Recombinant human serum albumin production method

Abstract

The present invention relates to a recombinant human serum albumin production method, and specifically to a method enabling simple expression and purification for producing a recombinant human serum albumin in an oxidative cytoplasm bacterial expression system. In addition, the present invention relates to a drug carrier comprising a recombinant human serum albumin produced by the method, the drug carrier being long-lasting in the human body through the binding ability of FcRn and an albumin binding domain (ABD).

Description

Method for producing recombinant human serum albumin

[0001] The present invention relates to a method for producing recombinant human serum albumin, and more particularly, to a method for producing recombinant human serum albumin in an oxidative cytoplasmic bacterial expression system, which enables easy expression and purification. In addition, the present invention relates to a drug delivery system that lasts long in the body through the binding ability of FcRn and an albumin binding domain (ABD), comprising recombinant human serum albumin produced by the method.

[0002] Human serum albumin (HSA), the most abundant protein in plasma, performs functions such as pH buffering and maintaining osmotic pressure. It also stores and transports metabolic substances, such as hormones and fatty acids, and possesses antioxidant properties. HSA is a major component of plasma proteins and is used medicinally to treat conditions such as massive bleeding, shock, burns, mild hypoproteinemia, and erythroblastosis.

[0003] In addition to its importance, HSA has the unique property of avoiding lysosomal degradation through FcRn (neonatal Fc receptor)-mediated recycling, significantly extending its in vivo half-life to approximately 19 days. This property has a significant impact on drug delivery, as HSA can bind to short-lived drugs and serve as a durable drug delivery scaffold. This binding enhances the half-life of the drug in the systemic circulation, thereby extending its duration of action. A prime example of the therapeutic application of plasma-derived HSA is the case of the FDA-approved drug Abraxane. Nanoparticle-based formulations have demonstrated enhanced efficacy and solubility while reducing toxicity in cancer treatment by complexly combining paclitaxel and albumin.

[0004] However, the primary source of albumin is human plasma fractionation, raising concerns about availability and safety. The primary source of HSA is plasma fractionation, which primarily produces the product from hematopoietic fractions. However, this method is not only uneconomical, but the supply of blood for HSA production is not always secure. Furthermore, blood extraction methods can pose various problems, as blood contains unwanted substances, such as hepatitis viruses and blood-borne infectious diseases such as HIV. These concerns necessitate stringent quality control when extracting HSA from human blood. Therefore, alternative methods for large-scale production of HSA that can replace plasma fractionation are needed.

[0005] As an alternative, methods for expressing recombinant human serum albumin (rHSA) in various cell systems, including yeast, transgenic animals, and plants, are attracting attention. Among these hosts, the Escherichia coli (E. coli) expression system offers several advantages for protein production. In particular, compared to other hosts, it grows rapidly and is cost-effective, allowing for the rapid production of recombinant proteins in large quantities within a short period of time. Furthermore, E. coli is a well-established host system with a wealth of knowledge and genetic tools, facilitating the optimization of expression conditions and genetic manipulation. However, the reductive environment of E. coli makes disulfide bond formation very difficult, making it difficult to form the 17 disulfide bonds contained in HSA. This environment disrupts the structural stability of HSA and leads to the formation of insoluble aggregates.

[0006] Previous studies have attempted to address these issues using specific strategies, such as fusing solubility-enhancing tags to the N- or C-terminus of HSA or co-expressing chaperones. The goal of these approaches was to induce structural folding of rHSA in Escherichia coli and improve its solubility, thereby increasing the yield of rHSA. However, it should be noted that these strategies require additional expression and purification steps, such as removal of the solubility-enhancing tag or co-expression with chaperones, which complicate the protein production process. Furthermore, rHSA produced through these processes has been characterized using standard analytical methods, such as esterase activity assays, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), and dynamic light scattering. However, these methods revealed insufficient biological similarity between rHSA and plasma-derived HSA (pHSA), which has raised questions about the suitability of bacterial hosts and genetic engineering approaches for rHSA production.

[0007] Therefore, rigorous biological assays are essential to demonstrate that E. coli-derived rHSA folds correctly and exhibits its unique structure and function as an effective sustained-release drug delivery system. Crucially, rHSA must maintain specific binding affinity for FcRn and other albumin-binding moieties.

[0008] Against this backdrop, the inventors of the present invention have developed a method for expressing and purifying human serum albumin easily and rapidly, at low cost, utilizing an oxidative cytosolic E. coli expression system without additional tools such as co-expression of a tag protein or chaperone, and have completed the present invention. Additionally, the present invention demonstrates the previously unexplored properties of human serum albumin expressed in E. coli as a drug delivery system.

[0009] The purpose of the present invention is to provide a method for producing recombinant human serum albumin.

[0010] An object of the present invention is to provide recombinant human serum albumin produced by the above production method.

[0011] The purpose of the present invention is to provide a drug delivery system comprising human serum albumin expressed in Escherichia coli.

[0012] To achieve the above object, the present invention provides a method for producing recombinant human serum albumin, comprising a step of culturing a mutant E. coli transformed with a vector containing a nucleic acid encoding human serum albumin.

[0013] The above mutant E. coli can be characterized as E. coliSHuffle, which is characterized by suppression of expression of thioredoxin reductase (trxB) and glutaredoxin reductase (gor) coding genes of E. coli and overexpression of cytoplasmic disulfide bond isomerase (DsbC) coding genes.

[0014] It may be characterized by further including a step of purifying the culture after the above culturing.

[0015] The above purification may be characterized by subjecting the culture to nickel affinity chromatography and anion exchange chromatography.

[0016] The present invention also relates to recombinant human serum albumin produced by the production method.

[0017] The present invention further relates to a drug delivery system comprising recombinant human serum albumin.

