponding immunostaining. The supernatant is applied to the lanes marked with "Ü", the lanes marked with "PP" show pellets containing pigment. The pigment could be separated from the recombinant antibody without suffering a significant loss of yield. Interference from the pigment in the further process is reduced to a minimum. Immunostaining was carried out with mouse anti-Strep antibody and rabbit anti-mouse IgG-AP, which is why only the heavy chain is detected in the immunostaining, since the light chain does not contain a Strep tag. The crude lysate of diatoms was cooled. After centrifugation or filtration, 2 fractions were formed. The fraction “Ü” was the soluble supernatant and the fraction “PP” was the pigment-containing pellet. It is crucial that the pigment could be separated and that the majority of the antibody remained in the soluble supernatant.

Figures 21A-D show the correlation between fluorescence and expression rate in 9 of a total of 30 independent clones. Figure 21A shows tGFP fluorescence units (Turbo-Green Fluorescent Protein, tGFP) for the 9 clones. Figure 21B shows corresponding protein extracts separated by Coomassie-stained SDS-PAGE, with the numbers above the lanes representing different, independent clones (black arrow marks the heavy chain of the antibody). Figure 21C shows the immunostaining of the expressed antibodies in the corresponding samples, the labeling of which corresponds to that in Figure 21B (black arrow marks the heavy chain of the antibody). Figure 21 D shows a strong correlation between the GFP signal from 21 A and the pixel intensity of the heavy chain from 21 B with an R ² value of 0.94 and Figure 21 E shows the corresponding bar graph in which the values of tGFP signal and SDS band strength (antibody band, measured densitometrically in the Bio-Doc (Bio-Rad)) are plotted side by side.

The product produced by the production process according to the invention is therefore not only cheaper to produce, but the production process has a number of advantages over conventional processes: The antibodies produced have the same avidity as the counterpart from Expi ^293F cells (Error! Reference source could not be found. 22). Furthermore, the process presented here is sustainable, animal-free and - depending on the origin of the antibody sequence to be produced - even vegan. In comparison to antibody sera from animals, the antibodies presented here are permanently available. Production can be increased very easily and the products are free of human pathogens.

Figure 22 shows a diagram comparing the binding affinity of two diatom antibodies (formats) (light gray, graphs with triangles) with those with the same sequence from human cell culture (Expi293F) (dark gray, graphs with squares). In a competitive titration assay, the avidities of antibodies of different formats (IgG or scFv-Fc) against equine interleukin 31 were analyzed at increasing antibody concentrations. There are no differences in the IgGs with the same sequence (solid lines) from diatoms or Expi ^293F cells, and in the scFv-Fcs (dashed lines) from the different starting lines, the scFv-Fc from diatoms is even more affine than the original from human cell culture.

Concrete implementation example

The embodiment, the sequence of which is outlined in Fig. 1, shows the production of an antibody using the method according to the invention. This antibody in IgG format is directed against equine interleukin 31 and was produced with more than 160 mg of purified eqlgG * L ^-1 cell culture within a 14-day culture period.

In a first step, the codon usage is adapted to P. tricornutum. In the present case, the starting sequence comes from a human scFv bank and was codon-optimized before use in the diatoms. The required genetic elements, typically the variable light chain (V _L ) and the variable heavy chain (V _H ) were synthesized by IDT-DNA (Coralville, Iowa) so that they fit optimally into the created vectors (Fig. 2B). Interfering restriction sites were also removed or modified. A variant of the vectors according to the invention for the production of antibodies in scFv-Fc format is shown in Fig. 2B. Here, the variable light chain (kappa or lambda format (V _LK or V _L A)) and the variable heavy chain (V _H ) can be used. In this example, the constant regions of both chains come from the horse. Constructs were also generated that contain constant antibody regions from other host organisms (e.g. mouse or human). Other variants of the vectors according to the invention have been produced and successfully used for the heterologous production of other formats such as Fab or scFv-Fc. The example shown in Fig. 2B contains the finished vector construct and, in addition to the elements to be introduced into P. tricornutum, also bacterial genetic elements that are used for cloning in E. coli. (colE1 origin and gentamycin resistance gene). The gene of interest was inserted in three copies in the construct. After the ballistic or electroporation-based transformation of P. tricornutum, the clones obtained must not only be checked for the uptake of the antibody genes, but also the rapid identification of clones that express the introduced antibody gene particularly strongly is a central innovation of this invention. The underlying screening method according to the invention enables the correlation of the fluorescence caused by tGFP (Turbo-Green Fluorescent Protein), which is measured in a special microtiter plate reader, with the later expected amount of antibody formed (Figure 21 shows the correlation between fluorescence and expression rate in 9 independent clones).

In comparison to production rates of a maximum of 3 mg of antibody * L ¹ culture described in the prior art, 160 mg of purified antibody could be obtained from 1 L of culture in the system according to the invention. To achieve this, new media compositions, aeration with more than 3 L * min ^-1 of compressed air and lighting with a light intensity of 100 - 1000 W * nr ² in a specially developed reactor column were used.

