JP7820943B2 - Method for producing VHH antibody - Google Patents

Description

The present invention relates to a method for producing a VHH antibody.

Antibodies in the immune response are known to include immunoglobulin M (IgM), which increases in the patient's blood relatively early after the body comes into contact with an antigen due to infection, and IgG, which increases thereafter.
While typical immunoglobulins (Ig) are composed of light chains and heavy chains, camelids are known to produce heavy-chain antibodies that do not contain light chains. Furthermore, a single domain containing the variable region of a heavy-chain antibody is a naturally occurring single domain, and functions as an antibody by itself. For this reason, it is called a "single-domain antibody" (hereinafter sometimes abbreviated as "VHH antibody").

When a viral three-dimensional structural epitope includes, for example, a hole-like structure like an enzyme pocket or a structural portion of a cleft between domains, conventional IgG is unable to bind to such epitopes due to its size. In contrast, the above-mentioned VHH antibodies have a molecular weight one-tenth that of IgG antibodies, and are therefore able to bind to epitopes with structures such as those described above, as well as to the surface of virus particles modified with many sugar chains, thereby broadening the range of molecules that can be targeted.

Furthermore, VHH antibodies have excellent acid resistance and heat resistance, and unlike IgG, they do not need to be produced in cultured cells but can be produced in E. coli, yeast, etc. This gives them the advantage of being easy to mass-produce and purify. Furthermore, because VHH antibodies are composed of a single-chain peptide, their function can be easily modified using protein engineering techniques or chemical modification techniques, making them suitable for the production of antibody-drug conjugates (ADCs).

Affinity chromatography is commonly used to purify antibodies, which utilizes specific affinity for the target substance to achieve high purity in one step (see, for example, Patent Document 1). A substance called a ligand, which has high specificity and affinity for the target substance, is immobilized on the surface of the affinity support. Typically, an antibody-binding protein called protein A is used as the ligand. In affinity chromatography, an antibody stock solution is passed through a column packed with protein A affinity support, allowing the antibody to specifically adsorb, and then the antibody is desorbed with an acidic buffer solution to recover highly pure antibody.

Special Publication No. 2009-508521

Recently, various protein A affinity supports have been developed that exhibit high antibody binding capacity, allowing for rapid processing of large amounts of antibody stock solutions in a single chromatographic purification run, and are expected to significantly improve antibody productivity.
However, it was found that increasing the loading (adsorption) amount of VHH antibody stock solution onto the column and eluting under normal acidic conditions may result in a decrease in antibody activity.
Therefore, an object of the present invention is to provide a method for producing a VHH antibody that can maintain antibody activity even at high loading levels.

The inventors investigated the cause of the decline in antibody activity and concluded that a high loading amount causes the VHH antibody to become unstable due to the combined effects of the collapse of the VHH antibody's molecular structure under acidic conditions and increased density, making the VHH antibody more susceptible to denaturation and aggregation. Further investigation revealed that VHH antibody aggregation can be suppressed and the decline in antibody activity can be avoided by eluting under conditions that increase the partition coefficient between the affinity support and the VHH antibody.

That is, the present invention provides a method for producing a pharmaceutical composition comprising the following steps (1) and (2):
The present invention provides a method for producing a VHH antibody, comprising: (1) passing a solution containing a VHH antibody through an affinity carrier having Protein A as a ligand such that the amount of VHH antibody adsorbed (g) is 4 to 20 g/L relative to the packed layer volume (L) of the affinity carrier; and (2) after step (1), passing an eluate such that the partition coefficient between the affinity carrier and the VHH antibody is in the range of 0.07 to 20.

According to the present invention, purified VHH antibodies can be obtained with high productivity while maintaining antibody activity.

The method for producing the VHH antibody of the present invention comprises the following steps (1) and (2):
(1) passing a solution containing a VHH antibody through an affinity carrier having Protein A as a ligand so that the amount of VHH antibody adsorbed (g) is 4 to 20 g/L relative to the packed layer volume (L) of the affinity carrier; and (2) after step (1), passing an eluate such that the partition coefficient between the affinity carrier and the VHH antibody is in the range of 0.07 to 20.

[Step (1)]
This step involves passing a solution containing a VHH antibody through the affinity carrier using Protein A as a ligand so that the amount of VHH antibody adsorbed (g) is 4 to 20 g/L relative to the packed bed volume (L) of the affinity carrier using Protein A as a ligand. This step allows the VHH antibody to be adsorbed onto the affinity carrier using Protein A as a ligand.

(VHH antibody)
VHH antibodies are antibodies that utilize the variable region of a heavy chain antibody. The target molecule of a VHH antibody is not particularly limited. VHH antibodies include antibodies produced in animal cells, humanized antibodies, and chimeric antibodies. Humanized antibodies can be administered to humans and therefore can be used as pharmaceuticals.
Preferably, the VHH antibody is a VHH antibody that binds to SARS-CoV-2. SARS-CoV-2 is a SARS-associated coronavirus that causes acute respiratory disease (COVID-19), and its viral genome is a single-stranded positive-sense RNA virus with approximately 29,903 bases. Specifically, the VHH antibody that binds to SARS-CoV-2 is an antibody that binds to the S1 subunit of the spike protein of SARS-CoV-2.
In the present invention, more preferred VHH antibodies are the SARS-CoV-2 VHH antibodies CoVHH1 and VHH-COVE1 to VHH-COVE9 exemplified below. CoVHH1 and VHH-COVE1 to VHH-COVE9 are antibodies discovered by the present applicant and are described in applications by the present applicant (Japanese Patent Application Nos. 2021-076809 and 2021-162820).

(CoVHH1)
CoVHH1 is an antibody that binds to SARS-CoV-2 and has one or more structural domains including CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one amino acid in said amino acid sequence has been substituted with another amino acid, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one amino acid in said amino acid sequence has been substituted with another amino acid, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which one amino acid in said amino acid sequence has been substituted with another amino acid.

CDRs (Complementarity Determining Regions) contain sequence-variable antigen recognition sites or random sequence regions, and are also called hypervariable regions. In the structural domain of an antibody, the three CDRs are located in the following order from the N-terminus: CDR1, CDR2, and CDR3.

Here, the amino acid sequence shown in SEQ ID NO: 1 is GSTFSDYVMA, the amino acid sequence shown in SEQ ID NO: 2 is TISRNGGTTT, and the amino acid sequence of CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3 is VGGDGDS.

