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Membrane Science and Technology, Vol. 19, No. 3, 1999, pp. 8-12
Lou Fule, Mao Weigang, Lu Xiaofeng, Liang Guoming
(Shanghai Institute of Nuclear Science, Chinese Academy of Sciences, Shanghai 201800)
Lu Wenda, Huang Meiju
(Shanghai Fuda Pharmaceutical Co., Ltd., Shanghai 201400)
Abstract: This paper introduces the nature of pyrogens, their quantitative representation, and determination methods. With the rapid development of membrane separation technology, ultrafiltration for removing pyrogens from injectable solutions has been practically applied in the pharmaceutical industry. Several international application examples are listed, and the use of spiral wound ultrafiltration devices produced by the Shanghai Institute of Nuclear Science, Chinese Academy of Sciences, in the process of removing pyrogens from pharmaceutical solutions is introduced.
Keywords: Pyrogens, Ultrafiltration, Spiral wound ultrafiltration device
Classification Number: TQ028.8
With the rapid development of membrane separation technology, the application of ultrafiltration membrane separation technology in the pharmaceutical industry to remove (or reduce) the pyrogen content in injectable drugs (liquids) to meet pharmacopoeia requirements is becoming increasingly widespread. For example, the Japanese and American Pharmacopeias allow the use of reverse osmosis and ultrafiltration units for the removal of pyrogens from large-volume parenteral solutions. In China, methods and processes for removing pyrogens using domestically produced ultrafiltration equipment are being explored and sought. The Shanghai Institute of Nuclear Research of the Chinese Academy of Sciences has developed and produced ultrafiltration membranes of various materials and specifications, as well as plate and frame and spiral wound ultrafiltration equipment, which have been applied in many fields. In order to promote the use of pyrogens removal projects in the pharmaceutical industry, the author conducted application experiments based on the research of relevant literature and achieved success, thereby promoting the application and development of the process of removing pyrogens from drug solutions by ultrafiltration.
The nature of pyrogens
M.Thmas et al. [1] pointed out that pyrogens (Pyrogen), also known as endotoxins, are produced on the outer wall of Gram-negative bacteria, that is, fragments of bacterial corpses. It is a lipopolysaccharide (LPS), with a relative molecular mass ranging from several thousand to hundreds of thousands, depending on the type of bacteria that produces it. In aqueous solution, its relative molecular mass can range from hundreds of thousands to millions. It has recently been revealed that lipid A (Lipid) is also a pyrogen, which constitutes an endotoxin that is harmful to the human body, with a relative molecular mass of about 2000.
Yagi et al. pointed out [2] that there are two types of pyrogens: one is low relative molecular mass pyrogen (Pyretice), and the other is high relative molecular mass pyrogen (Pyrogen), which are collectively referred to as endotoxins. It is generally believed that pyrogens refer to bacterial pyrogen, which is composed of the outer membrane of the cell wall of Gram-negative bacteria. Further analysis shows that its main components are lipopolysaccharide (LPS) and lipid A, which are the active parts of pyrogens. Their relative molecular mass is generally 10,000 to 25,000, and they form an association in aqueous solution with a relative molecular mass of 500,000 to 1,000,000. These substances are heat-resistant and chemically stable, making them difficult to eliminate.
Table 1 LPS heating deactivation time
Heating temperature ℃ | LPS inactivation time/min |
250 | 30 and above |
200 | 60 and above |
180 | 120 and above |
Quantitative Expression of Pyrogens
Pyrogens can be quantitatively scaled using concentration. Most literature uses grams per milliliter as the concentration unit, generally ng/ml (10⁻⁹ g/ml) or pg/ml (10⁻¹² g/ml). EU/ml is also used. The 1998 Chinese Pharmacopoeia adopted EU/ml as the pyrogen unit. The conversion between the two units is complex because the pyrogen toxicity of different bacterial species is not entirely consistent. US authorities, using probabilistic statistical methods on a large amount of sample data, have proposed the following results: for EC2 bacteria, 1 ng/ml = 5 EU/ml; for EKT bacteria, 1 ng/ml = 10 EU/ml. Japanese drug inspection agencies report that for EKT bacteria, 1 ng/ml = 8 EU/ml.
Methods for Pyrogen Determination
The main analytical and determination methods for pyrogens are the horseshoe crab reagent method and the rabbit method.
