Integrated Purification Process for 5-Aminolevulinic Acid Phosphate Produced via Biological Fermentation

Abstract

5-aminolevulinic acid (5-ALA) affords various positive health effects, including benefits for conditions such as diabetes. Biological fermentation holds potential for efficiently mass-producing biomolecules, including 5-ALA, yet it generally results in a mixture of target molecules and impurities, including byproducts. Pyrazine-2,5-dipropionic acid (PY), a dimer of 5-ALA, can easily form in 5-ALA production and is one of its major impurities. In this study, we developed an integrated purification process for 5-aminolevulinic acid phosphate (5-ALAP) produced via biological fermentation. The process consists of 16 stages, including impurity removal (ion-exchange resins) and crystallization. Three types of ion-exchange resin (IER) columns were combined to remove impurities such as byproducts and pigment. Comprehensive condition setting for crystallization was carried out to reduce the amount of residual poor solvent in the 5-ALAP crystals. The obtained crystals contained significantly fewer impurities (<0.05% vs. 5-ALAP), such as PY, compared with their commercially available counterparts. The residual poor solvent in the 5-ALAP crystals was reduced to below 1000 ppm under the crystallization conditions. We confirmed the high scalability of the purification method developed in this study. Therefore, this article provides an industrially applicable purification process for fermentatively produced 5-ALA.

1. Introduction

Awareness of health and wellness has experienced rapid growth recently due to the severity of lifestyle-related diseases. The International Diabetes Federation’s Diabetes Atlas reports that 11.1%—or one in nine—of the adult population (20–79 years old) is living with diabetes [1]. A. V. Benjafield et al. reported that more than 16% of the global population is estimated to have insomnia [2]. A decline in physical ability due to aging has also been widely reported [3,4]. Cancer is one of the most common diseases globally [5], and the number of people with cancer is estimated to exceed 35 million by 2050 [6]. Significant attention has been given to the use of medication or nutrient supplements—amino acids, vitamins, proteins, and so on—to reduce the incidence rates of lifestyle-related diseases [7].

Various reports have described the potential positive health impacts of 5-aminolevulinic acid (5-ALA). Its chemical structure in phosphate salt form is shown in Supplementary Figure S1a. Previous studies have discussed the various positive impacts of 5-ALA in the context of diabetes [8,9,10,11,12], stress [13,14], physical activity [15,16,17,18,19], sleep difficulty [20,21], cancer [22], late-onset hypogonadism [23,24], and COVID-19 infection [25]. However, M. H. Perez et al. reported that 5-ALA exists in very low amounts (<1 μg/g) in common foods such as banana and potato [26]. It can thus be difficult to consume a sufficient dose of 5-ALA from daily meals, and supplementation or specific medication can be a convenient way to reduce 5-ALA shortage.

5-ALA is sensitive to temperature and pH. P. A. McCarron et al. reported that 5-ALA was readily degraded to pyrazine-2,5-dipropionic acid (PY), a dimer of 5-ALA, at high temperature [27]. The chemical structure of PY is shown in Supplementary Figure S1b. Bunke et al. reported that 5-ALA was more quickly degraded in the presence of alkaline Na₂SO₃ compared with acidic phosphoric acid, and PY was detected after the degradation of 5-ALA [28]. From these reports, it can be concluded that a salt form of 5-ALA might be preferable regarding improved stability.

Biological fermentation has been previously discussed [29,30,31,32,33] for achieving the efficient production of 5-ALA. However, fermentation processes require broth nutrients and substrates, and byproducts produced by microorganisms can be detected using target molecules. Y.-C. Yi et al. reported the necessity to develop a purification process for 5-ALA produced via fermentation to achieve its biological production [31].

The process of purifying 5-ALA has been previously reported. G. D. Venosa et al. reported the separation of 5-ALA and derivatives using a single ion-exchange resin (IER, Dowex 50WX8, H⁺ type) [34]. H. Okada et al. used a cation-exchange resin (Amberlite IR-120B) to remove fermentative substrates such as glycine (Gly) from 5-ALA broth [35]. Reports on the crystallization of 5-ALA salts have also been published. H. Okada et al. reported crystallization of 5-ALA HCl salt by using several poor solvents such as ethanol, acetone, and normal propanol [35]. Lin et al. reported on the gradual-cooling crystallization of 5-ALA phosphate, the final temperature of which reached 0 °C [36].

However, these reports refer to single operations, such as using IER or crystallization. Generally, fermentation broth contains various residual nutrients, substrates, and byproducts produced by microorganisms. Therefore, it is necessary to develop an integrated purification process for 5-ALA produced from fermentation broth.

In this study, an integrated purification process for fermentatively produced 5-ALA was developed. The purification process features two-phase crystallization (crude crystallization and recrystallization). To achieve very-high-purity 5-ALA, we identified the optimum parameters for impurity removal processes. To remove impurities from 5-ALA broth, a combination of three types of IERs was used. 5-ALA was crystallized using EtOH, and the crystallization parameters, including the volume of added EtOH, the crystallization temperature, and the speed of EtOH addition, were examined to reduce residual EtOH in 5-ALA crystals. The applicability of the developed purification method to ton-scale manufacturing was also evaluated to provide knowledge on a purification process for 5-ALA that is applicable to industrial manufacturing.

2. Materials and Methods

Figure 1 shows the flow of the developed 5-ALA purification process, featuring dual crystallization phases (crude crystallization and recrystallization, comprising 7 and 9 stages, respectively). Considering the instability of 5-ALA, the phosphate form of 5-ALA (5-ALAP) was chosen as the target product. To transform 5-ALA to 5-ALAP, phosphoric acid was added in the middle of the process.