The CDRs consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 3, in which one amino acid is substituted with another amino acid, refer to CDRs in which a mutation has been introduced into the CDRs consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 3. Furthermore, even if a mutation has been introduced into CDR1, CDR2, or CDR3, it is still included in the CDRs of CoVHH1 as long as it has the ability to bind to SARS-CoV-2.
Here, the position of the amino acid to be substituted is not limited, but preferred positions include the 3rd, 4th, 5th, or 10th positions in CDR1, the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th positions in CDR2, and the 1st, 2nd, 3rd, or 4th positions in CDR3.
In addition, the type of amino acid after substitution is not limited, but preferred substitutions include substitution with amino acids of similar polarity or size.Preferred substitutions also include substitution of threonine residue with asparagine residue, isoleucine residue or methionine residue, substitution of phenylalanine residue with isoleucine residue, substitution of serine residue with arginine residue or asparagine residue, substitution of alanine residue with threonine residue, substitution of arginine residue with serine residue, substitution of asparagine residue with tyrosine residue, substitution of glycine residue with serine residue, substitution of valine residue with phenylalanine residue or isoleucine residue, and substitution of aspartic acid residue with glycine residue or valine residue.
Preferred combinations of the position of the amino acid to be substituted and the type of amino acid after substitution include, in CDR1, substitution of the third threonine residue with an asparagine residue, substitution of the fourth phenylalanine residue with an isoleucine residue, substitution of the fifth serine residue with an arginine residue, substitution of the tenth alanine residue with a threonine residue,
In CDR2, substitution of the third serine residue with an asparagine residue, substitution of the fourth arginine residue with a serine residue, substitution of the fifth asparagine residue with a tyrosine residue, substitution of the sixth glycine residue with a serine residue, substitution of the seventh glycine residue with a serine residue, substitution of the eighth threonine residue with a methionine residue, substitution of the ninth threonine residue with an isoleucine residue, substitution of the tenth threonine residue with a methionine residue,
In CDR3, substitution of the first valine residue with an isoleucine residue, the third glycine residue with a valine residue, and the fourth aspartic acid residue with a glycine or valine residue are preferred.

The binding ability to SARS-CoV-2 can be evaluated by methods known to those skilled in the art. Specifically, as shown in the Examples below, it can be evaluated by determining the equilibrium dissociation constant KD using biolayer interferometry. It can also be evaluated by methods using, for example, antigen-immobilized ELISA, immunochromatography, isothermal titration calorimetry, surface plasmon resonance measurement, etc. The dissociation constant KD in the present invention is determined by biolayer interferometry.

The structural domain of CoVHH1 may have framework regions on both sides of CDR1, CDR2, and CDR3.
The framework region refers to a region in the variable region of an antibody molecule excluding the complementarity determining regions, and is a highly conserved region.
That is, one embodiment of the structural domain of the present invention includes a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3, and a fourth framework region (FR4) in this order.
The amino acid sequences of the framework regions in the structural domains include the following:
FR1: the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 80% or more identity with said amino acid sequence; FR2: the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 80% or more identity with said amino acid sequence; FR3: the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 80% or more identity with said amino acid sequence; FR4: the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 80% or more identity with said amino acid sequence.

Framework regions consisting of amino acid sequences that share 80% or more identity with the amino acid sequences set forth in SEQ ID NOS: 4 to 7 include framework regions consisting of amino acid sequences that share preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more identity.

Here, amino acid sequence identity refers to the percentage (%) of the number of positions where identical amino acid residues exist in two amino acid sequences when the two sequences are aligned, relative to the total number of amino acid residues in both sequences. Sequence identity can be calculated, for example, by performing an analysis using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI).

Among CoVHH1s, antibodies having a structural domain (SEQ ID NO: 8) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO: 1, CDR2 is the amino acid sequence shown in SEQ ID NO: 2, CDR3 is the amino acid sequence shown in SEQ ID NO: 3, FR1 is the amino acid sequence shown in SEQ ID NO: 4, FR2 is the amino acid sequence shown in SEQ ID NO: 5, FR3 is the amino acid sequence shown in SEQ ID NO: 6, and FR4 is the amino acid sequence shown in SEQ ID NO: 7 are preferred.

(VHH-COVE1 to VHH-COVE9)
VHH-COVE1 to VHH-COVE9 are antibodies that bind to SARS-CoV-2 and have one or more structural domains each including CDR1, CDR2, and CDR3, and each consisting of the amino acid sequences shown in SEQ ID NOs: 9 to 35 below or amino acid sequences in which at least one amino acid in the amino acid sequences has been substituted with another amino acid.

Here, the position of the amino acid to be substituted and the type of amino acid after substitution are not limited, as long as the dissociation constant of these amino acid sequences with the antigen is not 100 nM or greater, but preferably a single substitution is made.

Of the above VHH-COVE1 to 9, VHH-COVE2, VHH-COVE5, VHH-COVE8, and VHH-COVE9 are preferred in terms of neutralizing activity against SARS-CoV-2.

The structural domains of VHH-COVE1 to VHH-COVE9 may have framework regions (FR1 to FR4) on both ends of CDR1, CDR2, and CDR3.
FR1 may be the amino acid sequence set forth in SEQ ID NOs: 36 to 42, and FR2 may be the amino acid sequence set forth in SEQ ID NOs: 43 to 50. FR3 may be the amino acid sequence set forth in SEQ ID NOs: 51 to 58, and FR4 may be the amino acid sequence defined by the nucleotide sequence set forth in SEQ ID NOs: 59 to 61.

Furthermore, FR1 to FR4 preferably contain amino acid sequences having 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 36 to 61. Here, the amino acid sequences having 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 36 to 61 are more preferably sequences having 85% or more identity, and even more preferably 90% or more identity, to the amino acid sequences shown in SEQ ID NOs: 36 to 61. It is even more preferable that the framework regions are composed of amino acid sequences having 99% or more identity.
The identity of the amino acid sequences is as described above.

VHH-COVE1 to VHH-COVE9 can be, for example, those in which, from the N-terminus to the C-terminus, the sequences are linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As described above, the amino acid sequences of these regions are shown in SEQ ID NOs: 9 to 61 in the Sequence Listing, and the amino acid sequences of the corresponding regions of VHH-COVE1 to VHH-COVE9 are shown in SEQ ID NO: 62 (VHH-COVE1), SEQ ID NO: 63 (VHH-COVE2), SEQ ID NO: 64 (VHH-COVE3), SEQ ID NO: 65 (VHH-COVE4), SEQ ID NO: 66 (VHH-COVE5), SEQ ID NO: 67 (VHH-COVE6), SEQ ID NO: 68 (VHH-COVE7), SEQ ID NO: 69 (VHH-COVE8), and SEQ ID NO: 70 (VHH-COVE9).