An example media composition is given in the following table:

After typically fourteen to twenty days of fed-batch culture, the diatoms can be harvested and digested. Purification follows the classic method, i.e. after lysis and centrifugation and/or ultrafiltration, purification is carried out either via protein A, protein G or via the tag sequences used, for example the 6xHis tag. This is another advantage of the method according to the invention, because unlike higher plants, which produce antibodies transiently or stably, the diatoms do not contain any fibers that make the purification of the antibodies extremely difficult. The purification therefore largely corresponds to the method used for animal cell cultures, such as CHO cells. REFERENCE SYMBOL LIST

1 Sequence optimization of the nucleic acid sequence according to the invention

2 Introducing the nucleic acid sequence according to the invention into a vector according to the invention

3 Transformation

4 screening procedures

5 Methods for the production of recombinant proteins

6 vector

7 cells

8 culture medium

9 expression cassette

9.1 Promoter element

9.2 first transcription unit

9.21 variable heavy chain

9.22 variable light chain

9.23 Hinge region

9.24 equine constant region (CH2 and CH3)

9.3 second transcription unit

9.4 Signal peptide

9.5 Selection markers

10.1 Length standard of gel electrophoresis

10.2 Wild-type extract in gel electrophoresis

10.3 equine lgG6 in gel electrophoresis

10.4 scFv-Fc in gel electrophoresis

10.5 murine lgG1 in gel electrophoresis

10.6 human IgG in gel electrophoresis

10.7 human lgG1 in gel electrophoresis

11.1 single cells of Phaeodactylum tricornutum

11.2 double cell chains of Phaeodactylum tricornutum

11.3 multiple cell chains of Phaeodactylum tricornutum

Claims

PATENT CLAIMS 1 . A nucleic acid sequence comprising at least one expression cassette for expressing a protein, wherein the expression cassette comprises at least one promoter element and at least one first transcription unit encoding at least one peptide, wherein the promoter element consists of the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having a homology of at least 70% to SEQ ID NO: 1. 2. The nucleic acid sequence of claim 1, wherein the expression cassette further comprises at least a second transcription unit encoding a reporter protein. 3. Nucleic acid sequence according to claim 1 or 2, wherein the expression cassette further comprises at least one sequence region encoding a selection marker. 4. Nucleic acid sequence according to one of claims 1 to 3, wherein the promoter element and/or the first transcription unit and/or a complete expression cassette is present repetitively. 5. Nucleic acid sequence according to one of claims 1 to 4, wherein the first transcription unit is present at least twice and the first two transcription units flank the sequence region encoding a resistance gene. 6. Nucleic acid sequence according to one of claims 1 to 5 further comprising at least one replicative viral unit. 7. Nucleic acid sequence according to one of claims 1 to 6, further comprising at least one sequence region encoding a retention signal. 8. A nucleic acid sequence according to any one of claims 1 to 7, wherein the first transcription unit comprises a polynucleotide encoding an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having a homology of at least 70% to SEQ ID NO: 2. 9. Vector or isolated nucleic acid comprising at least one nucleic acid sequence according to one of claims 1 to 8 in simple or repetitive form. 10. Vector or isolated nucleic acid according to claim 9, comprising at least two, preferably at least three expression cassettes. 11. Vector or isolated nucleic acid according to claim 9 or 10, further comprising at least one sequence region encoding a selection marker. 12. A nucleic acid sequence encoding an amino acid sequence comprising SEQ ID NO: 2 or an amino acid sequence having a homology of at least 70% to SEQ ID NO: 2. 13. An amino acid sequence comprising SEQ ID NO: 2 or an amino acid sequence having a homology of at least 70% to SEQ ID NO: 2. 14. Cell comprising a vector or an isolated nucleic acid according to any one of claims 9 to 11, or a nucleic acid sequence according to any one of claims 1 to 8, or a nucleic acid sequence according to claim 12, or an amino acid sequence according to claim 13, wherein the cell is a photosynthetically active cell, in particular a viridiplantae or a unicellular plant, preferably a diatom. 15. Screening method for selecting cells with an increased expression rate of a nucleic acid sequence according to one of claims 1 to 8, comprising the steps of a) providing cells which have been transformed with a vector or an isolated nucleic acid according to one of claims 9 to 11 and/or with a nucleic acid sequence according to one of claims 1 to 8, b) isolating and transferring the cells into a culture medium, c) enriching the culture medium with a phosphate concentration in the range of 0.1 pM to 200 pM and d) examining the cells for an expression rate of the nucleic acid sequence. 16. Screening method according to claim 15, wherein the expression rate of the nucleic acid sequence according to step (d) is determined by the activity of a reporter protein or by the described ELISA method. 17. Screening method according to claim 15 or 16, wherein the examination according to step (d) is carried out after a period of 1 to 21 days, preferably 1.5 to 21 days, particularly preferably 2 to 21 days after the implementation of step (c). 18. A screening method according to any one of claims 15 to 17, wherein the examination according to step (d) is carried out at a cell count of 20 to 120 million cells * ml_ ^-1 . 19. A method for producing recombinant antibodies using a vector or an isolated nucleic acid according to one of claims 9 to 11 and/or a nucleic acid sequence according to one of claims 1 to 8, comprising the steps of a) providing cells with a nucleic acid sequence according to one of claims 1 to 8, preferably determined by a screening method according to one of claims 15 to 18, b) culturing the transformed cells in a culture medium, wherein the culturing is initially carried out in a pre-culture and subsequently in a main culture and wherein the culture conditions are adapted in such a way that the yield of heterologously produced proteins is maximized, and c) extracting the heterologously produced proteins. 20. The method according to claim 19, wherein the preculture according to step (b) is cultivated without external gassing of the culture medium. 21. The method according to claim 20, wherein the preculture with the described division rate is transferred to the main culture after a period of at least 20 days. 22. The method according to claim 19, wherein the preculture according to step (b) is cultivated with external gassing of the culture medium. 23. The method of claim 22, wherein the preculture is transferred to the main culture at the time of the highest division rate after a period of at least 16 days. 24. The method according to any one of claims 19 to 23, wherein the culture medium contains phosphate in an amount of 20 pM to 300 pM, preferably 20 pM to 400 pM, particularly preferably 20 pM to 500 pM.