Limulus Amebocyte Lysate (LAL) Reagent Gel Method This method is a method of detecting the concentration of bacterial endotoxins that may exist in or on the surface of a test sample using LAL reagent [3]. Currently, the bacterial endotoxin test methods included in the pharmacopoeias of various countries all include the gel method. The bacterial endotoxin test of pharmaceuticals is based on the gel method. After the test proves that a certain concentration of the test sample does not interfere with the gel method, the test is conducted within the effective concentration range of the test sample according to the endotoxin limit of the test sample [4]. 1.3.2 Rabbit Method This method is the standard test method specified in the pharmacopoeias of various countries. It can be briefly described as follows: take 3 healthy rabbits and inject each of them with a specified amount of sample. If the body temperature of a single rabbit does not rise by more than 0.6℃ in one day and the total temperature rise of the 3 rabbits does not exceed 1.4℃, the pyrogen content in the test is considered to be qualified. 1.4 Harm of Pyrogens to Humans According to the literature [2], when a sample with a pyrogen concentration of 5 ng/ml is injected into a rabbit, and the total amount reaches 50 ng/kg (body weight), the body temperature of the rabbit rises by 0.6℃. Humans are three times more sensitive to fever than rabbits. Even trace amounts of pyrogens mixed into medications and injected into the human bloodstream can cause severe fever and even death. Therefore, minimizing the pyrogen content in medications is crucial, especially when using large volumes of injectable solutions (such as large-volume parenteral solutions), where the concentration requirements for pyrogens must be even stricter. For example, Shanghai Changzheng Pharmaceutical Factory controls the pyrogen content of large-volume parenteral solutions to 0.25 EU/ml.
Ultrafiltration Membrane Separation for Pyrogen Removal from Medications Removing pyrogens from injectable medications (or water for injection) to meet pharmacopoeia testing requirements is a fundamental production step in the pharmaceutical industry. Currently, the methods for pyrogen removal are generally categorized into three types:
Distillation to produce depyrogenated water for use as water for injection, washing water, etc., but this method is costly.
Adsorption to remove pyrogens. One method involves a surface adsorbent adsorbing the pyrogenic substance, allowing the product to pass through. Another method involves the adsorbent adsorbing the product, allowing the pyrogens to flow out, and then recovering the product from the adsorbent surface. The adsorbents can be silica mud, activated carbon, and ion exchange resins. Asbestos is prohibited from being used as an adsorbent.
Membrane separation for pyrogen removal is a new process and technology that is being promoted and applied in the pharmaceutical industry.
Ultrafiltration for Pyrogen Removal
Ultrafiltration is a physical separation method. The appropriate ultrafiltration membrane depends on the relative molecular mass, properties, and concentration of the pyrogens in the solution.
Reference [1] suggests that because the terminal structure of lipopolysaccharide, lipid A, has a relatively small relative molecular mass, an ultrafiltration membrane with a molecular weight cutoff of less than 5000 should be selected. If an ultrafiltration membrane with a molecular weight cutoff of 10,000 to 200,000 is used to remove pyrogens with a molecular weight cutoff of tens of thousands to millions, then a pyrogen adsorbent should be used to remove pyrogens with a molecular weight cutoff of approximately 2000. The average pore size of these different filter membranes ranges from 2 nm to 0.1 μm. Inami Yoshiaki et al. reported [5] that when using ultrafiltration to remove pyrogens, an ultrafiltration membrane with a pore size of 1 nm and a molecular weight cutoff of about 6000 must be used. However, when the yield is low and the processing volume is large, the equipment is large and the pressure requirement is also high. Secondly, using an ultrafiltration membrane with a molecular weight cutoff of 5000 or 10000 to remove pyrogens is not suitable for some drug solutions containing components with a large molecular weight. Because, while removing pyrogens, the effective components in the drug solution will be blocked and adsorbed, which will greatly affect the product yield. Therefore, some filter membranes with a molecular weight cutoff of 100,000 to 300,000 are used to remove most of the pyrogenic substances (the product substances basically pass through the filter membrane), and then a pyrogen adsorbent is used to remove the remaining pyrogens, so that the product yield is high and the pyrogen removal effect is good. To increase production, a microporous filter membrane made of polyamide (nylon) was used to filter tap water with a pyrogen concentration of 20 ng/ml. Due to the material's unique pyrogen adsorption properties, the filtered water quality met the pharmacopoeia requirements, and its production reached 2000 L/(m2·h). K. Mueeller reported [6] that the molecular weight cutoff of the ultrafiltration membrane for pyrogen removal was 6000. In his patent [2], Nagata Hiko stated that for general injection solutions, an ultrafiltration membrane with a molecular weight cutoff of 10,000 is used to remove low molecular weight pyrogenic substances. Tahara Osamu et al. [7] used an ultrafiltration membrane with a molecular weight cutoff of 10,000 and a flat-plate device to remove pyrogens from sodium lactate. Hashimoto Masafumi [8] pointed out that a filter membrane with a molecular weight cutoff of 10,000 is generally used to remove pyrogens from low molecular weight drug solutions, including pyrogen removal from intravenous injection solutions. Solin reported [9] that charged microporous membranes made of nylon 66 with pore sizes of 0.1, 0.2 μm, and 0.45 μm can also be used to remove pyrogens from dextran solutions with a relative molecular mass of 40,000. The 0.45 μm microporous membrane was used for pretreatment, while the 0.1 μm and 0.2 μm microporous membranes underwent further pyrogen removal treatment.