There are no particular limitations on the method for producing VHH antibodies, and they can be easily produced using techniques known in the art. For example, they can be produced by combining solid-phase peptide synthesis with native chemical ligation (NCL) or by genetic engineering. However, a preferred method involves incorporating nucleic acid encoding the VHH antibody into an appropriate vector (e.g., a plasmid), introducing this into host cells, and producing the antibody as a recombinant antibody.

Host cells used for producing a recombinant antibody include, for example, Escherichia coli, Bacillus bacteria, fungi, animal cells, plant cells, baculovirus/insect cells, yeast cells, etc. Among these, Bacillus bacteria are preferred from the viewpoint of productivity.
Specifically, Bacillus genus bacteria include Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus stearotheromophilus, Bacillus coagulans, Bacillus megaterium, Bacillus halodurans, Bacillus brevis (Brevibacillus brevis), Bacillus choshinensis (Brevibacillus choshinensis), Bacillus pumilus, Bacillus alcalophilus, Bacillus amylolyticus, Bacillus amyloliquefaciens, Bacillus liqueniformis, Bacillus polymyxa (Paenibacillus polymyxa), Bacillus sphaericus, Bacillus firmus, Bacillus clausii, Bacillus macerans, and the like. Among these, Bacillus subtilis, Bacillus megaterium, and Bacillus brevis are preferred.
Note that Bacillus brevis is classified in the genus Brevibacillus and may be described as Brevibacillus brevis or Brevibacillus choshinensis depending on the strain, Bacillus polymyxa is classified in the genus Paenibacillus and may be described as Paenibacillus polymyxa, and Bacillus stearotheromophilus is classified in the genus Geobacillus and may be described as Geobacillus stearotheromophilus. However, in this specification, Bacillus brevis, Bacillus polymyxa, and Bacillus stearotheromophilus are all defined as bacteria belonging to the genus Bacillus. That is, in the present invention, reference to bacteria of the genus Bacillus is interpreted as including bacteria described as Brevibacillus brevis, Brevibacillus choshinensis, bacteria described as Paenibacillus polymyxa, and bacteria described as Geobacillus stearotheromophilus.

The expression vector for expressing an antibody can be a vector suitable for various host cells. Examples of the expression vector include vectors derived from Escherichia coli such as pBR322, pBR325, pUC12, and pUC13; vectors derived from Bacillus subtilis such as pUB110, pTP5, and pC194; shuttle vectors that can be used in common with both Escherichia coli and Bacillus subtilis such as pHY300PLK; yeast-derived vectors such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and baculovirus; and vectors modified from these.
These expression vectors have a replication origin, a selection marker, and a promoter appropriate for each vector, and may also have, as necessary, an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, etc. Furthermore, in order to facilitate purification of the expressed polypeptide, the expression vector may have inserted therein a nucleotide sequence for expressing a fused tag such as a FLAG tag, a His tag, an HA tag, or a GST tag.

When expressing VHH antibodies from cultured bacteria or cells, the bacteria or cultured cells are collected after cultivation using a known method, suspended in an appropriate buffer, and disrupted by ultrasound, lysozyme, and/or freeze-thawing, followed by centrifugation or filtration to obtain a soluble extract.

In the present invention, it is preferable from the viewpoint of industrial productivity to use a solution derived from the culture medium, such as the above-mentioned soluble extract, as the solution containing the VHH antibody.
The content of VHH antibody in a solution containing VHH antibody is preferably 0.01 g/L or more, more preferably 0.1 g/L or more, even more preferably 0.3 g/L or more, and even more preferably 0.5 g/L or more, from the standpoints of adsorption and process time; and is preferably 50 g/L or less, more preferably 20 g/L or less, even more preferably 10 g/L or less, and even more preferably 5 g/L or less, from the standpoints of adsorption and stable solubility of VHH antibody molecules.

(Affinity carrier with protein A as a ligand)
The protein A used in this step is a membrane protein present in the cell wall of Staphylococcus aureus, and is known to specifically bind to the constant region of immunoglobulin G (antibody) and the like. Protein A may be either native or modified. For example, modified protein A with improved alkali resistance is known.
The protein A is not particularly limited, and protein A extracted and purified from Staphylococcus aureus, protein A produced by genetic recombination technology, etc. can be used.

Affinity carriers are insoluble carriers capable of specifically and reversibly adsorbing biomolecules used in affinity chromatography. Examples of carrier substrates include synthetic polymers, natural polysaccharide polymers, silica gel, and porous glass. Examples of synthetic polymers include styrene-based resins such as polystyrene and cross-linked polystyrene, acrylic resins, and methacrylic resins. Examples of natural polymers include polysaccharides such as agarose, dextran, cellulose, and pullulan.
The affinity carrier may be in the form of particles, fibers, membranes, monoliths, etc. Particulates are preferred. The carrier may be porous or non-porous.
The average particle size of the affinity carrier is preferably 1 to 1000 μm, more preferably 10 to 500 μm, from the viewpoints of adsorption/desorption properties and pressure loss. The average particle size can be measured by dispersing the carrier in distilled water and using a particle size distribution analyzer (for example, LA-920, Horiba, Ltd.).
The pore size is preferably 10 to 1000 nm, more preferably 20 to 200 nm, from the viewpoint of adsorption capacity and adsorption/desorption properties.

Protein A can be immobilized on an affinity support using standard methods for immobilizing proteins on supports. A spacer such as a polymethylene chain, polyethylene glycol chain, or sugar may also be introduced between the affinity support and the ligand, Protein A.

Affinity supports using Protein A as a ligand can be commercially available. Examples of commercially available products include Ampshere A3 (JSR Life Sciences), MabSelect Sure (GE Healthcare), Praesto AP (Purolite), and KanCapA 3G (Kaneka).

(equilibrium)
Before passing a solution containing a VHH antibody through the column, it is preferable to pass a solution of a neutral buffer solution such as phosphate-buffered saline (PBS) to which an inorganic salt such as sodium chloride or sodium sulfate has been added as needed through the column to pre-equilibrate the column.
The concentration of the buffer solution is preferably 0.5 mM or more, more preferably 1 mM or more, even more preferably 2 mM or more, and even more preferably 3 mM or more, from the viewpoints of adsorption and activity maintenance, and from the same viewpoints, is preferably 100 mM or less, more preferably 50 mM or less, even more preferably 25 mM or less, and even more preferably 15 mM or less.
The salt concentration is preferably 5 mM or more, more preferably 50 mM or more, and even more preferably 100 mM or more, from the viewpoints of adsorption and activity maintenance, and from the same viewpoints, is preferably 1500 mM or less, more preferably 1000 mM or less, and even more preferably 200 mM or less.