[0018] According to the present invention, human serum albumin produced in a bacterial expression system in oxidative cytoplasm exhibits the same structure and function as human serum albumin obtained through plasma fractionation, and can be easily expressed and purified because it does not utilize a solubility-enhancing tag protein or co-express a chaperone. Furthermore, it can also be utilized as a long-lasting drug delivery system in the body through the binding ability of FcRn and the albumin binding domain (ABD).

[0019] Figure 1. SDS-PAGE analysis of rHSA expressed as SHuffle at various temperatures. Samples for analysis were collected at appropriate time points during the main culture at 37°C (A), 30°C (B), and 18°C ​​(C), processed appropriately, and analyzed. Lane M, protein molecular weight marker; Lanes B-I, whole-cell extracts collected before IPTG induction; Lane S, soluble fraction after cell lysis; Lane I, insoluble fraction after cell lysis; Lanes 1-5, whole-cell extracts collected at 2, 4, 6, 8, and 8.5 h after induction in (A) or 3, 4.5, 6, 20, and 22 h after induction in (B), or 3, 6.5, and 20, 28.5, and 42 h after induction in (C).

[0020] Figures 2a to 2e. Purification of rHSA expressed in SHuffle. Figure 2a shows SDS-PAGE analysis of eluates collected from nickel-Histag affinity chromatography (lane 1) and anion exchange chromatography (lane 2). Figure 2b shows Western blot analysis of purified rHSA. Figure 2c shows SDS-PAGE analysis of rHSA purified under non-reducing (lane 3) and reducing conditions (lane 4). Lane M is a protein molecular weight marker. Figure 2d shows MALDI-TOF mass spectrometry analysis of purified rHSA. Figure 2e shows SDS-PAGE showing that the impurity sizes of rHSA after nickel-Histag affinity chromatography are similar to those of HSA. Lane M, protein molecular weight marker; Lane S, soluble fraction after solubilization; Lane I, insoluble fraction after solubilization; Lane E, eluate after nickel-Histag affinity chromatography.

[0021] Figures 3a-3e. Characterization of the rHSA structure. Figure 3a shows the SEC elution profiles of rHSA and pHSA. Figure 3b shows the circular dichroism spectra of rHSA and pHSA. The CD signal at 0.25 mg / mL was collected from each sample by measuring the difference in absorption between left and right circularly polarized light in the range of 197–260 nm. Figure 3c shows the intrinsic fluorescence spectra of rHSA and pHSA. Intensity data were collected by illuminating each sample (0.2 mg / mL) at 280 nm and measuring the emitted light at 320, 330, 355, 390, 405, 444, and 450 nm, and plotted against the corresponding emission wavelengths. Figure 3d shows the time course of the conversion of p-NPA to p-NP catalyzed by the esterase-like activity of rHSA. 10 μM rHSA and other controls were individually mixed with the substrate p-NPA and the increase in absorbance at 400 nm was monitored over time. Figure 3e shows the esterase-like activity of rHSA.

[0022] Figures 4a and 4b. Molecular interactions between rHSA and the albumin binding domain (ABD). Figure 4a shows the SEC elution profiles of rHSA (black), ABD (blue), and rHSA mixed with ABD (red). Figure 4b shows the SEC elution profiles of pHSA (black), ABD (blue), and pHSA mixed with ABD (red).

[0023] Figures 5a to 5d show the pH-dependent interaction between rHSA and FcRn.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0025] Human serum albumin (HSA), a polypeptide characterized by 17 disulfide bonds, serves as a key transport protein in human plasma. Its prolonged circulating half-life, mediated by recycling via the neonatal Fc receptor (FcRn), makes HSA an excellent carrier for sustained drug delivery. However, conventional methods for obtaining HSA from human blood face limitations due to its limited availability and potential contamination risks, such as blood-borne diseases.

[0026] It is important to recognize that common E. coli strains are not suitable for expressing HSA. One significant drawback is that the reducing environment of E. coli prevents proper disulfide bond formation in HSA, compromising folding and stability. This often leads to the formation of insoluble aggregates known as inclusion bodies, which require additional steps to reconjugate them into functional rHSA. This process is time-consuming and cost-intensive, and the protein yield is significantly lower compared to other methods.

[0027] The present invention aimed to evaluate the essential properties of HSA expressed in Escherichia coli to verify its potential as a long-acting drug delivery scaffold. Using the SHuffle strain of E. coli, which provides an oxidative environment, we expressed and purified soluble rHSA under optimized culture conditions. To assess the structural similarity between rHSA and pHSA, the purified rHSA underwent various characterizations. Circular dichroism (CD) and size exclusion chromatography (SEC) were used to assess the secondary structure content and oligomeric state of rHSA, respectively. Standard bioanalytical techniques, including esterase activity assays, fluorescence spectroscopy, and mass spectrometry, were also utilized. Specifically, we investigated the binding capacity of rHSA to the albumin-binding domain (ABD), a widely used HSA binding motif in drug delivery applications. Finally, biolayer interferometry (BLI) was used to investigate the pH-dependent binding properties of rHSA to FcRn, a key property for HSA to function as a long-acting drug carrier through FcRn recycling.

[0028] We produced a naturally self-folding recombinant HSA (rHSA) using the SHuffle, an oxidative Escherichia coli (E. coli) expression system. This system precisely forms disulfide bonds and ensures accurate folding of cysteine-rich rHSA, eliminating the need for chaperone co-expression or fusion of folding enhancer domains. Purified rHSA underwent rigorous physicochemical characterization, including mass spectrometry, circular dichroism, intrinsic fluorescence spectroscopy, esterase-like activity assays, and size-exclusion chromatography, to assess key quality attributes. Importantly, rHSA retained its intrinsic binding affinity for the FcRn and albumin-binding domains. Overall, our analysis demonstrated a high degree of similarity between rHSA and plasma-derived HSA. Expressing rHSA in E. coli with oxidative cytosolic activity provides a safe and cost-effective approach, enhancing the potential of rHSA for a variety of medical applications.

