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Recent years, endotoxin removal has been becoming the hot topic in the biopharmaceutical manufacturing process. Among them, the problem of excessive endotoxin levels after high times concentration and filtration has become a focus of concern for biopharmaceutical researchers.
Introduction of Endotoxin
Endotoxin is kind of lipopolysaccharide (LPS) with the unit of EU. It is the main component of external lipids in the outer membrane of Gram negative bacterial cell walls, which are released when cells die or decompose. When endotoxin enter the blood or CSF, they can cause fever and therefore known as pyrogens. According to relevant regulations, endotoxin removal measures must be taken during the production process of drugs, injectable biologics, and some tissue culture media.
How to remove endotoxin?
Due to the presence of negatively charged phosphate and carboxyl groups in endotoxins, their monomer isoelectric point is around 2, which is relatively low. In solutions with pH>2, endotoxins exhibit electronegativity. Therefore, ion exchange chromatography is one of the commonly used methods for protein depyrogenation.
However, traditional chrommatography methods have drawbacks, which also limit their application in exdotoxin removal steps. This include operational and usage issues such as turbulence in the packing, tedious operation, low flow rate, long regeneration time and limited chemical stability. So, low flow rates and sensitivity to fouling mean that implementing chromatography packing into large-scale purification steps is cumbersome and expensive.
In response to this challenging issue, Cobetter has innovatively developed and launched the high-capacity, scalable and ready-to-use Purcise™ Q strong anion exchange membrane adsorber technology. Our membrane adsorber features high flow rate, high loading capacity and simple-to-operate, which can meet the scale-up needs for endotoxin removal from lab research to production process.
Cobetter Case Study
We found that in the application of protein concentration using 10kDa TFF cassettes, endotoxins in the sample and washing buffer gradually enrich as the concentration and filtration process. And during the concentration process, endotoxins will increase their adsorption with proteins, making it hard to be removed finally.
Cobetter conducted internal research immediately based on the above situation and provided a new approach for endotoxin removal. That is, before concentration, the samples and exchange buffer are pretreated with endotoxins by using Purcise™ Q membrane adsorber. Therefore, control endotoxins from the source of the process to ensure that the residual endotoxins remain within the qualified range after concentration and buffer exchange.
Research Background
According to market research and feedback, there are cases of endotoxin exceeding or approaching the detection limit in high concentration preparations. Specifically, when using 10kDa TFF cassettes to obtain the final solutions by first concentration, filtration and then concentration, the residual endotoxin in the final solutions approaches the quality limit or exceeds the quality limit in multiple batches. What’s more, some projects use Purcise™ Q membrane adsorber directly to remove endotoxins from the concentrated solutions, showing no effect on exdotoxin removal.
According to preliminary analysis by Cobetter R&D team, this phenomenon is caused by the adsorption of high concentration proteins and endotoxins, which changes their charge distribution and ultimately leads to the failure of charge adsorption of endotoxins.
Based on the above situation, we conducted in-depth testing and research:
Experiment 1：the effect of buffer conditions
Prepare endotoxin challenge solution based on the same components(histidine, mannose, Tween system) in the protein buffer. Due to the presence of surfactant Tween in the buffer, the experiment also investigated the effect of Tween to Purcise™ Q membrane adsorber on endotoxin removal.
- Configure challenge buffer 1: UF/DF Buffer (remove Tween), add standard endotoxin solution and prepare a challenge solution with final concentration of 3 EU/ml
- Configure challenge buffer 2: UF/DF buffer (with Tween), add standard endotoxin solution and prepare a challenge solution with final concentration of 3 EU/ml
Both challenge buffers were filtered by 0.9ml Purcise™ Q syringe membrane adsorber at a flow rate of 9ml/min. Filtration volume: 90ml.
The test result are as follows:
The testing results show that the buffer components have no effect on the performance of endotoxin removal by Purcise™ Q membrane adsorber.
Experiment 2：the effect of trace endotoxin in Buffer
Concentrate the buffer (histidine, mannose, Tween system) directly using a 10kDa TFF cassette, and detect the residual endotoxins in the concentrated buffer.
Prepare 4L UF/DF exchange buffer and divide it into two:
- Experimental group: Filter 2L buffer by 5ml Purcise™ Q membrane adsorber at 10 MV/min (instantaneous sampling when filtering 1L and 2L to detect residual endotoxins). Concentrate the filtered solution and take samples to detect residual endotoxins at 10 and 40 fold concentration respectively
- Control group: Concentrate the 2L buffer directly, take samples to detect residual endotoxins at 10 and 40 fold concentration
The test result are as follows:
From the comparative experiment, when the buffer is made of pharmaceutical grade material and the solution is configured to meet the quality requirements, the trace endotoxins will gradually enrich during concentration process, thereby affecting the quality of final products. Especially in high-fold concentration process, the endotoxin exceeding risk will sharply increase. When using Purcisie ™ Q membrane adsorber, trace amounts of endotoxins in the buffer can be effectively controlled to reduce the risk of endotoxin exceeding the standard during the concentration process.
Experiment 3: Endotoxin control before ultrafiltration
Before concentration, exdotoxin control was performed on protein samples and buffer (histidine, mannose, Tween system) separately by Purcisie ™ Q membrane adsorber, then conducted concentration to obtain the final solution.
Sample Preparation:
- Protein sample preparation: filter 1L protein samples (concentration:~8.0g/L by 0.9ml Purcise™ Q syringe membrane adsorber at a flow rate of 4.5ml/min to remove the free endotoxin, residual endotoxin after filtration＜0.05EU/ml ;
- UF/DF buffer preparation: filter 5L preparation buffer by 5ml Purcise™ Q syringe membrane adsorber at a flow rate of 50ml/min（5 MV/min）to remove the trace endotoxin, residual endotoxin after filtration＜0.02 EU/ml.
Protein is concentrated, filtered and then concentrated:
Concentrate 1L protein solution 5 times and use UF/DF buffer for 8-fold exchange. After the exchange is complete, perform 3-fold concentration to obtain the final solution. The second concentration process after the exchange is to take instantaneous samples in segments to detect residual endotoxins.
The test result are as follows:
*Control group of this experiment: The protein is directly concentrated, filtered and then concentrated to obtain the final solution. After testing, the residual endotoxin in the original solution was found to be greater than 1 EU/ml.
The experiment results show that after using Purcise ™ Q membrane adsorber to control endotoxins in UF/DF process from the source, the final endotoxin in the original solution can be controlled within 0.5 EU/ml, with a significant decrease compared to before. This plan not only improves product quality but also reduces the risk of endotoxin contamination in the manufacturing process.
Therefore, in the high concentration and buffer exchange process, it is recommended to use Purcise™ Q membrane adsorber to perform endotoxin adsorption pretreatment separately for the sample and the exchange buffer before sample concentration. Controlling endotoxins from the process source to ensure that the residual endotoxins remain within the qualified range after final concentration and exchange.