The pH (20°C, same below) of the solution used for equilibration is preferably 5.5 or higher, more preferably 6.0 or higher, even more preferably 6.5 or higher, and even more preferably 7.0 or higher, from the standpoints of adsorption and activity maintenance. Also, from the same standpoints, it is preferably 9.0 or lower, more preferably 8.5 or lower, even more preferably 8.0 or lower, and even more preferably 7.5 or lower.

From the viewpoints of adsorption and activity maintenance, the column temperature is preferably 0°C or higher, more preferably 2°C or higher, and from the same viewpoints, it is preferably 35°C or lower, more preferably 30°C or lower.

The liquid passage rate (space velocity, SV) is preferably 0.1/hr or more, more preferably 1/hr or more, even more preferably 2/hr or more, and even more preferably 3/hr or more, from the viewpoints of adsorption ability, activity maintenance, and process time, and is preferably 25/hr or less, more preferably 20/hr or less, even more preferably 15/hr or less, and even more preferably 10/hr or less, from the viewpoints of adsorption ability and activity maintenance.
From the viewpoints of adsorption, activity maintenance, and process time, the flow volume (BV) is preferably at least 1, more preferably at least 1.5, even more preferably at least 2, and even more preferably at least 2.5. There are no particular upper limits, but from the viewpoint of the adsorption yield of VHH antibody, it is preferably at most 100, more preferably at most 30, and even more preferably at most 10.

(adsorption)
In this step, a solution containing a VHH antibody is passed through the affinity carrier using protein A as a ligand so that the amount of VHH antibody adsorbed (g) is 4 to 20 g/L relative to the packed bed volume (L) of the affinity carrier. Such chromatographic purification is expected to significantly improve the productivity of VHH antibodies.
The amount of VHH antibody adsorption (g) relative to the packed bed volume (L) of an affinity carrier using Protein A as a ligand is preferably 4 g/L or more, more preferably 5 g/L or more, and even more preferably 7 g/L or more, from the viewpoint of VHH antibody productivity, and is preferably 20 g/L or less, more preferably 15 g/L or less, and even more preferably 10 g/L or less, from the viewpoint of VHH antibody adsorption yield.

From the viewpoints of adsorption and activity maintenance, the column temperature is preferably 0°C or higher, more preferably 2°C or higher, and from the same viewpoints, it is preferably 35°C or lower, more preferably 30°C or lower.

The liquid passage rate (space velocity, SV) is preferably 0.1/hr or more, more preferably 1/hr or more, even more preferably 2/hr or more, and even more preferably 3/hr or more, from the viewpoint of adsorption ability and process time, and is preferably 25/hr or less, more preferably 20/hr or less, even more preferably 15/hr or less, and even more preferably 10/hr or less, from the viewpoint of adsorption ability.
The flow rate (BV) is preferably 0.1 or more, more preferably 1 or more, from the viewpoints of adsorption ability, activity maintenance, and process time, and from the same viewpoints, is preferably 200 or less, more preferably 100 or less, and even more preferably 50 or less.

(Washing)
After step (1), and before step (2), it is preferable to carry out a step of passing a washing solution through the packed layer of the affinity carrier to wash it. The number of washings may be one or more.
Examples of the washing solution include a neutral buffer solution such as phosphate buffered saline (PBS) to which inorganic salts such as sodium chloride and sodium sulfate are added as needed.
The concentration of the buffer solution is preferably 0.5 mM or more, more preferably 1 mM or more, even more preferably 2 mM or more, and even more preferably 3 mM or more, from the viewpoints of yield, activity retention, and purity, and from the same viewpoints, is preferably 100 mM or less, more preferably 50 mM or less, even more preferably 25 mM or less, and even more preferably 15 mM or less.
The salt concentration is preferably 5 mM or more, more preferably 50 mM or more, and even more preferably 100 mM or more, from the viewpoints of yield, activity maintenance, and purity, and from the same viewpoints, is preferably 1500 mM or less, more preferably 1000 mM or less, and even more preferably 200 mM or less.

The pH (20°C) of the washing solution is preferably 4.5 or higher, more preferably 5.5 or higher, even more preferably 6.5 or higher, and even more preferably 7.0 or higher, from the standpoints of yield and activity maintenance. Also, from the same standpoints, it is preferably 9.0 or lower, more preferably 8.5 or lower, even more preferably 8.0 or lower, and even more preferably 7.5 or lower.

From the standpoints of yield and activity maintenance, the column temperature is preferably 0°C or higher, more preferably 2°C or higher, and from the same standpoints, preferably 35°C or lower, more preferably 30°C or lower.

The liquid passage rate (space velocity, SV) is preferably 0.1/hr or more, more preferably 1/hr or more, even more preferably 2/hr or more, and even more preferably 3/hr or more, from the viewpoints of yield, purity, and process time; and is preferably 25/hr or less, more preferably 20/hr or less, even more preferably 15/hr or less, and even more preferably 10/hr or less, from the viewpoints of yield and purity.
In addition, from the viewpoints of yield, purity, and process time, the flow volume (BV) is preferably at least 1, more preferably at least 1.5, even more preferably at least 2, and still more preferably at least 3. There are no particular limitations on the upper limit, but from the viewpoints of process time and economy, it is preferably at most 100, more preferably at most 30, and still more preferably at most 10.

[Step (2)]
This step is a step of passing an eluate, which has a partition coefficient between the affinity carrier and the VHH antibody in the range of 0.07 to 20, after step (1), and by eluting the VHH antibody in this step and recovering the eluate, the target VHH antibody can be obtained without reducing its activity. The elution step can be carried out once or multiple times.

The partition coefficient between the affinity carrier and the VHH antibody is, from the standpoints of yield and activity retention, preferably 0.07 or more, more preferably 0.1 or more, even more preferably 0.3 or more, even more preferably 0.5 or more, even more preferably 0.7 or more, and even more preferably 1 or more; from the same standpoints, it is preferably 20 or less, more preferably 10 or less, even more preferably 7 or less, even more preferably 5 or less, even more preferably 3 or less, even more preferably 2 or less, and even more preferably 1.4 or less.
The method for calculating the partition coefficient between the affinity carrier and the VHH antibody is described in detail in the Examples.