In summary, the selection of ultrafiltration membrane pore size and material depends on the relative molecular mass and characteristics of the drug being treated, as well as the pyrogen content in the drug. The most suitable ultrafiltration membrane specifications and treatment process should be selected through process experiments. 2.2 Efficiency of Ultrafiltration for Pyrogen Removal
Because the active layer of the ultrafiltration membrane is very thin, pinholes may appear after prolonged use, reducing the rejection rate and causing leakage of pyrogens and bacteria. Furthermore, unreasonable equipment structure may create unsanitary areas, affecting the quality of the filtrate. If pyrogens such as lipid A are also present, the pyrogen removal efficiency of ultrafiltration often does not reach 100%. Table 2 summarizes relevant data reported in the literature.
Table 2: Pyrogen Removal Efficiency of Ultrafiltration
Project | Pyrogen concentration / (ng·ml⁻¹) Original solution filtrate | Removal rate /% | Filter membrane specifications Membrane pore size / μm | Relative molecular mass cutoff / 10,000 | |
SOD solution removes pyrogens | Approximately 10,000 | 1-10 | 99.9%～99.99% | — | 10 |
HAS Refined | 1300 | 3 | 99.8 | — | 8 |
Sodium lactate removes pyrogens | __ | __ | >97 | — | 1 |
Preparation of ultrapure water | 7.1 | <0.1 | >98.6 | 0.01 | — |
Tap water pyrogen removal | 20 | qualified | ~90 | 0.2 | — |
Medicine | 500 | — | >97 | 0.1 | — |
As shown in Table 2, the ultrafiltration membrane has a high pyrogen removal rate with a wide range, ranging from as high as 99.99% to as low as over 90%. The reason for this is that pyrogens are substances with uncertain forms and relative molecular masses. The type and concentration of pyrogens vary depending on the drug solution, therefore the pyrogen removal rate is determined by multiple factors.
SOD Drug Solution Pyrogen Removal [2] Using a two-stage treatment process, ultrafiltration + adsorption, the recovery rate, yield, repeatability, reliability, and economy of the SOD drug solution are excellent.
The filter membrane has a relative molecular mass cutoff of 100,000, and a cutoff rate of 60-80% for bovine serum albumin (relative molecular mass 67,000). The secondary pyrogen removal adsorbent was determined after screening, and appropriate adjustments to the chemical composition of the drug solution are beneficial to improving the yield. The ultrafiltration membrane is regenerated by soaking in 1 mol/L NaOH, and the adsorbent regeneration involves three steps. The SOD solution tested negative using the rabbit method. The results are listed in Table 3.
Table 3: Removal of pyrogens from SOD solution by ultrafiltration and adsorption methods
Methods for removing pyrogenic substances | Pyrogen concentration / (ng·ml)⁻¹ Before processing After processing | SOD recovery rate / % | |
Membrane filtration + pyrogen adsorbent treatment | Approximately 10,000 | 0.01 or less | 96 |
Membrane filtration | Approximately 10,000 | 1-10 | 98 |
Adsorption method (comparative example) | Approximately 10,000 | 10～100 | — |
Pyrogen Removal from Prourikinase, a New Drug for Treating Cerebral Thrombosis [7] This drug is a highly effective treatment for cerebral thrombosis. It is administered by injection. The relative molecular mass of the protein in the drug is about 50,000. Therefore, an ultrafiltration membrane with a relative molecular mass cutoff of 300,000 was developed for primary ultrafiltration treatment. The secondary treatment also uses a pyrogen removal adsorbent. The main results after treatment are shown in Table 4.