2.1. Quantification of 5-ALA (As 5-ALAP) for Crude Process

The 5-ALA concentrations in each liquor for each stage of the crude purification process were quantified using an HPLC (High Performance Liquid Chromatography) device connected to a reverse-phase column (YMC Triart-C18, 3 μm, 3.0 × 150 mm, YMC Co., Ltd., Kyoto, Japan), which was stabilized at 40 °C. In total, 3.325 g of K₂HPO₄ and 1.45 g of sodium lauryl sulfate were dissolved in 3.5 L of ultrapure water, sourced from MilliQ (Advantage A10, MILLIPORE, Darmstadt, Germany). The pH was adjusted to 2.5 by adding 85% H₃PO₄. The mobile phase was prepared by adding 1.5 L of MeOH (HPLC-grade, Kanto Chemical Co., Inc., Tokyo, Japan) and mixing the components. The mobile phase was filtered using a membrane filter (OmniporeTM Membrane Filters, 0.45 μm JH, MERCK, Darmstadt, Germany). We used the post-column ortho-phthalaldehyde (OPA) derivatization method to selectively detect compounds with amino functional groups, such as 5-ALA. In total, 54.0 g of boric acid, 32.4 g of NaOH, and 4.1 mL of Briji-35 (22%, BL TEC K.K. Osaka, Japan) were dissolved in 2.7 L of ultrapure water. The derivatization solution was prepared by adding 0.6 of ortho-phthalaldehyde and 4.6 g of N-acetyl-l-cysteine. The mobile phase was supplied in isocratic mode. The flow rates for the mobile phase and the derivatization solution were set at 0.5 and 0.2 mL/min, respectively. We employed an HPLC device (NEXERA XR, SHIMADZU CORPORATION, Kyoto, Japan) connected to a fluorescence detector (RF-20A, SHIMADZU CORPORATION). The excitation and fluorescent wavelengths were set at 360 and 440 nm.

Hydrochloric salt of 5-ALA (5-ALAH, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was dissolved in ultrapure water with a 0.1 g/L concentration, and the 5-ALAH std solution was used for single-point calibration. Samples were prepared at approximately 0.1 g/L 5-ALAP concentrations with ultrapure water. Samples of 5 μL (or the 5-ALAH standard) were injected into the HPLC.

The 5-ALAP concentrations in each crude process liquor were quantified according to Equation (1), where A₁ is the detected area value of a 5-ALAP peak in the process liquor, D₁ is the detected area value of the 5-ALAH std peak, C₁ is the 5-ALAH concentration (g/L) used for the single-point calibration, and DR₁ is the dilution ratio of the samples. The detected area value of the 5-ALAH std (D₁) is calibrated as the ratio of the molecular weight of 5-ALAP (229.125 g/mol) to that of 5-ALAH (167.59 g/mol).

$$5\text{-}\mathrm{ALAP} \mathrm{concentration}={\displaystyle \frac{A_1}{D_1}}\times C_1\times {DR}_1\times {\displaystyle \frac{229.125}{167.59}}$$

(1)

2.2. Quantification of 5-ALAP for Recrystallization Process

The 5-ALA concentrations in each liquor for each stage of the recrystallization process were quantified using an HPLC method. A reverse-phase column (Inertsil ODS-3V, 5 μm, 4.6 × 150 mm, GL-Science Inc., Tokyo, Japan) was used; its temperature was stabilized at 25 °C. Samples were diluted to approximately 1 g/L 5-ALAP concentrations with 0.05 mol/L HCl. To prepare mobile phase A, 6.0 g of sodium 1-heptanesulfonate was dissolved in 5.0 L of ultrapure water, and the pH was adjusted to 2.0 by adding 85% H₃PO₄. It was then filtered using a membrane filter (OmniporeTM Membrane Filters, 0.45 μm JH, MERCK). CH₃CN was used as mobile phase B. The flow rate was set at 1.0 mL/min. The gradient conditions were as follows: start at 10% (B) and hold for 5 min, linearly increase to 30% (B) for 25 min, hold at 30% (B) for 5 min, and return to 10% (B) for 0.01 min. We employed an HPLC device (NEXERA XR, SHIMADZU CORPORATION) connected to a UV detector (SPD-20A, SHIMADZU CORPORATION). The detection wavelength was set at 210 nm.

5-ALAP (KIYAN PHARMA Co., Ltd., Tokyo, Japan) was dissolved in 0.05 mol/L HCl with a concentration of approximately 1 g/L, and the 5-ALAH std solution was used for single-point calibration. A 20 μL volume of the samples or the 5-ALAH standard was injected into the HPLC.

The 5-ALAP concentrations in each liquor of the recrystallization process were quantified according to Equation (2), where A₂ is the detected area value of a 5-ALAP peak in the process liquor, D₂ is the detected area value of the 5-ALAP std peak, C₂ is the 5-ALAP concentration (g/L) used for single-point calibration, and DR₂ is the dilution ratio of the samples.

$$5\text{-}\mathrm{ALAP} \mathrm{concentration} (\mathrm{g}/\mathrm{L})={\displaystyle \frac{A_2}{D_2}}\times C_2\times {DR}_2$$

(2)

2.3. Quantification of Related Impurities

PY concentrations in the liquor for each stage of the crude and recrystallization processes were quantified using the HPLC method described in Section 2.2.

PY (NIPPON RIKA Co., Ltd., Tokyo, Japan) was dissolved in 0.05 mol/L HCl with a concentration of 0.01 g/L, and the PY std solution was used for single-point calibration. A 20 μL volume of the samples or the PY standard was injected into the HPLC. The detection limit of this analysis method for PY is 10 ppm.

The PY concentrations in each liquor of the recrystallization process were quantified according to Equation (3), where A₃ is the detected area value of a PY peak in the process liquor, D₃ is the detected area value of the PY std peak, C₃ is the PY concentration used for single-point calibration, and DR₃ is the dilution ratio of the samples.

$$\mathrm{P}\mathrm{Y} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} (\mathrm{g}/\mathrm{L})={\displaystyle \frac{A_3}{D_3}}\times C_3\times {DR}_3$$

(3)

2.4. Crystal Structure Analysis of 5-ALAP Crystals

We analyzed the crystal structure of the obtained 5-ALAP using X-ray diffraction (XRD; D8 ADVANCE, Bruker, MA, USA) with a Cu anode.

2.5. Comparison Targets

Three commercially available 5-ALAP crystals were used as comparison targets for each quality assessment. The names of the manufacturers are withheld; they are referred to as Manu A, Manu B, and Manu C.