The elution solution is one that can release and elute the VHH antibody adsorbed to the affinity carrier. Examples of elution solutions include acetate buffer, citrate buffer, tartrate buffer, glycine-hydrochloric acid buffer, and phosphate buffer. A buffer containing glycine is preferred.
The concentration of the buffer solution is preferably 5 mM or more, more preferably 50 mM or more, and even more preferably 80 mM or more, from the viewpoints of yield and activity maintenance, and is preferably 200 mM or less, more preferably 150 mM or less, and even more preferably 120 mM or less, from the same viewpoints.
From the viewpoints of yield and activity maintenance, the salt concentration is preferably 5 mM or more, more preferably 10 mM or more, even more preferably 50 mM or more, and even more preferably 80 mM or more; from the same viewpoints, it is preferably 1500 mM or less, more preferably 1000 mM or less, even more preferably 200 mM or less, and even more preferably 120 mM or less.

From the standpoints of yield and activity retention, the pH of the eluate is preferably 2.8 or higher, more preferably 3.0 or higher, even more preferably 3.2 or higher, and even more preferably 3.3 or higher. From the same standpoints, it is preferably 4.0 or lower, more preferably 3.8 or lower, even more preferably 3.7 or lower, even more preferably 3.6 or lower, and even more preferably 3.5 or lower.

From the standpoints of yield and activity maintenance, the column temperature is preferably 0°C or higher, more preferably 2°C or higher, and from the same standpoints, preferably 35°C or lower, more preferably 30°C or lower.

The liquid passage rate (space velocity, SV) is preferably 0.1/hr or more, more preferably 1/hr or more, even more preferably 2/hr or more, and even more preferably 3/hr or more, from the viewpoints of yield, purity, and process time; and is preferably 25/hr or less, more preferably 20/hr or less, even more preferably 15/hr or less, and even more preferably 10/hr or less, from the viewpoints of yield and purity.
Furthermore, taking into consideration the yield, process time, activity maintenance, and liquid volume, the flow volume (BV) is preferably 1 or more, more preferably 1.5 or more, even more preferably 2 or more, and even more preferably 2.5 or more; and from the viewpoints of process time and the burden on subsequent purification steps due to a decrease in VHH antibody concentration, the BV is preferably 100 or less, more preferably 30 or less, and even more preferably 10 or less.

In the present invention, the yield of VHH antibodies in step (2) can be as high as 80% by mass or more.

(neutralization)
After step (2), it is preferable to carry out a step of neutralizing the eluted VHH antibody by mixing it with a neutral or alkaline solution.
Examples of neutral or alkaline solutions include buffer solutions such as Tris-HCl buffer, etc. The pH of the solution is preferably 6 or higher, more preferably 7 or higher, even more preferably 8 or higher, and even more preferably 8.5 or higher, from the viewpoint of activity maintenance, and from the same viewpoint, is preferably 14 or lower, more preferably 11 or lower, even more preferably 10 or lower, and even more preferably 9.5 or lower.

The pH of the eluate containing the neutralized VHH antibody is preferably 6 or higher, more preferably 7 or higher, from the viewpoint of maintaining activity, and from the same viewpoint, is preferably 10 or lower, more preferably 9 or lower.

(reproduction)
A regeneration step of the affinity carrier may also be carried out. Affinity purification columns are usually washed and equilibrated with an alkaline solution before repeated use.
Examples of the alkaline solution include solutions of sodium hydroxide, potassium hydroxide, triethylamine, tetrabutylammonium hydroxide, and the like.
The concentration of the alkaline solution is preferably 0.05 M or more, more preferably 0.1 M or more, from the viewpoints of yield and purity, and is preferably 1 M or less, more preferably 0.5 M or less, from the same viewpoints.
The liquid passage rate (space velocity, SV) is preferably 0.1/hr or more, more preferably 1/hr or more, and even more preferably 1.5/hr or more, from the viewpoints of yield, purity, and process time, and is preferably 25/hr or less, more preferably 20/hr or less, even more preferably 15/hr or less, and even more preferably 10/hr or less, from the viewpoints of yield and purity.
In addition, from the viewpoints of yield, purity, and process time, the flow volume (BV) is preferably at least 1, more preferably at least 1.5, and even more preferably at least 2. The upper limit is not particularly limited, but from the same viewpoints, it is preferably at most 100, more preferably at most 30, and even more preferably at most 10.

As a result of such treatment, the VHH antibody is purified to obtain a purified VHH antibody.
The VHH antibodies obtained according to the present invention can be used as they are, but can also be further purified by ion exchange chromatography, hydrophobic interaction chromatography, or the like, if necessary, before use.

[Materials for producing VHH-expressing Bacillus subtilis mutant strains]
(1) Strains Defective strains of extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX) were prepared from Bacillus subtilis strain 168 (hereinafter referred to as strain 168) according to the method described in Japanese Patent Publication No. 2006-174707. Hereinafter, the resulting strains deficient in a single extracellular protease are referred to as Δepr, ΔwprA, Δmpr, ΔnprB, Δbpr, ΔnprE, Δvpr, ΔaprE, and ΔaprX, respectively. Furthermore, according to the method described in Japanese Patent No. 4336082, the sigF gene involved in sporulation was deleted from Bacillus subtilis strain Dpr9, which is deficient in all nine of the above extracellular protease genes. The resulting strain multiple-deficient in extracellular proteases is referred to as Dpr9ΔsigF.

(2) Culture Medium: LB medium: 1% Bacto (registered trademark) Tryptone (Difco), 0.5% Bacto Yeast Extract (Difco), 1% sodium chloride. 1.5% agar was added to the plate medium. Tetracycline hydrochloride (50 ppm) was added as needed.
SMMP solution: Antibiotic Medium 3 (Difco) (35 g/L), sucrose (171.5 g/L), disodium maleate (3.2 g/L), magnesium chloride hexahydrate (4.06 g/L)
PEG solution: sucrose (85.75 g/L), disodium maleate (1.6 g/L), magnesium chloride hexahydrate (2.03 g/L), PEG 8000 (400 g/L)
DM3 medium: 1% CMC (Kanto Chemical), 0.5% Bacto Casamino Acids (Difco), 0.5% Bacto Yeast Extract (Difco), 8.1% disodium succinate hexahydrate, 0.35% dipotassium hydrogen phosphate, 0.15% potassium dihydrogen phosphate, 0.5% glucose, 20 mM magnesium chloride, 0.01% BSA, 50 ppm tetracycline hydrochloride. 1% agar was added to the plate medium.

(3) Reagents Reagents manufactured by Wako were used unless otherwise specified.