[0029] The amino acid sequence and DNA sequence of the above albumin binding domain are as follows:

[0030] LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP;

[0031] CTGGCGGAAGCGAAAGTTCTGGCGAACCGTGAACTGGATAAATACGTGTTTCTGATTACTACAAAAACCTGATCAACAACGCGAAAACCGTTGAAGGCGTTAAAGCGCTGATCGATGAAATCCTGGCGGCGCTGCCG.

[0032] In one aspect, the present invention relates to a method for producing recombinant human serum albumin, comprising the step of culturing a mutant Escherichia coli transformed with a vector containing a nucleic acid encoding human serum albumin.

[0033] Escherichia coli is currently the most widely used host cell for mass production of various useful proteins due to its rapid growth rate and accumulated fermentation and genetic engineering technologies. In order to produce recombinant proteins with high efficiency, it is necessary to cultivate recombinant E. coli at high concentrations. To this end, various media compositions such as complex medium, synthetic medium, and semi-synthetic medium, and various media feeding methods such as constant feeding, stepwise increase, exponential growth, specific growth rate control, pH and dissolved oxygen control (pH-stat, DO-stat), and glucose and acetic acid concentration control have been developed.

[0034] For human serum albumin, in vitro refolding steps and cloning are essential to secrete the active protein form through one or more biofilms. However, the in vitro refolding step accounts for a significant portion of the overall manufacturing cost and is a time-consuming process that takes approximately 120 hours (approximately 5 days). Furthermore, multiple disulfide-containing proteins can refold incorrectly during transport of the expressed protein to the oxidative periplasm for disulfide bond formation, leading to aggregation and inclusion body formation.

[0035] The above human serum albumin may comprise the following amino acid sequence:

[0036] .

[0037] The DNA sequence encoding recombinant human serum albumin for expression in the above mutant E. coli is as follows:

[0038] ATTACTCCGTTGTACTGTTGCTTAGACTGGCAAAAACGTATGAGACCACCTTGGAGAAATGTTGTGCGGCGGCAGACCCCCATGAATGTTACGCCAAAGTATTCGACGAGTTTAAGCCCCTTGTCGAGGAGCCACAAAACTTGATTAAACAGAACTGCGAATTGTTTGAACAATTGGGAGAGTACAAATTTCAGAACGCTCTGTTAGTGCGTTACACGAAAAAGGTCCCCCAAGTGAGTACACCTACGTTAGTCGAAGTTTCTCGTAACCTTGGAAAAGTCGGATCAAAATGCTGTAAACATCCCGAGGCGAAACGGATGCCATGTGCAGAAGACTACTTATCTGTCGTACTTAATCAGTTATGTGTATTACACGAGAAGACACCAGTGTCTGATCGCGTTACAAAGTGCTGTACAGAGTCTTTAGTAAACCGGAGACCCTGCTTTTCGGCGCTTGAAGTTGACGAGACGTATGTCCCAAAAGAGTTCAATGCTGAAACATTCACGTTCCACGCGGATATCTGCACGCTTAGCGAGAAAGAGAGACAAATAAAGAAACAGACCGCCCTTGTAGAACTTGTCAAGCATAAACCCAAGGCGACAAAAGAGCAACTGAAAGCCGTCATGGACGATTTCGCGGCGTTCGTGGAGAAATGTTGCAAGGCAGATGATAAGGAGACGTGCTTCGCCGAAGAGGGTAAAAAGTTAGTAGCAGCGTCCCAGGCGGCTTTAGGACTGCACCACCACCACCACCAC

[0039] 본 발명에 따르면, 변이 대장균을 이용하여 재조합 인간 혈청 알부민을 생산할 수 있다.

[0040] The above mutant E. coli may be E. coliSHuffle, which is characterized by suppression of expression of thioredoxin reductase (trxB) and glutaredoxin reductase (gor) coding genes of E. coliK12 and overexpression of cytoplasmic disulfide bond isomerase (DsbC) coding genes.

[0041] The above thioredoxin reductase (trxB) coding gene sequence is as follows:

[0042] ATGGGCACGACCAAACACAGTAAACTGCTTATCCTGGGTTCAGGCCCGGCGGGATACACCGCTGCTGTCTACGCGGCGCGCGCCAACCTGCAACCTGTGCTGATTACCGGCATGGAAAAAGGCGGCCAACTGACCACCACCACGGAAGTGGAAAACTGGCCTGGCGATCCAAACGATCTGACCGGTCCGTTATTAATGGAGCGCATGCACGAACATGCCACCAAGTTTGAAACTGAGATCATTTTTGATCATATCAACAAGGTGGATCTGCAAAACCGTCCGTTCCGTCTGAATGGCGATAACGGCGAATACACTTGCGACGCGCTGATTATTGCCACCGGAGCTTCTGCACGCTATCTCGGCCTGCCCTCTGAAGAAGCCTTTAAAGGCCGTGGGGTTTCTGCTTGTGCAACCTGCGACGGTTTCTTCTATCGCAACCAGAAAGTTGCGGTCATCGGCGGCGGCAATACCGCGGTTGAAGAGGCGCTGTATCTGTCTAACATCGCTTCGGAAGTGCATCTGATTCACCGCCGTGACGGTTTCCGCGCGGAAAAAATCCTCATTAAGCGCCTGATGGATAAAGTGGAGAACGGCAACATCATTCTGCACACCAACCGTACGCTGGAAGAAGTGACCGGCGATCAAATGGGTGTCACTGGCGTTCGTCTGCGCGATACGCAAAACAGCGATAACATCGAGTCACTCGACGTTGCCGGTCTGTTTGTTGCTATCGGTCACAGCCCGAATACTGCGATTTTCGAAGGGCAGCTGGAACTGGAAAACGGCTACATCAAAGTACAGTCGGGTATTCATGGTAATGCCACCCAGACCAGCATTCCTGGCGTCTTTGCCGCAGGCGACGTGATGGATCACATTTATCGCCAGGCCATTACTTCGGCCGGTACAGGCTGCATGGCAGCACTTGATGCGGAACGCTACCTCGATGGTTTAGCTGACGCAAAATAA.