Table 4 Pyrogen Removal from Prourikinase Solution by Ultrafiltration and Adsorption Methods
Methods for removing pyrogenic substances | Endotoxin concentration/(ng·ml)⁻¹ Before processing After processing | Drug component yield /% | |
Membrane filtration + pyrogen adsorbent treatment | 0.1 to 10,000 | 0.01 | >90 |
Membrane filtration | 0.1 to 10,000 | 1-10 | 98 |
Depyrogen adsorption column (comparative example) | 0.1 to 10,000 | 10～100 | — |
Sodium lactate for pyrogen removal [6] Because sodium lactate with a mass fraction of 70% has high viscosity, the flux is low when using a filter membrane with a molecular weight cutoff of 10,000. In order to increase the yield, the equipment and pipeline jacket heating method is adopted to raise the temperature of the liquid to 50-60℃. This measure significantly improves the processing yield. As shown in Figure 1, the filtration speed at 58℃ is 2.5 times that at 40℃, while the pyrogen removal rate remains above 97%. After the temperature exceeds 60℃, the membrane pore size increases and the membrane retention rate decreases, resulting in a significant decrease in the pyrogen removal effect. Therefore, the ultrafiltration pyrogen removal platform has a test process with specific process conditions for different liquids.
Preparation of Ultrapure Water [14] A miniaturized ultrapure water preparation device is constructed using a three-stage treatment process of ultrafiltration module + ion exchange + ion exchange fiber, which meets the requirements of departments such as electronics, pharmaceuticals, and precision chemical analysis. Among them, the ion exchange fiber is a patented achievement. The separation pore size of the ultrafiltration membrane is 10nm, the inner diameter is 250μm, the membrane thickness is 30μm, the filtration area is 1.6m2, the filling is 1.8L of mixed resin MB-2 type, 0.2L of fiber mixture, the flow rate is 50L/h, the device volume is 45cm×45cm×25cm, the mass is 20kg, and 600L of pure water can be produced at one time. The water quality indicators are detailed in Table 5.
Table 5 Overview of water quality for ultrapure water preparation
project | resistivity /(MΩ·cm) | sodium /(ng·g-1) | Silicon dioxide / (ng·g⁻¹) | Bacteria/(cells·ml⁻¹) | Pyrogen/(ng·ml⁻¹) | >0.2μm particles/(particles·ml⁻¹) | Organic matter / (ng·g⁻¹) |
raw water | 0.01 | >600 | 980 | 0.6 | 7.1 | 1×10⁵ | 900 |
Ultrapure water | 18 | 1 | <5 | 0.1 | <0.1 | 15 | 70 |
Ultrafiltration + Adsorption Process
M. Thomas[1] established a new process for removing pyrogens using "ultrafiltration + adsorption" after extensive experimentation. The ultrafiltration membrane used in this process has a molecular weight cutoff of 10,000 to 200,000 and an average pore size of 0.002 to 0.1 μm, typically 3 to 20 nm. This membrane has a large porosity and high flux, and can remove pyrogens with a molecular weight of tens of thousands to millions. Then, a pyrogen-removing adsorbent is used to remove pyrogens with a molecular weight of less than tens of thousands and lipid A substances with a molecular weight of approximately 2,000. After the ultrafiltration membrane has been used for a long time and the retention rate has decreased, pyrogens can also be removed by the adsorbent, and lipid A substances with a molecular weight of 2,000 can also be effectively removed. This process allows the active ingredients of medicines to easily permeate through the membrane, thereby improving the yield of medicines.
The ultrafiltration membrane materials used in this process include polysulfone, polyacrylonitrile, and polyamide, and the equipment can be in the form of hollow fiber, spiral wound, pleated, or plate and frame filters. Each of these different types of equipment has its own characteristics. Spiral wound ultrafiltration units have a large membrane area and are easy to replace, but require more stringent pretreatment of the feed solution. Hollow fiber ultrafiltration units have high membrane packing density and small pump capacity, but cannot replace individual membranes and also require more stringent pretreatment of the feed solution. Plate and frame ultrafiltration units offer easy feed solution pretreatment and allow for individual membrane replacement, but the equipment itself is relatively large, and pre-cleaning can be challenging.
This process utilizes adsorbents of various materials and types. The concentration of pyrogens in the stock solution should ideally be below 104 ng/ml.
Applications of this process include the preparation of enzyme preparations, the synthesis of pharmaceuticals containing proteins, peptides, hormones, and polysaccharides, and large-volume parenteral solutions such as dextran infusions, fructose infusions, and glucose infusions. This process can also be used in the production of sodium citrate injection.
In an application example, the purification of human serum albumin (HSA) involves first filtering with a hollow fiber ultrafiltration membrane module with a molecular weight cutoff of 100,000, followed by passing the sample through an adsorption column packed with adsorbent. The HAS mass fraction decreased from 5% to 3.2% or 3%, and the pyrogen content decreased from 1300 ng/ml to 3 ng/ml and 0.016 ng/ml, respectively. The product recovery rate was 60%, the ultrafiltration pyrogen removal rate was 99.8%, and the overall process efficiency was 99.998%.