[Monitoring of the column outlet solution]
A detector of a chromatography system (BioLogic LP (Bio-Rad Laboratories) or BioLogic DuoFlow (Bio-Rad Laboratories)) was attached to the outlet of the column, and the time trends of ultraviolet absorbance at 280 nm and electrical conductivity were measured.

[pH measurement method]
The pH was measured at room temperature (20° C.) using a pH meter TPX-999i (Toko Chemical Research Institute Co., Ltd.).

[Method for measuring VHH protein concentration]
The samples were applied to Any kD precast polyacrylamide gels (Bio-Rad Laboratories) and analyzed by electrophoresis (SDS-PAGE). The band patterns of the gels stained with Bio-Safe Coomassie G-250 Stain (Bio-Rad Laboratories) were analyzed for brightness using ImageJ, an imaging software developed by the National Institutes of Health (NIH). Bovine serum albumin (BSA) (Fujifilm Wako Pure Chemical Industries, Ltd.) was applied to the same gel as a standard protein to create a calibration curve, and the VHH protein concentrations were quantified by analyzing the band patterns of the samples.

[Method for measuring VHH protein adsorption rate]
The VHH adsorption rate after chromatographic purification was calculated by the following formula.
VHH adsorption rate (mass%)=[(amount (g) of VHH in load solution (crude VHH solution))−(amount (g) of VHH in column effluent in adsorption step)]/[amount (g) of VHH in load solution (crude VHH solution)]×100

[Method for measuring VHH protein recovery rate]
The recovery rate of VHH by chromatographic purification was calculated by the following formula.
VHH recovery rate (mass%)=[amount (g) of VHH in eluate (purified VHH solution)]/[amount (g) of VHH in load solution (crude VHH solution)]×100

[Method for measuring binding activity of VHH to S1 protein by sandwich ELISA]
100 μL of 20 μg/mL His-tagged VHH was added to each well of Pierce® Nickel Coated Plates, Clear, 96-Well (Thermo Fisher Scientific) and allowed to stand overnight at 4°C. After carefully removing any VHH antibodies that had not adsorbed to the wells using a pipette, 200 μL of 5% skim milk/PBST (PBS containing 0.05% Tween 20) was added and incubated at room temperature for 1 hour. After carefully removing the blocking agent using a pipette, 200 μL of PBST was added and carefully removed using a pipette. This washing procedure was performed three times. To each well containing immobilized VHH antibodies, 100 μL of 0.1 μg/mL SARS-CoV-2 (2019-nCoV) Spike S1-Fc Recombinant Protein (Fc-tagged S1 protein, Sino Biological)/PBST was added and incubated at room temperature for 50 minutes. After carefully removing the Fc-tagged S1 protein using a pipette, 200 μL of PBST was added and carefully removed using a pipette. This washing procedure was performed three times. Goat Anti-Human IgG Fc (HRP) (Abcam) was used as the detection antibody. The primary antibody was diluted 1/5,000 in PBST. 100 μL of detection antibody was added to each well and incubated at room temperature for 50 minutes. After carefully removing the detection antibody using a pipette, 200 μL of PBST was added and carefully removed using a pipette. This washing procedure was repeated three times. The chromogenic substrate was prepared by dissolving an OPD tablet (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of chromogenic substrate was added to each well, incubated for 20 minutes in the dark, and then the absorbance at 450 nm (OD450) was immediately measured using a microplate reader, Multiskan Sky (Thermo Fisher Scientific).

[Method for measuring the rate of retention of binding activity of VHH to S1 protein]
The retention rate of binding activity of VHH to S1 protein after chromatographic purification was calculated using the following formula.
Percentage of retention of binding activity to S1 protein (%)=[Binding activity (OD450) of VHH in eluate (purified VHH solution) to S1 protein]/[Binding activity (OD450) of VHH in eluate (purified VHH solution) obtained by batch chromatography purification to S1 protein]×100

Example 1
(1) Construction of a Bacillus subtilis mutant strain expressing VHH (CoVHH1) (CoVHH1)
1. Artificial synthesis of gene The VHH gene (SEQ ID NO: 71 (translated sequence 73)) was artificially synthesized by Thermo Fisher Scientific. To facilitate insertion of the gene into a plasmid vector, GCAGCTCTTGCAGCA (SEQ ID NO: 75) was added to the 5' end of each artificially synthesized gene, and TCTATTAAAACTAGTT (SEQ ID NO: 76) was added to the 3' end of each artificially synthesized gene.

2. Construction of VHH Expression Plasmids Using recombinant plasmid pHY-S237 (JP 2014-158430 A) as a template, the plasmid sequence was amplified by PCR using a primer set of 5'-TGCTGCAAGAGCTGCCGGAAATAAAA-3' (SEQ ID NO: 77) and 5'-TCTATTAAAACTAGTTATAGGG-3' (SEQ ID NO: 78) and PrimeSTAR Max DNA polymerase (TaKaRa). DNA containing the artificial synthetic gene described in 1 above was inserted into the resulting PCR fragment using an In-Fusion HD Cloning Kit (Takara), to construct VHH expression plasmids containing each of the artificial synthetic VHH genes shown in 1 above.
The promoter region was changed to that derived from the Bacillus subtilis spoVG gene by the following procedure.
The plasmid sequence was amplified by PCR using the VHH expression plasmid as a template and the primer set of 5'-GATCCCCGGGGAATTCCTGTTATAAAAAAAAAAGG-3' (SEQ ID NO: 79) and 5'-ATGATGTTAAGAAAGAAACAAAGCAG-3' (SEQ ID NO: 80) with PrimeSTAR Max DNA polymerase (TaKaRa). The promoter DNA derived from the spoVG gene was amplified by PCR using the genome of strain 168 as a template and the primer set of 5'-GAATTCCCGGGGATCTAAGAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 81) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3' (SEQ ID NO: 82). The resulting promoter DNA was integrated into a plasmid sequence using an In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid containing a VHH gene linked to the spoVG promoter. The constructed plasmid was introduced into Dpr9ΔsigF according to the procedure described in 3 below.

3. Construction of Recombinant Bacillus subtilis Plasmids were introduced into Bacillus subtilis strains by the protoplast method described below.
Various Bacillus subtilis strains stocked in glycerol were inoculated into 1 mL of LB liquid medium and cultured overnight at 30°C with shaking at 210 rpm. The next day, 10 μL of this culture was inoculated into a new 1 mL of LB liquid medium and cultured for approximately 2 hours with shaking at 37°C and 210 rpm. This culture was collected in a 1.5 mL tube and centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed and the pellet was suspended in 500 μL of SMMP containing 4 mg/mL Lysozyme (SIGMA) and incubated at 37°C for 1 hour. The mixture was then centrifuged at 3,500 rpm for 10 minutes. The supernatant was removed and the pellet was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with various plasmids, and 100 μL of 40% PEG was added and vortexed. 350 μL of SMMP was added to this solution, mixed by inversion, and shaken at 30°C and 210 rpm for 1 hour. The entire amount was then spread onto a DM3 agar medium plate and incubated at 30°C for 2 to 3 days to obtain colonies of transformants.