[0043] The above glutaredoxin reductase (gor) coding gene sequence is as follows:

[0044] CCGTGGTCTTCAGCCATCCGCCGATTGGTACTGTTGGTTTAACGGAACCGCAGGCGCGCGAGCAGTATGGCGACGATCAGGTGAAAGTGTATAAATCCTCTTTCACCGCGATGTATACCGCCGTCACCACTCACCGCCAGCCGTGCCGCATGAAGCTGGTTGCGT TGGATCGGAAGAGAAGATTGTCGGTATTCACGGCATTGGCTTTGGTATGGACGAAATGTTGCAGGGCTTCGCGGTGGCGCTGAAGATGGGGGCAACCAAAAAAGACTTCGACAATACCGTCGCCATTCACCCAACGGCGGCAGAAGAAGTTCGTGACAATGCGTTAA

[0045] The above cytoplasmic disulfide bond isomerase (DsbC) coding gene sequence is as follows:

[0046] .

[0047] "Nucleic acid" is a comprehensive term encompassing DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequence of human serum albumin as a nucleic acid may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0048] DNA can be readily isolated or synthesized using conventional molecular biological techniques (e.g., by using oligonucleotide probes that can specifically bind to DNA encoding the antibody and its heavy and light chains), and the nucleic acid can be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or further expression.

[0049] "Vector" means a nucleic acid product having a nucleic acid sequence operably linked to suitable regulatory sequences capable of expressing the nucleic acid in a suitable host, as a means for expressing a target gene in a host cell, and "recombinant vector" means a recombinant nucleic acid molecule containing a target coding sequence and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a specific host organism.

[0050] The above vector includes a viral vector such as a plasmid vector, a cosmid vector, a bacteriophage vector, an adenovirus vector, a retrovirus vector, an adeno-associated virus vector, etc. Components of the vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, a transcription termination sequence. A nucleic acid encoding an antibody is operably linked to the promoter and the transcription termination sequence, etc.

[0051] "Operably linked" means a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0052] It usually includes a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation initiation, and a transcription / translation termination sequence.

[0053] The above vector contains antibiotic resistance genes commonly used in the art as selectable markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0054] The mutant E. coli is transformed with a vector containing a nucleic acid encoding the above human serum albumin. "Transformation" may mean that the characteristics of an organism or cell are genetically changed by a nucleic acid, which is genetic material provided from outside.

[0055] Any commercially available medium can be used as a culture medium. Any other necessary supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with the host cells selected for expression and will be apparent to those skilled in the art. The medium can be, for example, any chemically defined medium.

[0056] The above culturing can be performed at a temperature of 16°C to 20°C. Specifically, the above culturing can be performed at a temperature of 18°C.

[0057] To optimize the expression conditions of recombinant human serum albumin, temperature conditions were compared and tested. 18°C ​​was selected as the temperature condition that resulted in the highest proportion of stable, water-soluble proteins after expression under various temperature conditions (37°C, 30°C, and 18°C).

[0058] In some cases, a step of purifying the culture after the above culturing may be additionally included.

[0059] After the above culture, the culture can be recovered, for example, by centrifugation or ultrafiltration to remove impurities, and the result can be purified.

[0060] The above purification refers to a process for removing or reducing unwanted substances, such as HCPs, DNA, or salts, from a mixture containing, for example, a protein of interest. "Purification" may be part of an overall purification process that results in a "homogeneous" composition.

[0061] Purification according to the present invention may involve subjecting the culture to nickel affinity chromatography and anion exchange chromatography. The nickel affinity chromatography and anion exchange chromatography may be applied sequentially.

[0062] The present invention also relates to recombinant human serum albumin produced by the above production method.

[0063] The albumin production technology using the E. coli-based recombinant protein technology proposed by the present invention can overcome the shortcomings of existing methods for obtaining albumin from human blood, such as cross-infection with pathogenic substances from donor blood and limited supply.

[0064] The present invention further relates to a drug delivery system comprising the recombinant human serum albumin.

[0065] FcRn is a receptor present on endothelial cells of the human lung and kidney that can bind to albumin and IgG. Albumin has a long half-life due to FcRn-mediated recycling. Endothelial cells form endosomes with a slightly acidic environment (pH < 6.5) to transport albumin into the cell. During this process, albumin binds to FcRn, preventing its degradation in lysosomes. Albumin then moves out of the cell and is separated from albumin in a neutral environment (pH > 7.0).

[0066] It was confirmed that the recombinant human serum albumin according to the present invention can have a half-life similar to that of human serum-derived albumin in the body.

[0067] ABD (Albumin binding domain) is a peptide that binds to albumin through hydrophobic and electrostatic interactions. After reacting recombinant human serum albumin with ABD, analysis confirmed that recombinant human serum albumin had an affinity for ABD, and it is expected that it will be possible to load biologically active substances using this.

[0068] Analysis of the binding capacity of FcRn and ABD confirms that recombinant human serum albumin can be utilized as a scaffold for long-term biologically active substances, such as drug delivery.

[0069] The above biologically active substance is a drug that regulates all physiological phenomena in a living body, and may be, but is not limited to, DNA, RNA, siRNA, aptamer, protein, antibody, or cytotoxic compound.