2.6. HPLC Analysis of Hydrophobic Impurities

Hydrophobic impurities in the obtained 5-ALAP were investigated using an HPLC method with a reverse-phase column (InertSustain C18(UP), 5 μm, 4.6 × 250 nm, GL-Science Inc., Tokyo, Japan), the temperature of which was stabilized at 30 °C. Trifluoroacetic acid (TFA) was dispensed into ultrapure water at a concentration of 0.1%. CH₃CN was added to the TFA solution at a concentration of 15%; upon mixing, this was used as mobile phase A, which was filtered using a membrane filter (OmniporeTM Membrane Filters, 0.45 μm JH, MERCK). The mobile phase was supplied in isocratic mode. The flow rate was set at 1.0 mL/min. We employed an HPLC device (NEXERA XR, SHIMADZU CORPORATION) connected to a UV detector (SPD-20A, SHIMADZU CORPORATION). The detection wavelength was set at 216 nm. Samples were diluted with the mobile phase. Here, 50 μL of the samples was injected into the HPLC. The detection limit of this analysis is 0.02% vs. 5-ALAP area, and the quantification limit is 0.05% vs. 5-ALAP area.

5-ALAP samples were dissolved in the mobile phase at a concentration of 5 g/L and analyzed using the HPLC device. The 5-ALAP samples were also diluted 100-fold using the mobile phase and subsequently used for analysis. It was impossible to detect HPLC peaks of major and minor 5-ALAP impurities at the same dilution rate. Therefore, impurity peaks were quantified using prepared samples with a concentration of 5 g/L, and the 5-ALAP peak was quantified using 100-fold-diluted samples with a concentration of 0.05 g/L. Concentrations of the detected impurities in the 5-ALAP samples were quantified according to Equation (4), where A₄ is the detected area value of an impurity peak and D₄ is the detected area value of the 5-ALAP peak in the 100-fold-diluted samples. The linearity of this HPLC method is shown in Figure S2 of Supplementary Material S2.

$$\mathrm{Impurity} \mathrm{concentration} (\% \mathrm{vs}. 5\text{-}\mathrm{ALAP})={\displaystyle \frac{A_4}{{(D}_4\times 100)}}\times 100$$

(4)

2.7. HPLC Analysis of Hydrophilic Impurities

We investigated hydrophilic impurities in the obtained 5-ALAP crystals using an HPLC method with a HILIC column (Lichrospher 100 NH₂, 5 μm, 4.0 × 250 nm, SPELCO, Inc., Bellefonte, PA, USA), the temperature of which was stabilized at 30 °C. In total, 11.9 g of KH₂PO₄ was dissolved in 1.75 L of ultrapure water. The pH was adjusted to 4.0 by adding 85% H₃PO₄. A total of 3.25 L of CH₃CN was added to the KH₂PO₄ solution and used as a mobile phase, which was subsequently filtered using a membrane filter (Omnipore^TM Membrane Filters, 0.45 μm JH, MERCK). It was supplied in isocratic mode with a flow rate set to 0.7 mL/min. We employed an HPLC device (NEXERA XR, SHIMADZU CORPORATION) connected to a UV detector (SPD-20A, SHIMADZU CORPORATION). The detection wavelength was set at 216 nm. Then, 50 μL of the samples, diluted with the mobile phase, was injected into the HPLC. The detection limit of this analysis is 0.02% vs. 5-ALAP area, and the quantification limit is 0.05% vs. 5-ALAP area.

5-ALAP samples were dissolved in the mobile phase at a concentration of 2.5 g/L and analyzed using the HPLC device. The 5-ALAP samples were also diluted 200-fold using the mobile phase and subsequently used for analysis. It was impossible to detect HPLC peaks of major and minor 5-ALAP impurities at the same dilution rate. Therefore, impurity peaks were quantified by using prepared samples with a concentration of 2.5 g/L, and the 5-ALAP peak was quantified by using the 200-fold-diluted samples at a concentration of 0.05 g/L. Concentrations of the detected impurities in the 5-ALAP samples were quantified according to Equation (5), where A₅ is the detected area value of an impurity peak and D₅ is the detected area value of the 5-ALAP peak in the 200-fold-diluted samples. The linearity of this HPLC method is shown in Figure S3 of Supplementary Material S3.

$$\mathrm{Impurity} \mathrm{concentration} (\% \mathrm{vs}. 5\text{-}\mathrm{ALAP})={\displaystyle \frac{A_5}{{(D}_5\times 100)}}\times 100$$

(5)

2.8. Absorption Spectroscopy

The transmittance at 430 nm and 525 nm was measured to evaluate the color quality of the obtained 5-ALAP crystals. The former was used as an indicator of yellow coloration, since yellow matter absorbs visible blue light with a wavelength of 400–450 nm. The latter was used as an indicator of purple coloration. The obtained 5-ALAP was dissolved in ultrapure water at a concentration of 100 g/L. The 5-ALAP solution was filtered using a syringe filter (MillEX-LH, pore size of 0.45 μm, MERCK). A double-beam spectrophotometer (U-3900H, Hitachi, Ltd., Tokyo, Japan) was used to measure the absorbance of the 5-ALAP solution at 430 nm and 525 nm (Abs). The absorbance of ultrapure water without 5-ALAP was used as a reference sample for the double-beam measurement. Ultrapure water was used as a blank (Abs_BLANK). The transmittance was calculated according to Equation (6), where C is the concentration of 5-ALAP.

$$\mathrm{T}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{s}\mathrm{m}\mathrm{i}\mathrm{t}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\left(\mathrm{\%}\right)={10}^{-\left\{(Abs-{Abs}_{BLANK})/(C \times 100))\right\}}\times 100$$

(6)

2.9. Quantification of Residual EtOH in 5-ALAP Crystals

The concentration of residual EtOH in the obtained 5-ALAP was quantified using a gas chromatograph (GC, GC-2014, SHIMADZU CORPORATION) incorporating a flame ionization detector (FID). A separation column (DB-WAX, 30 m × 0.530 mm × 1.0 μm, Agilent, Santa Clara, CA, USA) was employed, with the column temperature stabilized at 35 °C. He was used as the carrier gas, flowing at a rate of 25.0 mL/min; 1.0 μL of the samples was injected into the GC.

EtOH was dissolved in MilliQ water with concentrations of 0.008, 0016, 0.040, 0.080, and 0.160 g/L and these were used as standard solutions for calibration.

2.10. Quantification of Residual Metal Elements

The concentrations of metal elements in the pure 5-ALAP crystals were quantified using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900, Agilent Technologies). In total, 0.2 g of 5-ALAP was dissolved in 1 M HNO₃ containing 1% HCl. The 5-ALAP solutions were filtered using a syringe filter (MillEX-LH, pore size of 0.45 μm, MERCK). A total of 0.1 mL of the filtered samples was diluted with 9.9 mL of 1 M HNO₃ containing 1% HCl.

