Blood products primarily refer to biologically active preparations derived from the healthy human plasma or plasma from individuals with specific immunity, using separation and purification techniques. These include human serum albumin (HA), immunoglobulin (IgG), human coagulation factor VIII, prothrombin complex, and fibrinogen.

Blood products represent a special dosage form that integrates both active pharmaceutical ingredient (API) production and final formulation manufacturing. The core of the manufacturing process involves extracting the aforementioned proteins from plasma through various separation and purification methods. These include plasma thawing, protein separation, ultrafiltration, pasteurization, and sterile filtration.

Figure 1 Components of blood

Traditional blood product manufacturing relies on stainless steel equipment. However, in recent years, single-use technologies (such as single-use storage bags, filters, and tubing assemblies) have gradually emerged, providing manufacturers with more options.

Single-use technology effectively eliminates the need for repeated cleaning and sterilization of stainless steel equipment between batches, thereby reducing the risk of cross-contamination, simplifying operations, and enhancing sterility assurance throughout the production process. Additionally, its design flexibility helps minimize product loss, making it particularly suitable for small-scale production and applications with high safety requirements.

This article will introduce the advantages of Cobetter single-use products in blood product manufacturing from three aspects: sterility assurance, foreign particle control, and reduction of product residue.

Figure 2 Single-Use System vs. Stainless Steel System

Sterility Assurance of Single-use Systems

Single-use Systems have the advantages of shortening time-to-market, reducing costs, minimizing cross-contamination and improving sterility assurance. For the sterility assurance of Single-use Systems, it mainly relies on the comprehensive application of design, production process and sterilization methods to ensure that the whole production process is free from contamination in order to achieve the requirements of aseptic production.

Radiation Sterilization Validation

Firstly, it is necessary to confirm the radiation sterilization dose according to the international standard ISO 11137, which provides specific verification methods and dose setting procedures for radiation sterilization. The main process for confirming the radiation sterilization dose for single-use systems is as follows:

1. Establish the Maximum Acceptable Dose

The maximum acceptable dose for the product is established: The product should be treated with the maximum acceptable dose and must meet its specified functional requirements during its designated shelf life.
The basic technical requirements for establishing the maximum acceptable dose should include:

• Equipment for evaluating the product’s intended function;
• Products that represent standard production processes;
• A suitable radiation source capable of delivering an accurate dose.

2. Establishing Sterilization Dose (Dose Setting Validation)

You can choose one of the following methods to establish the sterilization dose:

• Obtain and utilize information on biological load quantity and/or resistance to establish the sterilization dose;
• Select and confirm 15 kGy or 25 kGy as the sterilization dose that meets the specified sterility requirements, using the VDmax25 and VDmax15 methods to achieve a sterility assurance level of 10^-6.

The basic technical requirements for establishing the sterilization dose should include:

• A microbiology laboratory to determine the biological load and conduct sterility testing;
• Products that represent standard production processes;
• A suitable radiation source capable of delivering an accurate dose.

3. Dose Distribution Validation

The product should be loaded according to the prescribed loading pattern to:

• Determine the maximum and minimum dose values and their locations;
• Determine the relationship between the maximum and minimum doses and the doses at regular monitoring locations.

The sterilization method for the product should be specified, including:

• The size and density of packaged products;
• The positioning of products within the packaging;
• A description of the irradiation container (if multiple irradiation containers are used in one irradiation device);
• A description of the transport path (if multiple transport paths are used in the irradiation device).

Records of dose distribution testing should include descriptions of the irradiation containers, loading patterns, transport paths, irradiation device operating conditions, dose measurements, and conclusions drawn.

4. Sterilization Dose Audit Validation

The continuous effectiveness of the sterilization dose is demonstrated by performing a sterilization dose audit to monitor the radiation resistance of the biological load on the product. The irradiation sterilization dose audit validation interval is generally quarterly to ensure continuous achievement of the sterility assurance level.

Aseptic Connection and Disconnection

Another focus of aseptic assurance in the blood product process is how aseptic connections and disconnections are achieved between single-use units.

 

When using TPE tubing, it is usually necessary to consider the performance as well as welding performance. TPE tube performance can be seen in Table 1 below.

Table 1 Cobetter TPE Tube Performance

To evaluate its welding performance, we will test the pressure resistance, burst resistance, and tensile strength of the Cobetter Lifemeta STF thermoplastic tube (STFH150S) and other different brands of thermoplastic tubes (C-F*x 374 thermoplastic tube, A*F*x thermoplastic tube) after welding under three conditions: non-sterilized, 25-45 kGy gamma irradiation sterilization, and autoclave sterilization.