(2) Cultivation The transformant (recombinant Bacillus subtilis) obtained above was inoculated into 360 mL of LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) and cultured in a 2 L pleated Erlenmeyer flask for 20 hours at 30°C with a shaking speed of 180 r/min to prepare an inoculum (seed culture).

Next, the seed culture was inoculated into 18 L of 2xL/mal medium containing tetracycline (20 g/L tryptone, 10 g/L yeast extract, 10 g/L sodium chloride, 75 g/L maltose monohydrate, 7.5 ppm manganese sulfate pentahydrate, 15 ppm tetracycline hydrochloride), and cultured with aeration and agitation for 72 hours at 30°C, an aeration flow rate of 0.2 vvm, 300 rpm, and a pressure of 0.04 MPa (main culture).

(3) Microfiltration The culture medium was sterilized using a microfiltration membrane module (hollow fiber microfiltration membrane, Asahi Kasei Corporation, PSP-113L) to obtain a crude VHH solution. The VHH concentration in the crude VHH solution was 0.66 g/L.

(4) Chromatographic Purification All subsequent solutions were passed through the column from the top at room temperature (20°C). A column (inner diameter 11 mm, height 250 mm, volume 22.5 mL) was packed with Amsphere® A3 (JSR Life Sciences Corporation), a protein A affinity chromatography carrier, and equilibrated with phosphate-buffered saline (4 mM phosphoric acid, 154 mM sodium chloride, pH 7.2, PBS(-)) at SV = 6.7/hr and BV = 3.3. Next, the crude VHH solution was passed through at SV = 6.7/hr and BV = 11.1 (loading amount 7.3 g VHH/L of packed bed), and the VHH in the crude VHH solution was adsorbed onto the chromatography carrier (adsorption step). Next, washing was performed by passing PBS(-) at SV = 6.7/hr and BV = 5.1 (washing step). Next, VHHs were eluted using glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 3.0) as an elution buffer at SV = 6.7/hr and BV = 2.8 to obtain a purified VHH solution (elution step). The purified VHH solution was mixed with 1/20th the volume of 1 M Tris-hydrochloric acid buffer (pH 9.0) and neutralized to pH 8. The VHH adsorption yield was 100%, the VHH adsorption amount was 7.3 g-VHH/L-packed layer, the VHH recovery rate was 96%, and the retention of VHH binding activity to S1 protein during the chromatographic purification step was 85%.
After elution, the carrier column was regenerated by passing 0.1 M sodium hydroxide solution through it at SV = 3.7/hr and BV = 2.2, and then washed by passing PBS(-) through it at SV = 6.7/hr and BV = 3.3. The acid was then replaced with a storage buffer (16% ethanol, 50 mM phosphate buffer, pH 7.5) and stored at 5°C until the next use.

(5) Measurement of the partition coefficient between affinity support and CoVHH1 The elution buffer (0.1 M glycine, 0.1 M sodium chloride, pH 3.0) used in (4) was passed through a column packed with the Protein A affinity chromatography support Amsphere A3 used in (4) at SV = 6.7/hr until the column was replaced. The neutralized purified VHH solution obtained in (4) was passed through at SV = 6.7/hr and BV = 0.019, and then the elution buffer was passed through again at SV = 6.7/hr, and the residence time (t _R ) of the VHH was measured from the UV absorbance at the column outlet. The partition coefficient K for VHH was calculated using the following formula, and the partition coefficient K' of the elution buffer was measured in the same manner. The partition coefficient K _d between the affinity carrier and CoVHH1 under pH 3.0 conditions, calculated from the difference, was 3.2×10 ⁻¹ .

Reference Example 1 Batch Chromatography Purification (CoVHH1)
50 μL of Amsphere A3 protein A affinity chromatography carrier was placed in a 1.5 mL microtube. 1 mL of PBS(-) was added, and the carrier was centrifuged (3,000 × g, 2 minutes) followed by removal of the supernatant. This procedure was repeated twice to equilibrate the carrier. Next, 0.56 mL of the crude VHH solution obtained in Example 1(1) to (3) was added and mixed for 5 minutes, allowing the VHHs in the crude VHH solution to be adsorbed onto the chromatography carrier (loading amount: 7.4 g VHH/L resin layer). After centrifugation (3,000 × g, 2 minutes), the supernatant was removed, and the carrier was washed with PBS(-) using the same procedure as for equilibration described above. Next, 0.14 mL of glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 2.5) was added as an elution buffer, and the mixture was mixed for 5 minutes to elute the VHHs. After centrifugation (3,000 × g, 2 minutes), the supernatant was collected to obtain a purified VHH solution. This elution procedure was performed twice. The purified VHH solution was mixed with 1/20th the volume of 1 M Tris-hydrochloric acid buffer (pH 9.0) and neutralized to pH 8. The VHH adsorption yield was 100%, the VHH adsorption amount was 7.4 g-VHH/L-packed layer, and the VHH recovery rate was 99%.

Reference example 2
The same procedure as in Example 1(5) was carried out, except that the elution buffer was changed to glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 2.5). The partition coefficient _Kd between the Protein A carrier and CoVHH1 at pH 2.5 was 1.1 × ^10-2 . Next, the crude VHH solution was passed through at a BV of 5.6 (loading amount of 3.7 g VHH/L packed bed), and the same procedure as in Example 1(1) to (4) was carried out, except that the elution buffer was changed to glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 2.5).
The VHH adsorption yield was 100%, the VHH adsorption amount was 3.7 g-VHH/L-packed layer, the VHH recovery rate was 97%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 81%.

Comparative Example 1
The same procedures as in Example 1 (1) to (4) were carried out except that the elution buffer was changed to a glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 2.5).
The VHH adsorption yield was 100%, the VHH adsorption amount was 7.3 g-VHH/L-packed layer, the VHH recovery rate was 97%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 48%.

Example 2
The same procedure as in Example 1(5) was carried out, except that the elution buffer was changed to a glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 3.5). The partition coefficient _Kd between the affinity carrier and CoVHH1 under pH 3.5 conditions was 1.4. Next, the same procedure as in Example 1(1) to (4) was carried out, except that a glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 3.5) was used as the elution buffer and the solution was passed through at a BV of 3.3.
The VHH adsorption yield was 100%, the VHH adsorption amount was 7.3 g-VHH/L-packed layer, the VHH recovery rate was 94%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 99%.