A standard solution (XSTC-13B, SEISHIN, containing Na⁺ 10 mg/L) was diluted and used for calibration. A total of 0.5 mL of the standard solution was diluted with 1 M HNO₃ containing 1% HCl to 0.005, 0.01, 0.1, 0.25, and 0.5 mg/L. Standard solutions with 0 (1 M HNO₃ containing 1% HCl), 0.005, 0.01, 0.1, 0.25, and 0.5 mg/L of Na⁺ were used for calibration.

2.11. Ion-Exchange Resins

Three types of IERs were used in this study. A strong cation-exchange resin (SCER, DIAIONTM UBK04, Na⁺ type, Mitsubishi Chemical Corporation, Tokyo, Japan) was used to remove neutral impurities such as Gly. The recovery criteria of the UBK04 eluates are based on the results shown in Figure S4 of Supplementary Material S4. The UBK04 process requires NaOH as an eluent for 5-ALA (cationic molecule); therefore, a weak cation-exchange resin (WCER, DIAIONTM WK40L, H⁺ type, Mitsubishi Chemical Corporation) was used to remove contaminated Na⁺ ions from the UBK04 filtrate. The fermentation process is reportedly accompanied by pigment components [37,38]; therefore, a strong anion-exchange resin (SAER, DIAIONTM PA412, CH₃COO⁻ type, Mitsubishi Chemical Corporation) was used to remove pigment components. Additionally, combining these three IERs considerably affects the color of the 5-ALAP power, as shown in Figure S5 of Supplementary Material S5; therefore, a combination of the three IERs was integrated into the purification process.

2.12. Necessity of Nanoparticle Removal Using Activated Carbon

Figure S6 of Supplementary Material S6 shows that the permeation flux in the ultrafiltration process was drastically reduced with no nanoparticle removal using powdered activated carbon compared with nanoparticle removal, which negatively impacts the efficiency of the purification process. The US Food and Drug Administration (FDA) and other regulatory agencies provide clear guidance on maximum endotoxin levels for different types of therapies [39]. Therefore, ultrafiltration is an essential process to remove endotoxins from medicines. Based on this guidance and our results, carbon filtration of nanoparticles was deemed essential to improve permeation flux in the ultrafiltration process.

2.13. Recrystallization Process

The solubility of 5-ALAP was sufficiently reduced after the first addition of EtOH. A seeding crystal of 5-ALAP was added as a primary crystal nucleus. Finally, secondary nucleation was promoted by adding EtOH a second time. As shown in Supplementary Material S7, we optimized the recrystallization parameters to reduce the concentration of residual EtOH in the 5-ALAP. In the recrystallization process, a two-phase process of EtOH addition and seeding was carried out. We optimized several crystallization parameters for 5-ALAP, such as the first EtOH addition volume and the crystallization temperature, as shown in Figures S7–S9 of Supplementary Material S7.

2.14. Integrated Production of 5-ALAP

The detailed conditions of the developed purification process are described in Supplementary Material S8.

Corynebacterium glutamicum (C. glutamicum) KY10995/pAM7-122 was used for the fermentative production of 5-ALA [40].

The composition of the fermentation broth is shown in Table S8. 5-ALA was biologically produced by adding glucose monohydrate (90.0 g/L) and Gly (33.3 g/L) to the cell suspension as substrates. To inactivate C. glutamicum, the pH of the cell suspension was adjusted to 2.88 using H₂SO₄. Overall, we prepared 60.1 L of fermentation broth with a 5-ALA (as 5-ALAP) concentration of 45.4 g/L.

The dead cell bodies in the suspension were removed using a cross filter (Pall Corporation, Washington, USA; 0.1 μm; membrane area: 0.35 m²). In total, we obtained 150.05 L of cross-filter filtrate with a 5-ALA concentration of 17.3 g/L (as 5-ALAP).

Impurities were removed from the cross-filter filtrate using three IERs (UBK04, WK40L and PA412) in separated columns.

The pH of the PA412 filtrate was adjusted to 2.75 using 85.0% H₃PO₄. As a result, the 5-ALA in the filtrate was converted to 5-ALAP. Following treatment using three types of IER, we obtained a total of 59.86 L of the filtrate, with a 5-ALAP concentration of 37.0 g/L. The pH-adjusted solution was concentrated under 40 °C and 72 hPa using a rotary evaporator. In total, we obtained 3.52 L of crude crystallization mother liquor (cCML) with a 5-ALAP concentration of 627.7 g/L.

The temperature of the 5-ALAP crude concentrate was controlled at 17 °C in a crystallizer, and the concentrate was softly mixed during crystallization. A total of 0.6 v/v of 99.8% MeOH was introduced into the 5-ALAP concentrate at a speed of 0.8 v/v/h to sufficiently reduce the solubility of 5-ALAP. A seed crystal of 5-ALAP was added to the CML and incubated for 2 h below 20 °C, causing primary nucleation. Subsequently, 2.4 v/v of 99.8% MeOH was added to the crystallization mother liquor (CML) at a speed of 0.8 v/v/h and then reacted for 2 h at 17 ± 3 °C to cause secondary nucleation.

The 5-ALAP crude extract was recovered from the CML using a centrifuge (H-110F TOKU, KOKUSAN Co. Ltd., Saitama, Japan). Centrifugation was operated at 2687 rpm for 10 min. Then, 100 v/w% of 99.8% MeOH (versus 5-ALAP weight) was added, followed by washing for 1 min, and centrifugation was operated once again under the same conditions. Finally, the 5-ALAP wet crude extract was obtained.

The extract was dissolved in DI water, resulting in 2.25 L of a 5-ALAP dissolution with a 5-ALAP concentration of 441.3 g/L. Nanoparticles were removed from the 5-ALAP solution using powdered activated carbon (CT-SW50, FUTAMURA CHEMICAL Co., Ltd., Nagoya, Japan) at 1.0 wt% of the contained 5-ALAP weight. The activated carbon was removed via vacuum filtration with a cellulose filter aid (BC-200, J. RETTENMAIER & SÖHNE GMBH + CO KG, Rosenberg, Germany).

Metal impurities such as iron (Fe) in the nanoparticle-free liquor were removed using a chelate resin (Lewatit^® MonoPlus TP260, H⁺ type, LANXESS, Cologne, Germany).