Pyrogen removal test of streptomycin sulfate (Shuangqing)
Shanghai Fourth Pharmaceutical Co., Ltd. used a spiral wound ultrafiltration device with a membrane column having a molecular weight cutoff of 20,000 to conduct a pyrogen removal test on streptomycin sulfate (Shuangqing). The pyrogen removal effect is shown in Table 6.
Table 6 Results of pyrogen testing using the rabbit method
Test Serial Number | Rabbit temperature rise/℃ Before treatment After treatment Ⅰ Ⅱ Ⅲ Ⅰ Ⅱ Ⅲ | medicine Recovery rate /% | |||||
1 | 1.00 | 1.05 | 0.70 | 0.35 | 0.30 | 0.25 | 94.0 |
2 | 0.60 | 1.75 | 0.95 | 0.40 | 0.25 | 0.15 | 92.3 |
3 | 0.80 | 0.60 | 1.30 | 0.40 | 0.25 | 0.20 | 97.1 |
4 | 1.20 | 1.30 | 0.80 | 0.25 | 0.25 | 0.20 | 94.9 |
The experimental results show that it is feasible to use ultrafiltration to replace the traditional activated carbon adsorption of pyrogens for the production of streptomycin sulfate.
Ultrafiltration for pyrogen removal in Astragalus injection, a traditional Chinese medicine preparation
Shanghai Fuda Pharmaceutical Co., Ltd.'s HPL-type plate and frame ultrafiltration unit, equipped with a PES membrane with a molecular weight cutoff of 10,000, is used for pyrogen removal ultrafiltration of Astragalus injection, a traditional Chinese medicine preparation. Following adsorption with an appropriate amount of activated carbon, the pyrogen compliance rate of the finished product (whether measured by the Limulus amebocyte lysate (LAL) gel electrophoresis or rabbit assay) has improved from fluctuating levels to 100%. Because the effective components of traditional Chinese medicine solutions, such as flavonoids, alkaloids, and total glycosides, have molecular weights below 1000, ultrafiltration is the most suitable method for pyrogen removal and sterilization in traditional Chinese medicine preparations, especially injections, enabling the product to meet the quality standards for intravenous injections.
Depyrogenation test of amino acid products
In collaboration with Shanghai Tianchu MSG Factory, a pyrogen removal test was conducted on amino acid products produced using ultrafiltration. The test was conducted at one-eighth of the actual production volume (500L), using an HW-type spiral wound ultrafilter and an SPES-type ultrafiltration membrane with a molecular weight cutoff of 10,000. Based on previous experiments investigating the effects of operating pressure, flow rate, temperature, and concentration factor on yield, the pyrogen removal test was conducted with optimal operating parameters, achieving good results. The results are shown in Table 7. Building on the success of the pilot-scale test, the factory installed an HW4-2 type spiral wound ultrafilter (effective membrane area 48m²) produced by the Shanghai Institute of Nuclear Research, which has been in operation for over a year with excellent performance.
Table 7 Results of amino acid pyrogen removal test
test date | Sample Name | Sample dilution Explanation of multiples | Limulus amebocyte lysate (LAL) method Test Results | Rabbit farming Test Results |
05-28 | Alanine stock solution Alanine ultrafiltration permeate | 5 | Positive Negative | qualified |
05-29 | Glutamate stock solution Glutamic acid ultrafiltration permeate | 5 | Positive Negative | qualified |
06-21 | Glutamate stock solution Glutamic acid ultrafiltration permeate | 5 | Positive Negative | Unqualified qualified |
06-22 | Lysine stock solution Glutamic acid ultrafiltration permeate | 5 | Positive Negative | qualified |
07-16 | Alanine stock solution Alanine ultrafiltration permeate (min 1) Alanine ultrafiltration permeate (min 40) Alanine ultrafiltration permeate (min 67) Alanine ultrafiltration permeate (min 110) | 5 | Positive Negative Negative Negative Negative |
Membrane separation technology, especially ultrafiltration technology, has been internationally recognized as one of the most promising major production technologies from the late 20th to the mid-21st century. Currently, it is not only being researched and developed, but is also experiencing a surge in application across various fields, particularly the pharmaceutical industry. The various specifications of ultrafiltration and sodium filtration membranes, as well as HW-type spiral wound and HP-type plate and frame ultrafiltration membranes produced and under development by the Institute of Nuclear Physics will strive to meet the needs of various fields, enabling my country's membrane separation technology to catch up with and reach international standards.
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Application of ultrafiltration technology to eliminating pyrogen in pharmaceutical industry