Comparative Example 2
The same procedures as in Example 1 (1) to (4) were carried out, except that the crude VHH solution was passed through at a BV of 8.3 (loading amount: 5.5 g - VHH/L - packed bed) and the elution buffer was changed to a glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 2.5).
The VHH adsorption yield was 100%, the VHH adsorption amount was 5.5 g-VHH/L-packed layer, the VHH recovery rate was 92%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 60%.

Example 3
The same procedure as in Comparative Example 2 was carried out except that the elution buffer was changed to a glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 3.0).
The VHH adsorption yield was 100%, the VHH adsorption amount was 5.5 g-VHH/L-packed layer, the VHH recovery rate was 99%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 74%.

Example 4
The same procedure as in Comparative Example 2 was carried out, except that a glycine-hydrochloric acid buffer solution (0.1 M glycine, 0.1 M sodium chloride, pH 3.5) was used as the elution buffer and the solution was passed through at a BV of 3.3.
The VHH adsorption yield was 100%, the VHH adsorption amount was 5.5 g-VHH/L-packed layer, the VHH recovery rate was 98%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 74%.

Example 5
The same operations as in Example 1 (1) to (4) were carried out, except that the crude VHH solution was passed through at a BV of 22.2 (loading amount 14.7 g - VHH/L - packed bed).
The VHH adsorption yield was 93%, the VHH adsorption amount was 13.6 g-VHH/L-packed layer, the VHH recovery rate was 92%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 99%.

Example 6
The same procedure as in Example 5 was carried out, except that a glycine-hydrochloric acid buffer solution (0.1 M glycine, 0.1 M sodium chloride, pH 3.5) was used as the elution buffer and the solution was passed through at a BV of 3.3.
The VHH adsorption yield was 93%, the VHH adsorption amount was 13.7 g-VHH/L-packed layer, the VHH recovery rate was 93%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 82%.

Example 7
(1) Construction of a Bacillus subtilis mutant strain expressing VHH (VHH E2) (VHH E2)
Colonies of transformants of the Bacillus subtilis mutant strain expressing VHH E2 were obtained in the same manner as in Example 1(1), except that the artificially synthesized VHH gene of SEQ ID NO: 72 (translated sequence 74) was incorporated into the PCR fragment.

(2) Culturing The same procedure as in Example 1(2) was carried out except that the transformant obtained above was used.

(3) Microfiltration The culture medium obtained above was subjected to the same procedure as in Example 1(3). The VHH concentration in the obtained crude VHH solution was 0.33 g/L.

(4) Chromatographic Purification The same procedure as in Example 1(4) was carried out, except that the crude VHH solution obtained above was passed through the column at SV=6.7/hr and BV=14.4 (loading amount 4.8 g - VHH/L - packed bed).
The VHH adsorption yield was 100%, the VHH adsorption amount was 4.8 g-VHH/L-packed layer, the VHH recovery rate was 90%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 90%.

(5) Measurement of the partition coefficient between affinity carrier and VHH E2 The purified VHH solution obtained above was used in the same manner as in Example 1(5). The partition coefficient _Kd between the affinity carrier and VHH E2 at pH 3.0 was 2.3 × ^10-1 .

Reference Example 3 Batch chromatographic purification (VHH E2)
The same procedure as in Reference Example 1 was carried out, except that the crude VHH solution obtained in Example 7 (1) to (3) was used and the VHH in the solution was adsorbed onto the affinity carrier (loading amount: 3.7 g VHH/L resin layer). The VHH adsorption yield was 100%, the VHH adsorption amount was 3.7 g VHH/L packed layer, and the VHH recovery rate was 91%.

Example 8
The same procedure as in Example 7(5) was carried out, except that the elution buffer was changed to a glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 3.5). The partition coefficient _Kd between the affinity carrier and VHH E2 under pH 3.5 conditions was 1.4. Next, the same procedure as in Examples 7(1) to (4) was carried out, except that a glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 3.5) was used as the elution buffer and the solution was passed through at a BV of 3.3.
The VHH adsorption yield was 100%, the VHH adsorption amount was 4.8 g-VHH/L-packed layer, the VHH recovery rate was 83%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 93%.

Comparative Example 3
The same procedures as in Example 7(5) were carried out, except that the elution buffer was changed to glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 2.5). The partition coefficient _Kd between the affinity carrier and VHH E2 under pH 2.5 conditions was 5.7 × ^10-3 . Next, the same procedures as in Examples 7(1) to (4) were carried out, except that the elution buffer was changed to glycine-hydrochloric acid buffer (0.1 M glycine, 0.1 M sodium chloride, pH 2.5).
The VHH adsorption yield was 100%, the VHH adsorption amount was 4.8 g-VHH/L-packed layer, the VHH recovery rate was 88%, and the retention rate of VHH binding activity to S1 protein during the chromatographic purification step was 38%.

Claims (6)

The following steps (1) and (2):
(1) A method for producing a VHH antibody, comprising: (1) passing a solution containing a VHH antibody through an affinity carrier having Protein A as a ligand such that the amount of VHH antibody adsorbed (g) is 4 to 20 g/L relative to the volume (L) of the packed layer of the affinity carrier; and (2) after step (1), passing an eluate which has a partition coefficient between the affinity carrier and the VHH antibody in the range of 0.07 to 20, and which is a buffer solution containing 5 to 200 mM glycine and has a pH in the range of 2.8 to 4.0. 2. The method for producing a VHH antibody according to claim 1, wherein in step (2), the partition coefficient between the affinity carrier and the VHH antibody in the eluate passed through the eluate is in the range of 0.1 to 2. The method for producing a VHH antibody according to claim 1 or 2 , further comprising, after step (1) and before step (2), a step of passing a washing solution through the packed layer of the affinity carrier to wash it. The method for producing a VHH antibody according to any one of claims 1 to 3 , further comprising, after step (2), a step of neutralizing the eluted VHH antibody by mixing it with a neutral or alkaline solution. The method for producing a VHH antibody according to any one of claims 1 to 4 , wherein the VHH antibody is an antibody that binds to SARS-CoV-2. The method for producing a VHH antibody according to any one of claims 1 to 5 , wherein the solution containing the VHH antibody is a solution derived from a culture medium obtained by culturing Bacillus subtilis, Bacillus megaterium, or Bacillus brevis.