To remove high-mass impurities (such as endotoxins) contaminating the DI water, the TP260 eluent passed an ultrafiltration module (Microza SIP-0013, Asahi Kasei Corporation, Tokyo, Japan). We obtained 7.50 L of the ultrafiltrate with a 5-ALAP concentration of 114.7 g/L.

The ultrafiltrate was concentrated under 40 °C, resulting in 1.50 L of a 5-ALAP pure CML (pCML) with a 562.7 g/L 5-ALAP concentration. After concentration, the 5-ALAP pCML was treated at 40 °C for 21.6 h.

The crystallization was carried out based on the parameters optimized in S5. The temperature of the pure 5-ALAP pCML was controlled at 15 °C in a crystallizer, and the pCML was softly mixed during crystallization. A total of 0.3 v/v of 99.5% EtOH was introduced into the 5-ALAP concentrate to sufficiently reduce the solubility of 5-ALAP at a speed of 0.7 v/v/h. 5-ALAP (IWATA CHEMICAL Co., Ltd., Shizuoka, Japan) was added to the pCML as a seed crystal and reacted for 10 min at 15 °C to cause primary nucleation. Subsequently, 1.7 v/v of 99.5% EtOH was added to the pCML at a speed of 0.7 v/v/h and then incubated for 2 h at 15 °C to cause secondary nucleation.

The pure 5-ALAP product was recovered from the pCML using a centrifuge (H-110F TOKU, KOKUSAN Co., Ltd.). Centrifugation was operated at 2687 rpm for 10 min. A total of 758.1 g of the 5-ALAP wet product was obtained, 40 g of which was sampled. Then, 100 v/w% of 99.5% EtOH (versus 5-ALAP weight) was added to the 5-ALAP crystal, followed by washing for 1 min. Subsequently, centrifugation was operated once again under the same condition. A total of 703.1 g of the pure 5-ALAP wet product was obtained, 40 g of which was sampled, and the remaining crystals were dried using a rotary evaporator at 53 hPa and 30 °C for 5 h. Finally, 658.0 g of the pure 5-ALAP dried product was obtained. The qualities of the obtained 5-ALAP dried product, such as impurity content, crystallinity, color, and residual poor solvent, were investigated using the methods explained in Section 2.1, Section 2.2, Section 2.3, Section 2.4, Section 2.5, Section 2.6, Section 2.7, Section 2.8 and Section 2.9.

3. Results

3.1. Concentration Behavior of 5-ALA in Purification Process

The concentrations of 5-ALAP and PY in each process liquor were quantified by using the HPLC methods described in Section 2.1 and Section 2.2. The chromatograms are shown in Figures S10–S12 of Supplementary S8–S10, and the results are summarized in

Table 1. Concentration behavior of 5-ALAP in crude crystallization process.

Process	Volume [L]	5-ALAP			PY
		[g/L]	[g]	Yield [%]	[g/L]	[g]	[% vs. 5-ALAP]
Fermentation broth (after inactivation)	60.07	45.43	2728.73	100.0	0.65	39.19	1.4364
Cross filter	150.05	17.30	2595.27	95.1	0.25	37.02	1.4265
PA412 after pH adjustment	59.86	37.02	2215.73	81.2	0.03	1.78	0.1010
Crystallization with mother liquor	3.52	627.73	2207.75	80.9	0.25	0.87	0.0393
Wet crude extract	-	-	1953.40	71.6	-	0.02	0.0010

and

Table 2. Concentration behavior of 5-ALAP in recrystallization process. N.D. stands for not detected. Values in blanks () are estimated numbers without experimental sampling.

Process	Volume [L]	5-ALAP			PY
		[g/L]	[g]	Yield [%]	[g/L]	[mg]	[ppm vs. 5-ALAP]
Dissolution	2.25	441.3	992.9	100.0	0.02	51	51
Nanoparticle removal	6.97	140.8	981.7	98.9	0.03	209	213
Chelate resin (TP260)	7.05	123.9	873.3	88.0	0.01	90	103
Ultrafiltration	7.50	114.7	860.1	86.6	0.01	80	93
Crystallization with mother liquor	1.50	562.7	844.1	85.0	0.19	292	346
Wet product before crystal washing	-	-	758.1	76.4 (80.4)	-	N.D.	N.D.
Wet product after crystal washing	-	-	703.1	70.8 (78.9)	-	N.D.	N.D.
Dried pure product	-	-	658.0 (738.0)	66.3 (74.3%)	-	N.D.	N.D.

.

Initially, 2728.7 g of 5-ALA was contained in the fermentation broth as 5-ALAP. Nearly 20% of 5-ALAP yield was lost through the three IERs in the crude process. In total, we obtained 1953.4 g of the 5-ALAP wet crude extract; therefore, the total yield from the crude process was 71.6%. A total of 39.2 g (1.4% vs. 5-ALAP) of PY was contained in the fermentation broth and drastically reduced through the three IERs. PY was reduced to 0.0010% (10 ppm) vs. 5-ALAP by the end of the crude process. The concentration behavior of PY in the crude crystallization process showed that PY was mainly removed by the IERs and the crystallization process.

In the recrystallization process, the 5-ALAP wet crude extract was initially dissolved in DI water, and approximately 44% of the 5-ALAP was lost by the end. However, the major point of loss of 5-ALAP was found to be during the crystallization process. PY was quantified at 51 ppm vs. 5-ALAP in the dissolution of the 5-ALAP wet crude extract. The concentration of PY increased to 213 ppm through the interaction between 5-ALAP and the activated carbon, and again reduced to half upon use of the chelate resin. It increased to 346 ppm vs. 5-ALAP during the concentration process due to heating. More than 300 ppm of PY was removed during the EtOH recrystallization process, and the remaining concentration was under the detection limit (<10 ppm). Therefore, the concentration behavior of PY in the recrystallization process showed that it was mainly removed as a result of crystallization.

We quantified the PY concentrations in the commercially available 5-ALAP powders; they are summarized in

Table 3. Concentrations of PY in obtained and commercially available 5-ALAP powders. N.D. stands for not detected.