[0070] The biologically active substance can bind to recombinant human serum albumin and can be linked via a non-covalent bond or a covalent bond. The non-covalent bond can be, for example, at least one selected from the group consisting of a hydrogen bond, an electrostatic interaction, a hydrophobic interaction, a van der Waals interaction, a pi-pi interaction, and a cation-pi interaction. The covalent bond can be a degradable or non-degradable bond, and the degradable bond can be a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzymatically degradable bond, and the non-degradable bond can be, but is not limited to, an amide bond or a phosphate bond.

[0071] Example

[0072]

[0073] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0074] Manufacturing Example 1. Materials

[0075] 1-1. Materials

[0076] Escherichia coli SHuffle T7 was purchased from New England Biolabs (Ipswich, MA). Ni-NTA agarose was provided by Qiagen (Valencia, CA). Amicon Ultra centrifugal filters with a cutoff of 10 kDa were purchased from Merck Corporation (Germany). PD-10 desalting columns were purchased from GE Healthcare (Piscataway, NJ). The Mini-PROTEAN system for polyacrylamide electrophoresis was purchased from Bio-Rad (Hercules, CA). Hand-cast gels were prepared according to the standard protocol for 12% polyacrylamide gels. Biotinylated human FcRn was purchased from ACROBiosystems (Newark, DE). Plasmid extraction and gel extraction kits were purchased from Bioneer (Daejeon, Korea). Sodium chloride, sodium phosphate monobasic, and sodium phosphate dibasic were purchased from Samchun Chemical (Seoul, Korea). Purified anti-6×His primary antibody was purchased from BioLegend. Goat anti-mouse IgG (H+L)-HRP was purchased from GenDEPOT (USA). Albumin extracted from human serum was purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were provided by Sigma-Aldrich (St. Louis, MO).

[0077] 1-2. Plasmids and bacterial strains

[0078] The gene encoding HSA fused to a C-terminal hexahistidine (UniProt ID, P02768-1, amino acid sequence from D25 to L609) was synthesized (Bionics, Korea) and cloned into a modified pQE-80L expression vector (Qiagen, Germany) with the T5 promoter replaced, which is compatible with the T7 RNA polymerase expressed in E. coli SHuffle T7 or E. coli BL21 (DE3) used in this study. The resulting expression vector was named pQE-T7-rHSA. The gene encoding the C-terminal His-tagged ABD was synthesized (Bionics, Korea) and cloned into the modified pQE-80L expression vector, resulting in pQE-T7-ABD.

[0079] [Amino acid sequence]

[0080] MDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHP YFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHH

[0081] [DNA 서열]

[0082] ATTACTCCGTTGTACTGTTGCTTAGACTGGCAAAAACGTATGAGACCACCTTGGAGAAATGTTGTGCGGCGGCAGACCCCCATGAATGTTACGCCAAAGTATTCGACGAGTTTAAGCCCCTTGTCGAGGAGCCACAAAACTTGATTAAACAGAACTGCGAATTGTTTGAACAATTGGGAGAGTACAAATTTCAGAACGCTCTGTTAGTGCGTTACACGAAAAAGGTCCCCCAAGTGAGTACACCTACGTTAGTCGAAGTTTCTCGTAACCTTGGAAAAGTCGGATCAAAATGCTGTAAACATCCCGAGGCGAAACGGATGCCATGTGCAGAAGACTACTTATCTGTCGTACTTAATCAGTTATGTGTATTACACGAGAAGACACCAGTGTCTGATCGCGTTACAAAGTGCTGTACAGAGTCTTTAGTAAACCGGAGACCCTGCTTTTCGGCGCTTGAAGTTGACGAGACGTATGTCCCAAAAGAGTTCAATGCTGAAACATTCACGTTCCACGCGGATATCTGCACGCTTAGCGAGAAAGAGAGACAAATAAAGAAACAGACCGCCCTTGTAGAACTTGTCAAGCATAAACCCAAGGCGACAAAAGAGCAACTGAAAGCCGTCATGGACGATTTCGCGGCGTTCGTGGAGAAATGTTGCAAGGCAGATGATAAGGAGACGTGCTTCGCCGAAGAGGGTAAAAAGTTAGTAGCAGCGTCCCAGGCGGCTTTAGGACTGCACCACCACCACCACCAC

[0083] 1-3. 재조합 인간 혈청 알부민 발현 조건 최적화

[0084] Expression hosts were generated by transforming E. coli SHuffle or E. coli BL21(DE3) with the expression vector pQE-T7-rHSA or pQE-T7-ABD. E. coli SHuffle containing pQE-T7-rHSA or E. coli BL21(DE3) containing pQE-T7-rHSA were cultured to saturation in 2×YT medium containing 100 μg / mL ampicillin at 37°C with vigorous shaking at 220 rpm. The saturation culture was diluted 100-fold into fresh 2×YT medium and inoculated with the main culture. When the optical density at 600 nm (OD) reached 0.8, protein expression was induced with 1 mM IPTG and the temperature was adjusted to 18, 30, or 37°C. After 12–48 h of incubation, cells were harvested and pelleted by centrifugation at 6000 rpm for 10 min. To extract and purify proteins, cell pellets were resuspended in lysis buffer containing 50 mM phosphate buffer (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 1 mg / mL lysozyme, and incubated with rotation at 4°C for 30 min. After 10 min of sonication (10 s on / 10 s off), the supernatant was centrifuged at 12,000 rpm for 30 min, filtered, mixed with Ni-NTA agarose for 30 min, and washed on a gravity flow column with phosphate buffer (pH 8.0) containing 30 mM imidazole to remove impurities. Proteins were eluted with phosphate buffer (pH 8.0) containing 250 mM imidazole. Further purification was performed by anion exchange chromatography using a HiTrap Q HP column equilibrated with 20 mM Bis-Tris buffer at pH 7.0. Elution was performed with a gradient of NaCl. The eluate was buffer-exchanged from the PD-10 column into PBS (pH 7.4) and stored in aliquots at -20°C. E. coli BL21(DE3) containing pQE-T7-ABD and plasmid-free E. coli BL21(DE3) were cultured and treated similarly to extract ABD and host cell proteins.