After the appearance and size of the above TPE tubes are confirmed to be correct, they will be subjected to 25-45 kGy gamma irradiation sterilization and autoclave sterilization at 121°C for 1 hour. The TPE tubes will be welded using sterile tube welding equipment, and the welded samples will be visually inspected to ensure the interface is smooth. Following this, burst testing, pressure resistance testing, and tensile strength testing will be conducted. (Due to space limitations, only the burst data is shown; for more information, please contact Cobetter).

Table 2 Burst Test After TPE Tube Welding

And the selection of aseptic connection components should consider their ability to achieve aseptic connection, which can be confirmed by evaluating the product aerosol bacterial challenge test or direct bacterial challenge test.

For example, aseptic connectors, dis-connectors, which have a large number of applications in aseptic production processes, can effectively prevent microbial penetration or entry, and we can verify their connection sterility assurance by the following methods.

Microbial Barrier Validation of the Tube Welder

The validation aims to assess the sterile assurance capability of the Lifemeta Tube Welder during and after the welding process. The specific procedure is as follows:

a. Lifemeta STF and C-F*x 374 TPE tubes are inspected for appearance and dimensions, then sterilized using steam or irradiation.

b. Sterile Tryptic Soy Broth (TSB) is injected into the sterile tubing, and both ends of the tubing are sealed using the Lifemeta Tube Sealer handheld. The outer surface of the tubing and the blade of the tube welder are inoculated with >1×10? CFU/mL of Geobacillus stearothermophilus. The welding process is then performed using the Lifemeta Tube Welder automatic sterile tube welder.

c. The welded samples are incubated at 55–60°C for 14 days, and the presence of Geobacillus stearothermophilus growth inside the tubing is observed.

Note: "+" indicates bacterial growth; "-" indicates no bacterial growth.

Table 3 Tube Welder Bacterial Challenge Results

Metallic Sleeve Microbial Barrier Validation  

The pre-assembled tubing was disconnected by using metallic sleeve, and the tubing was sealed by injecting Tryptic Soy Broth (TSB) into the tubing from the other end of the metallic sleeve until the liquid level of the medium was just above the top end of the metallic sleeve.

Completely immerse one end of the test tube containing TSB into the challenge medium containing defective Brevundimonas diminuta at room temperature for 2 hours. Take out the metallic sleeve that has been soaked in the SLB challenge medium containing defective Brevundimonas diminuta, disinfect its surface, and allow it to dry completely. Then, incubate it at 30±1°C for 7 days as the test group.

The bacterial challenge results for metallic sleeve the  are shown in Table 4 below:

Note: Challenge bacterial suspension concentration is 2.1x10? cfu/ml.

Table 4 Results of Bacterial Challenge of Aseptic Metallic Sleeve

Aseptic Connector Microbial Barrier Validation

The membrane of aseptic connector component is dipped into a suspension of Brevundimonas diminuta (ATCC® 19146). The two components are then connected and locked to the first stage. After removing the membrane, the components are locked to the second stage, and it’s immersed in the bacterial liquid. Tryptic Soy Broth (TSB) is injected from the upstream of the component, while the downstream collects the medium in a sterile bottle.

The bacterial challenge results for the aseptic connector are shown in Table 5 below.

Table 5 Aseptic Connector Bacterial Challenge Results

Sterility Barrier for Single-Use Systems

Sterility barrier is directly related to the risk of microbial contamination of the product or operation, especially in the production of biologics, where any external contamination may lead to product failure or impact on patient health. Therefore, ensuring the sterility of single-use systems is very important. We designed specifications based on the bag production process to microbiologically challenge the single use bag with the following protocol and results.

The Cobetter Lifecube, single-use storage bag was sterilized using not less than 50 kGy gamma irradiation, and after the appearance and dimensions were checked for correctness, the bag was filled with the bag's labeled volume of Tryptic Soy Broth (TSB) liquid medium in a biosafety cabinet. After sealing the bag, it was completely immersed in SLB challenge suspension containing Brevundimonas diminuta with a content of not less than 1010 cfu, and immersed at room temperature for 24 h.

Take out the single-use bag immersed in SLB challenge suspension, and incubate the bag at 30~35°C for 7 days, and observe whether there is any bacterial growth in the bag.

The results of single-use system bacterial challenge are shown in Table 6 below.

Note: Challenge bacterial suspension concentration is 1.1x10^11cfu/ml.

Table 6 The Results of Single-use System Bacterial Challenge