5-ALAP Powders	PY (ppm vs. ALAP)
Obtained 5-ALAP crystal	N.D.
Manu A	N.D.
Manu B	20
Manu C	526

. Overall, we measured 20 and 526 ppm of PY in the commercially available 5-ALAP powders, with some remaining undetected. From these results, regarding the PY content, it can be deduced that the 5-ALAP obtained in this study is comparable or superior to the commercially available powders.

The Na⁺ concentrations in the obtained and commercially available 5-ALAP powders were quantified and are summarized in

Table 4. Concentrations of Na⁺ in obtained and commercially available 5-ALAP powders.

Sample	Na+ [ng/g]
Obtained 5-ALAP crystal	N.D.
Manu A	545
Manu B	N.D.
Manu C	794,000

. In the obtained 5-ALAP, Na⁺ was undetectable. On the other hand, the 5-ALAP manufactured by Manu A and Manu C contained Na⁺. From these results, it can be deduced that the obtained 5-ALAP powder is superior to the commercially available powder regarding residual Na⁺.

3.2. X-Ray Diffraction Spectroscopy

Figure 2 shows the XRD pattern of the obtained 5-ALAP dried product. Distinctive XRD peaks were detected and are summarized in

Table 5. XRD 2θ pattern.

2θ	Intensity
(°)	Counts
7.9	661
12.4	410
15.4	370
15.8	3266
18.6	637
19.0	2558
19.2	687
20.3	1097
20.6	250
20.8	3460
21.2	6184
21.4	3464
22.5	1411
22.7	2304
23.0	7757
23.9	902
24.3	907
25.3	482
25.8	793
26.6	466
27.0	1674
27.9	1758
28.1	376
30.1	501
30.2	1193
30.8	893
31.3	1689
31.6	170
31.7	168
32.0	1703
32.6	692
32.8	274
33.2	3833
33.5	144
34.4	298
34.8	2056
35.2	68
35.9	502
36.9	142
37.6	216
37.9	1563
38.2	1167
38.5	118
38.8	1385
38.9	771
39.2	988
39.6	560

. N. Tachiya et al. reported an XRD pattern of 5-ALAP [41] that is identical to ours. Therefore, it is clear that the obtained 5-ALAP is crystalline.

3.3. Hydrophobic Impurities

Figure 3 shows the chromatogram of the obtained 5-ALAP crystal analyzed using the method described in Section 2.6. A distinct peak of 5-ALAP was detected at an RT of 3.0 min. Several smaller peaks were detected from the obtained 5-ALAP crystal and are summarized in

Table 6. Impurities detected using method described in Section 2.6. N.D. stands for “not detected”.

RT [min]	Manu A	Manu B	Manu C	Obtained 5-ALAP Crystal
2.8	N.D.	N.D.	0.32	N.D.
3.5	0.88	0.21	0.05	0.02
4.2	0.07	N.D.	N.D.	N.D.
4.4	N.D.	N.D.	0.02	N.D.
4.5	0.23	0.05	0.02	0.02
4.7	N.D.	N.D.	0.04	N.D.
4.9	0.05	N.D.	0.06	N.D.
5.4	0.03	N.D.	0.06	N.D.
5.9	0.02	N.D.	0.03	N.D.
6.2	0.06	0.13	5.09	0.03
6.7	0.29	0.02	N.D.	N.D.
7.1	0.06	N.D.	N.D.	N.D.
7.4	0.03	0.02	0.64	0.02
8.0	N.D.	0.02	0.03	N.D.
8.2	0.04	N.D.	N.D.	N.D.
8.8	0.02	N.D.	0.03	N.D.
9.1	N.D.	N.D.	0.03	N.D.
9.6	0.02	N.D.	N.D.	N.D.
9.7	N.D.	0.02	N.D.	N.D.
10.6	N.D.	N.D.	0.03	N.D.
11.1	N.D.	N.D.	0.35	N.D.
11.9	N.D.	N.D.	0.02	N.D.
12.8	N.D.	N.D.	0.02	0.03
13	0.02	N.D.	N.D.	N.D.
13.6	N.D.	N.D.	0.03	N.D.
14.8	N.D.	N.D.	0.48	N.D.
15.5	N.D.	N.D.	0.02	N.D.
16.7	N.D.	0.02	0.7	N.D.
18.9	N.D.	N.D.	0.03	N.D.
20.2	N.D.	N.D.	0.06	N.D.
22.3	0.05	N.D.	N.D.	N.D.
24.4	N.D.	N.D.	0.04	N.D.
25.3	N.D.	N.D.	0.03	N.D.
27.5	0.03	0.02	0.48	N.D.
29.2	N.D.	N.D.	0.02	N.D.
30	N.D.	N.D.	0.05	N.D.
38.4	N.D.	N.D.	0.06	N.D.
38.7	0.06	N.D.	N.D.	N.D.
Sum	1.75	0.39	8.40	0.00

. Common peaks between 5-ALAP samples and the mobile phase (blank) were rejected. Several impurity peaks higher than the detection limit (0.02% vs. 5-ALAP area) were detected from the obtained 5-ALAP crystal, yet none higher than the quantification limit (0.05% vs. 5-ALAP area) were observed. Therefore, the ratio of the total area value of the unidentified peaks to that of 5-ALAP was 0.00%. Impurities with higher concentrations were detected in the commercially available 5-ALAP, with those from Manu A, Manu B, and Manu C quantified as 1.96%, 0.51%, and 8.84%, respectively. From these results, it can be deduced that the obtained 5-ALAP crystal contains considerably fewer and lower concentrations of hydrophobic impurities compared with the commercially available 5-ALAP crystals.

3.4. Hydrophilic Impurities

Figure 4 shows the chromatogram of the obtained 5-ALAP crystal analyzed using the method described in Section 2.7. A distinct peak of 5-ALAP was detected at an RT of 15.7 min. Several smaller peaks were detected from the obtained 5-ALAP crystal and are summarized in

Table 7. Impurities detected using method described in Section 2.7. N.D. stands for “not detected”.