[0085] We initially evaluated rHSA expression in BL21(DE3), an Escherichia coli strain commonly used for recombinant protein production. BL21(DE3) strains containing pQE-T7-rHSA in seed culture exhibited retarded growth and low turbidity. Upon inoculation, cultures exhibited a non-exponential growth pattern, precluding conventional IPTG induction in the mid-log phase. Induction in the early growth phase at 37°C for 24 h resulted in moderate rHSA expression. However, this was problematic, as rHSA degradation occurred in the mid-phase and cell densities remained low, as evidenced by a 600 nm saturation optical density of less than 2.0. Lowering the culture temperature did not improve cell growth or rHSA expression. These results suggest that the robust expression of heterologous rHSA by T7 under reducing conditions may have induced abnormal disulfide bond formation, misfolding, aggregation, and / or degradation, which compromise E. coli fitness, and that temperature control alone is insufficient to effectively control rHSA self-folding. To stabilize the native form of rHSA by enhancing cross-translational formation of disulfide bonds, we decided to use E. coli SHuffle, which has an oxidative cytoplasm, for rHSA expression. Interestingly, SHuffle carrying pQE-T7-rHSA did not show growth delay in seed cultures. To investigate the effect of temperature on cell growth and productivity, main flask cultures were performed at 37°C, 30°C, and 18°C, and the results showed notable differences (Fig. 1). At 37°C and 30°C, rHSA expression was low, and most of it was in an insoluble form (Fig. 1A, B). However, at 18°C, a significant increase in the amount of soluble rHSA was observed (Fig. 1C), suggesting that the combination of slow translation at 18°C ​​and an oxidative cytoplasm promotes self-folding of the soluble form of rHSA.

[0086] Extraction of rHSA from cell lysate involved a two-step purification process, starting with nickel-Histag affinity chromatography and followed by ion-exchange chromatography. The first purification achieved a moderate rHSA purity of approximately 60% (Fig. 2a), and the second purification further improved the purity to over 95%, effectively removing impurities of similar size to rHSA observed after affinity purification (Fig. 2e). The specificity of the purified rHSA was confirmed by Western blotting (Fig. 2b). Notably, rHSA showed a faster migration under non-reducing conditions in SDS-PAGE than under reducing conditions, indicating the presence of intramolecular disulfide bonds and a more compact structure (Fig. 2c). Analysis of rHSA by MALDI-TOF mass spectrometry revealed a dominant peak corresponding to a molecular mass of 67402 Da with a mass-to-charge ratio (m / z) of 67403 [M+H]+ (Fig. 2d).

[0087] The temperature condition that gave the highest proportion of stable water-soluble state after expression under various temperature conditions (37 ℃, 30 ℃, 18 ℃) was selected (18 ℃).

[0088] Considering the presence of N-terminal N-formylmethionine (fMet), C-terminal hexahistidine, and 17 cysteines, unlike the native 34 cysteines due to disulfide bond formation, the expected molecular weight of rHSA at pH 6.0 was theoretically calculated to be 67405. The slight mass change could be attributed to the protonation of histidine residues, which is affected by pH close to the pKa of 6.0. In summary, the two-step purification process achieved an impressive productivity of 2.5 mg of soluble and disulfide-bonded rHSA per liter of SHuffle culture.

[0089] Manufacturing Example 2. Cell Expression and Purification

[0090] The quality of human serum albumin expressed and purified from cells was analyzed by SDS-PAGE and immunoblotting.

[0091] For SDS-PAGE, 1 mL of cells were collected at each designated time point. After appropriate dilution to an OD 600 value of less than 1, the diluted sample was centrifuged at 13,000 rpm for 1 min, and the supernatant was removed. Distilled water was added in an appropriate amount to achieve an OD 600 value of 10. A total cell sample was prepared using 10 μL of this suspension.

[0092] After sonication and centrifugation of the harvested cells, 5 μL of the supernatant was used as the soluble fraction. The pellet was resuspended in an equal volume of distilled water as lysis buffer, and 5 μL of this resuspension was used as the insoluble fraction.

[0093] Samples were incubated in 5× reducing sample buffer (0.25 M Tris-HCl pH 6.8, 50% glycerol, 10% SDS, 0.25% bromophenol blue, 0.5 M DTT) or non-reducing sample buffer (0.25 M Tris-HCl pH 6.8, 50% glycerol, 10% SDS, 0.25% bromophenol blue) at 95°C for 5 min and then run on polyacrylamide gels (12% resolving gel and 4% stacking gel) at 150 V for 80 min. The gels were then stained with Coomassie blue overnight and then destained with destaining solution (20% methanol and 10% acetic acid).

[0094] For immunoblotting, proteins were separated by 12% SDS-PAGE under reducing conditions, transferred to polyvinylidene difluoride (PVDF) membranes, and mounted on a Tetra Blotting Module (Bio-Rad, USA) at 100 V on ice for 90 min. The membranes were blocked with phosphate-buffered saline (PBS) containing 5% skim milk powder and 0.5% v / v Tween 20 for 2 h, followed by addition of anti-6×His primary antibody (1:2000) and incubation for 2 h at RT. After each step, the membranes were washed three times for 5 min with PBS containing 0.5% Tween 20 (PBST). Then, the appropriate HRP-conjugated secondary antibodies (1:5000) were added and incubated for 1.5 h at RT. The blots were washed three times with PBST for 10 min each, and HRP activity was detected with TMB (3,3',5,5'-tetramethylbenzidine substrate).