RT [min]	Manu A	Manu B	Manu C	Obtained 5-ALAP Crystal
5.7	0.05	N.D.	0.08	N.D.
5.9	N.D.	N.D.	0.1	N.D.
6.1	0.14	N.D.	N.D.	N.D.
6.2	N.D.	N.D.	0.03	0.03
6.5	0.07	0.02	N.D.	N.D.
6.6	N.D.	N.D.	0.33	N.D.
6.7	0.34	0.1	N.D.	N.D.
7.4	N.D.	N.D.	0.03	N.D.
7.6	N.D.	N.D.	0.02	N.D.
7.8	0.04	0.08	2.73	0.03
8.2	0.04	N.D.	N.D.	N.D.
9.4	N.D.	N.D.	0.04	N.D.
10.6	0.03	0.02	0.04	N.D.
10.7	0.03	N.D.	N.D.	N.D.
11.6	0.2	N.D.	N.D.	N.D.
18.8	N.D.	N.D.	0.02	N.D.
19.4	0.02	N.D.	N.D.	N.D.
19.9	0.02	N.D.	N.D.	N.D.
20.7	N.D.	N.D.	N.D.	0.02
21.5	N.D.	N.D.	0.02	N.D.
21.9	0.06	0.03	0.32	0.04
24.9	0.2	0.03	N.D.	N.D.
29.8	N.D.	N.D.	0.03	N.D.
36.3	N.D.	N.D.	0.02	N.D.
40.2	0.02	N.D.	N.D.	N.D.
44.4	0.08	0.03	0.02	N.D.
Sum	1.14	0.18	3.56	0.00

. Common peaks between the 5-ALAP samples and mobile phase (blank) were rejected. Several impurity peaks higher than the detection limit (0.02% vs. 5-ALAP area) were detected from the obtained 5-ALAP crystal, but none higher than the quantification limit (0.05% vs. 5-ALAP area) were observed. Therefore the ratio of the total area value of the unidentified peaks to that of 5-ALAP was 0.00%. On the other hand, impurities with higher concentrations were detected from the commercially available 5-ALAP powders, with those from Manu A, Manu B, and Manu C quantified as 1.34%, 0.31%, and 3.83%, respectively. From these results, it can be deduced that the obtained 5-ALAP crystal contains considerably fewer and lower concentrations of hydrophilic impurities compared with the commercially available 5-ALAP crystals.

3.5. Color Quality

Figure 5 compares the photographs of the obtained 5-ALAP crystal and the commercially available 5-ALAP powders. The obtained 5-ALAP crystals are individual and white. The 5-ALAP powders manufactured from Manu A and B are also white. A yellowish color is visible on the 5-ALAP powder obtained from Manu C.

Table 8. Transmittances at 430 and 525 nm.

Sample	430 nm [%]	525 nm [%]
Obtained 5-ALAP crystal	99.8	100.0
Manu A	98.1	99.6
Manu B	98.6	99.6
Manu C	82.4	93.9

summarizes the transmittance of the obtained 5-ALAP crystal at 430 nm and 525 nm. The former was measured as 99.8%. Almost no color is visible on the obtained 5-ALAP crystal. The same values for the powders from Manu A, Manu B, and Manu C were 98.1%, 98.6%, and 82.4%, respectively. The transmittance of the obtained 5-ALAP crystal at 525 nm was measured to be 100.0%. The same values for the powders from Manu A, Manu B, and Manu C were calculated as 99.6%, 99.6% and 93.6%, respectively. From these results, it can be deduced that the transmittance of the obtained 5-ALAP crystal is superior to that of the commercially available 5-ALAP powders.

3.6. Residual EtOH

Figure 6 shows the chromatogram of the GC analysis. EtOH and MeOH were detected at an RT of 1.3 and 1.7 min, respectively, in the EtOH and MeOH std, with a concentration of 8 mg/L.

Table 9. Residual EtOH and MeOH. N.D. stands for “not detected”.

Sample	Residual EtOH [ppm]	Residual MeOH [ppm]
Obtained 5-ALAP crystal	498	N.D.
Manu A	3357	N.D.
Manu B	52	N.D.
Manu C	1706	N.D.

shows the quantified concentrations of EtOH and MeOH. From the dried 5-ALAP crystal obtained, 498 ppm of residual EtOH was detected. Contrastingly, we measured 1706 and 3357 ppm of residual EtOH in the 5-ALAP powders from Manu A and Manu C. Only 52 ppm of residual EtOH was detected in the powder from Manu B. From these results, it can be deduced that under the set recrystallization conditions, residual EtOH was lower than in the powders from Manu A and Manu C (Figures S7–S9 of Supplementary Materials S7).

4. Discussion

Various studies have reported on the biological synthesis and production of 5-ALAP [29,30,31,32,33,40], yet those focused on purifying 5-ALA manufactured via the fermentation process are extremely limited. The purification process developed in this study can be used as an example. Various conditions were set to achieve superior quality compared with the commercially available 5-ALAP.

PY from the fermentation process was sufficiently removed by the IERs and the crystallization process. As shown in Table 2, the PY concentration increased upon nanoparticle removal using activated carbon, as well as during the concentration process of recrystallization. A. Bunke et al. reported on a PY generation mechanism involving dehydration, condensation and oxidation between two 5-ALA molecules [28]. It has also been reported that activated carbons contain metal impurities such as Fe [42]. The generation kinetics of PY depend on the collision frequency of 5-ALA molecules. Scholars have speculated whether the generation kinetics of PY increase due to local concentration buildup in porous structures of activated carbon. Moreover, the oxidation of 5-ALA can be accelerated via catalysis of metal impurities in activated carbon. In our study, the volume of the 5-ALAP solution was concentrated from 7.5 to 1.53 L, an approximately 4.9-fold concentration. The PY concentration increased from 93 to 346 ppm at the concentration stage of recrystallization, an approximately 3.7-fold compaction, as shown in Table 2. Although the increased magnitude of the PY concentration did not perfectly align with the reduction in the 5-ALAP solution, the effect of heating at 40 °C on the increase in the PY concentration during the concentration process was considered negligible. Therefore, we deemed the volume reduction in the 5-ALAP solution as the main cause of PY’s concentration.

As shown in Table 2, PY was removed to a notable degree during the crystallization process, seemingly resulting from an ordered arrangement of target molecules—the definition of crystallization. As a general understanding, impurities are largely eliminated as they fail to fit into the regular lattice of the growing crystals during crystallization.

Fermentation processes result in the generation of impurities, byproducts, and target molecules. Three different types of IERs (SCER, WCER, and SAER) were combined to sufficiently remove impurities. Gly was used as a broth substrate for 5-ALA fermentation and was mainly removed via an SCER (UBK04). UBK04 required NaOH as the eluent, and WK40L was used to remove Na⁺ from the process liquor. The fermentation process is reportedly accompanied by pigment components [37,38], which we utilized an SAER (PA412) to remove. A combination of these three IERs resulted in considerably fewer impurities when compared with the commercially available 5-ALAP.