[0095] Example 1. Characteristic Evaluation

[0096] 1-1. Mass spectrometry (MALDI-TOF)

[0097] : The mass of recombinant human serum albumin was measured by MALDI-TOF. The rHSA mass was confirmed by MALDI-TOF mass spectrometry using an auroflex maX TOF / TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a 200 Hz laser operating in linear positive ion detection mode. A saturated solution of sinapinic acid (10 mg / mL) in a 0.1% v / v TFA / ACN mixture was used for matrix preparation. Then, rHSA at a concentration of 1.0 mg / mL in deionized water was directly coupled to the MALDI target with 2 μL of matrix solution and vacuum-dried. The mass spectra of the samples were acquired in the m / z range of 2,000–200,000 with 2,000 laser shots.

[0098] 1-2. Circular dichroism (CD) spectroscopy

[0099] : The secondary structure was analyzed by comparing the wavelength changes by irradiating the sample with rotating light. CD spectra were recorded on a J-1500 (Jasco, Japan). The secondary structure changes of HSA were monitored in the far-infrared region (180–260 nm) using a cell with a 0.1 cm path length. Samples (pHSA or rHSA) were prepared in deionized water at concentrations of 0.5 or 0.25 mg / mL and then added to a 0.1 cm quartz cell for measurement. CD software (CD pro) was used to predict the secondary structure of the protein by statistical methods.

[0100] 1-3. Intrinsic fluorescence spectroscopy

[0101] : The 3D structure was analyzed by irradiating the samples with fluorescence at 280 nm and measuring the emitted fluorescence at various wavelengths. Intrinsic fluorescence spectroscopy was performed on a Hidex Sense multimodal microplate reader (Hidex, Finland) with a 1 cm pathlength. All samples were diluted to 0.2 mg / mL in PBS (pH 7.4). Samples were excited at 280 nm at 25°C, and the emission was recorded at wavelengths of 320, 330, 355, 390, 405, 444, and 450 nm.

[0102] 1-4. Esterase-like activity

[0103] Albumin exhibits enzymatic activity by reacting with several organophosphorus compounds and p-nitrophenyl acetate due to its 82 amino acid residues (59 lysines, 10 serines, 8 threonines, 4 tyrosines, and 1 asparagine). HSA exhibits esterase-like activity toward p-nitrophenyl acetate (p-NPA). The esterase activity toward p-NPA was performed in a 200 μL reaction mixture. Serial dilutions of the protein were prepared in PBS (pH 7.4) to a final concentration of 10–80 M and pre-incubated at 25°C for 5 min. After incubation, p-NPA was diluted in DMSO at a concentration of 0.1 M to a final concentration of 700 μM. The formation of p-nitrophenol (p-NP) was measured spectrophotometrically at a wavelength of 400 nm and recorded every minute for 15 min using the kinetics / time application of a Hidex Sense multimodal microplate reader (Hidex, Finland).

[0104] 1-5. pH-dependent FcRn binding check

[0105] Albumin exhibits a pH-dependent binding pattern, binding to FcRn in slightly acidic environments (pH < 6.5) and dissociating from FcRn in neutral environments (pH > 7.0). The binding affinity of FcRn to pHSA or rHSA was measured using biolayer interferometry on an OCTET QK384 instrument (Forte Bio, USA). Biotinylated human FcRn (hFcRn) was prepared at a concentration of 0.4 μg / mL in a binding buffer consisting of 25 mM sodium acetate, 25 mM NaH2PO4, and 150 mM NaCl at pH 5.5. The streptavidin biosensor surface was saturated with biotinylated hFcRn for 600 s, and then stabilized in a sodium phosphate buffer at pH 5.5 for 60 s. To evaluate the interaction between FcRn and pHSA or rHSA under slightly acidic conditions, hFcRn-coated biosensors were immersed in wells containing pHSA or rHSA samples at concentrations of 4 μM to 0.5 μM in binding buffer at pH 5.5. The binding step was initiated by shaking at 1,000 rpm for 300 s, and the dissociation of the bound HSA samples from the biosensor was monitored for 600 s in the same binding buffer. To evaluate the interaction between FcRn and pHSA or rHSA in a neutral environment, the stabilization, binding, and dissociation steps of the FcRn-coated biosensor were performed similarly, except that the pH of the binding buffer was adjusted to 7.4.

[0106] 1-6. Characteristic Evaluation

[0107] To elucidate the structural similarity between rHSA and pHSA, comprehensive comparative physicochemical analyses were performed. First, rHSA and pHSA were individually exposed to SEC to compare their hydrodynamic volumes. rHSA eluted at a retention time of 10.52 min, which was very similar to the 10.43 min observed for pHSA (Fig. 3a). Next, the secondary structure of rHSA was investigated using circular dichroism (CD). The far-ultraviolet (FUV) CD spectrum of rHSA, ranging from 195 nm to 260 nm, showed high similarity to that of pHSA (Fig. 3b).

[0108] Quantitative analysis of the secondary structural components derived from these spectra revealed that rHSA and pHSA were quite comparable in terms of the distribution of α-helices, β-sheets, β-turns, and random coil structures (Table 1). In addition, the tertiary structures of rHSA and pHSA were compared using intrinsic fluorescence spectroscopy. This analysis measures the fluorescence emitted upon excitation and is sensitive to the exposure of aromatic residues, primarily tryptophan, which vary depending on their position and orientation within the folded protein structure. Excitation at 280 nm revealed that the emission spectra of rHSA and pHSA were very similar, with a high degree of similarity in their profiles, providing strong evidence that these proteins have similar three-dimensional conformations (Fig. 3c).