This combination was also effective in ensuring the color quality of 5-ALAP. As shown in Figure S5 of Supplementary Material S5, the color of the recrystallized 5-ALAP was considerably improved as a result of the combination of the SCER, WCER, and SAER. G. D. Venosa et al. reported a 90% recovery of 5-ALA following purification using Dowex 50Wx8 [34]. As shown in Table 1, the 5-ALA yield after using the cross filter was 95%, and that following PA412 after pH adjustment was reduced to 81.2%, an approximately 15% loss. Overall, the combination of the three IERs negligibly affected the 5-ALA yield.

Residual EtOH in the 5-ALAP crystal was reduced under the set conditions of the recrystallization parameters. As shown in Figures S7–S9 of Supplementary Material S7, the volume of the first EtOH addition, the crystallization temperature, and the speed of the second EtOH addition were carefully set to suppress residual EtOH. We detected 498 ppm of residual EtOH in the obtained 5-ALAP crystal and 52, 1706, and 3357 ppm in the powders manufactured by Manu A, Manu B, and Manu C, respectively. Upon comparison, the obtained 5-ALAP crystal sits in the middle of this range. From these results, it can be ascertained that the purification process developed in this study can produce 5-ALAP with a commercially acceptable level of residual EtOH.

Lin et al. [36] reported gradual-cooling crystallization of 5-ALAP on a large scale. Lin’s crystallization required only stirring and cooling, not the addition of a poor solvent, making it another available option. However, the crystallization in this study was accurately controlled by cooling at 15 °C, as well as through the addition of EtOH and the seed crystal. These technical ingenuities achieved a high reproducibility of crystallization, not only on the lab scale but also in ton-scale manufacturing, as shown in Table S9 Supplementary Material S11. In ton-scale manufacturing, mixing and cooling can be considerably harder to achieve than in the lab. Insufficient stirring and temperature control might result in crystallization failure. Therefore, the addition of EtOH and the seed crystal were integrated into the crystallization process in our study.

We obtained 658.0 g of 5-ALAP pure dried crystal as a final product. Additionally, 40 g of a wet product was sampled before and after crystal washing, causing 80 g of experimental loss. The weight difference between the wet product after washing (703.1 g) and the pure crystal (658.0 g) was approximately 40 g; thus, the weight of the remaining EtOH on the wet 5-ALAP was negligible. From these data, the true total yield of this study can be estimated as 74.3%, according to Equation (7), where the denominator—992.9 g—is the initial amount of 5-ALAP in grams, as shown in Table 2.

$$\mathrm{True} \mathrm{total} \mathrm{yield} \left(\%\right)={\displaystyle \frac{(658.0+40+40)}{992.9}}=74.3\%$$

(7)

The crystallization yield was calculated by comparing the weight of 5-ALAP before and after crystallization. Since the volume of pCML was 1.50 L, containing 562.7 g/L of 5-ALAP, the weight of 5-ALAP before crystallization was calculated to be approximately 844.1 g. The estimated true weight of pure 5-ALAP was 658.0 + 40 = 738.0 g; therefore, the crystallization yield was calculated to be 87.4%. This value can be improved by increasing the amount of EtOH used in the crystallization. However, an excessive increase in EtOH addition could result in excessive accumulation of residual EtOH in the 5-ALAP crystal ≥ 1000 ppm. Therefore, a balance must be struck between optimizing the crystallization yield and considering possible residual EtOH in 5-ALAP.

We were unable to find other articles declaring a total yield of 5-ALAP following purification online, but the total purified yields of other amino acids were considered comparable with this study. L. Bonomi et al. reported total yields of 72% following tryptophan purification [43]. A tryptophan solution was treated using activated carbon, and crystallization was carried out through the addition of NH₄OH. Z. Liu et al. reported total yields of 85.9% following methionine purification [44]. Methionine was fermentatively produced, and impurities were removed using an IER. Consequently, the methionine solution was concentrated, and crystallization was carried out at 0 °C. Compared with these reports, the total yield achieved in this study (73.4%) lies in the middle of previously reported values and is thus concluded to be acceptably high.

We have confidence in the high generalizability of the developed purification process. The salt formation of 5-ALA was carried out after the impurity removal process using the three IERs by simply adding a target pair salt (H₃PO₄). Therefore, other types of 5-ALA salts, such as 5-ALAH, could be produced by adding hydrochloric acid instead of phosphoric acid without significant modification of the purification process. If the compositions of the fermentation broth are significantly different from those described in our study, modifications to the process may be required; otherwise, the developed purification process can be applied to different 5-ALA fermentation broths.

The developed purification process, described in Section 2.14 and hereafter referred to as lab-scale purification, was applied to the ton scale, as shown in Table S9 Supplementary Material S11. In total, 1563 kg of 5-ALAP crystals was obtained by the method described therein. Remarkably, the total yield and qualities of the 5-ALAP crystals obtained on both the lab and ton scales were almost identical. These results indicate the high scalability of the purification method developed in this study. Therefore, this article provides knowledge on a purification process for fermentatively produced 5-ALA that is applicable to industrial manufacturing.

5. Conclusions

To disclose knowledge essential for fermentative production of 5-ALA, an integrated purification process for 5-ALAP produced via biological fermentation was developed, producing highly pure 5-ALAP crystals compared with commercially available 5-ALAP. The importance of combining three different types of IERs (SCER, WCER, and SAER) was highlighted by the fact that their combination sufficiently removes impurities such as substrates and pigments from the 5-ALA solution. Comprehensive condition setting for crystallization was effective in reducing the amount of residual poor solvent in the 5-ALAP crystals. The quality of the obtained 5-ALAP crystals surpassed that of the commercially available 5-ALAP powders regarding impurities, residual poor solvent, and pigments. PY hardly remained in the obtained 5-ALAP crystals compared to commercially available 5-ALAP powders.

5-ALAP has reported benefits to human health. As an efficient production method, biological fermentation can be used to mass-produce 5-ALA. The applicability of the developed purification process to ton-scale manufacturing was confirmed; therefore, the purification process developed in this study can be used to effectively remove various impurities from the 5-ALA fermentation broth.