[0109] [Table 1]

[0110]

[0111]

[0112] Comparison of CD spectra results and secondary structure ratios confirmed similarity in secondary structures between the two types of albumin.

[0113] We investigated the esterase-like activity of rHSA and pHSA, focusing on the catalytic hydrolysis of ester bonds to carboxylic acids and alcohols. Sudlow I and II are known to be two distinct localized domains of HSA responsible for esterase-like activity (https: / / www.mdpi.com / 1422-0067 / 22 / 19 / 10593). Using p-NPA as a substrate ester, whose hydrolysis induces readily detectable p-NP absorbance at 400 nm, we observed that the initial reaction rate by rHSA was nearly identical to that by pHSA (Fig. 3d). Furthermore, the reaction was found to increase proportionally with rHSA concentration. Control experiments, including negative controls and reference samples (host cell proteins and lysozyme), showed no or very weak activity, highlighting the specificity of the enzymatic reaction toward HSA.

[0114] These results strongly suggest that the self-folded rHSA in the shuffle maintains an arrangement of active site residues surrounding tertiles I and II similar to the native arrangement of pHSA. In summary, a series of physicochemical analyses clearly confirmed the structural similarity between rHSA and pHSA.

[0115] 1-7. Molecular Interactions between rHSA and ABD

[0116] To investigate whether the high structural similarity of rHSA could translate into functional similarity in terms of molecular interactions with cognate receptors or ligands of HSA, we assessed the ability of rHSA to interact with ABD. ABD is a versatile albumin-binding motif widely used to extend the in vivo half-life of short-lived therapeutics. Using SEC, we compared the retention time of rHSA co-incubated with ABD with that of isobaric rHSA or ABD alone. The analysis revealed a characteristic peak with a significantly increased absorbance and decreased retention time compared to rHSA alone, indicating the formation of a heterodimeric complex between rHSA and ABD (Figure 4a). Simultaneously, pHSA incubated with ABD exhibited a shorter retention time and higher absorbance compared to pHSA alone (Figure 4b). Collectively, these results demonstrate the functional activity of rHSA binding to ABD and suggest its potential as an effective drug delivery system.

[0117] 1-8. pH-dependent interaction between rHSA and FcRn

[0118] The pH-dependent interaction with FcRn represents a key aspect of HSA function that is essential for extending the in vivo half-life when delivering therapeutic payloads. Using biolayer interferometry, we investigated whether rHSA could bind hFcRn in a pH-dependent manner similar to naturally occurring pHSA. Under mildly acidic conditions (pH 5.5) mimicking the endosomal environment, rHSA had a K of 48 nM. D It showed remarkable affinity for FcRn, which is characterized by a K of 17 nM at pH 5.5 (Fig. 5a). Conversely, at neutral pH, rHSA showed a reduced binding affinity for hFcRn (Fig. 5b). Parallel analysis using pHSA revealed a pH-dependent behavior, with a K of 17 nM at pH 5.5. D It showed affinity for hFcRn (Fig. 5c and d).

[0119] These results suggest that rHSA has the potential to evade endosomal degradation after cellular internalization and undergo FcRn-mediated recycling in vivo. The pH-dependent reversible interaction with FcRn is essential for rHSA's role as a persistent drug carrier. Specifically, we demonstrated that this pH-dependent property was maintained in HSA produced recombinantly in Escherichia coli.

[0120] Analysis of the binding capacity of FcRn and ABD confirmed that recombinant human serum albumin can be used as a scaffold for long-term drug delivery.

[0121] We successfully produced rHSA with high biological similarity using E. coli SHuffle under optimized culture conditions. These conditions provide an oxidative cytoplasm and a slow translation rate, promoting correct disulfide bond formation and self-folding of rHSA. Comprehensive analyses, including SEC, CD spectroscopy, intrinsic fluorescence spectroscopy, and esterase-like activity assays, confirmed that rHSA and pHSA have identical secondary and tertiary structures. The functional similarity of rHSA was further investigated by evaluating the interaction of rHSA with the ABD and the pH-dependent interaction with hFcRn. The results revealed the formation of a heterodimeric complex between rHSA and the ABD, suggesting its potential as an effective drug carrier. Furthermore, rHSA, like pHSA, exhibited pH-dependent binding to hFcRn and demonstrated the potential for recycling through FcRn in vivo. The approach presented herein not only ensures high structural similarity between rHSA and pHSA, but also ensures the ability of rHSA to specifically interact with ABD and FcRn, which are important for its role as a drug carrier.

[0122] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0123] According to the present invention, human serum albumin produced in a bacterial expression system in oxidative cytoplasm exhibits the same structure and function as human serum albumin obtained through plasma fractionation, and can be easily expressed and purified because it does not utilize a solubility-enhancing tag protein or co-express a chaperone. Furthermore, it can also be utilized as a long-lasting drug delivery system in the body through the binding ability of FcRn and the albumin binding domain (ABD).

Claims

1. A method for producing recombinant human serum albumin, comprising the step of culturing a mutant E. coli transformed with a vector containing a nucleic acid encoding human serum albumin.

2. A production method characterized in that in the first paragraph, the mutant E. coli is E. coliSHuffle, characterized in that the expression of the thioredoxin reductase (trxB) and glutaredoxin reductase (gor) coding genes of E. coli is suppressed, and the cytoplasmic disulfide bond isomerase (DsbC) coding gene is overexpressed.

3. A production method according to claim 1, characterized in that the culturing is performed under temperature conditions of 18°C.

4. A production method according to claim 1, further comprising a step of purifying the culture after the culturing.

5. A production method according to claim 4, characterized in that the purification is performed by subjecting the culture to nickel affinity chromatography and anion exchange chromatography.

6. Recombinant human serum albumin produced by a production method according to any one of claims 1 to 5.

7. A drug delivery system comprising the recombinant human serum albumin of clause 6.

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