In the biopharmaceutical industry, single-use systems are widely used in upstream cell culture, downstream purification and final formulation products due to their advantages such as flexibility, low cost and reduced risk of cross contamination. At different stages of biopharmaceutical processes, various applications have distinct requirements for sampling. Efficient and reliable sampling solutions are crucial for ensuring process accuracy and sterility. This article provides a brief introduction to the key design considerations for sampling in single-use systems.

1. Aseptic Sampling Design in Upstream Cell Culture

In upstream process such as mammalian cell culture, microbial fermentation, it is necessary to monitor parameters like cell density, metabolites (glucose, lactate), pH, dissolved oxygen, and product concentration. The design of aseptic sampling directly impacts the reliability of process monitoring and the safety of cell culture.

1. Sampling Design for Single-Use Bioreactor Bags

1.1 To assure the sterility of cell culture, there are several sampling design solutions for bioreactor bags:

① Check valve+Needle-free Sampling Ports：

The check valve allows unidirectional flow of media from the bioreactor bag while preventing backflow of external gas or liquid, thereby avoiding contamination. Additionally, the needle-free design eliminates the risk of diaphragm damage caused by needle puncture. Combined with proper disinfection before and after sampling, safe multi-sampling can be achieved during cell culture process. In the WAVE bag-based cultivation model, in addition to the check valve design, an extended inner tube is typically incorporated into the sampling design to ensure samples can be collected even under low-volume conditions.

② Sterile Connection by Thermoplastic Elastomer Tubing：

Thermoplastic elastomer tubing enables sterile connection and disconnection between bioreactor bag and sampling bag. Due to the inherent material safety of TPE tubing and its ability to facilitate flexible multiple connections and disconnections, this kind of sampling design perfectly balances sterility, flexibility and cost efficiency. 

③ On-line Monitoring as an Alternative to Off-line Sampling：

In additional to direct sampling design, the electrode ports on the bioreactor bag allow real-time monitoring pH, dissolved oxygen and temperature by connecting on-line sensors. This reduces the need for off-line sampling and helps maintain sterility throughout the cultivation process. 

④ Sampling Design of High Viscosity Cultivation System：

For high viscosity cultivation system, such as high density mammalian cell, the sampling tubing design requires a larger tube OD ≥6mm, typically #17 or 24# tube. Combined with the low-shear characteristics of peristaltic pump, it assures smooth sampling of high viscosity samples.

1.2 Sampling Design for Stainless Steel Reactors

For stainless steel reactors, the sampling design must meet the following requirements:

• Compatibility with online sterilization (SIP) 
• Maintenance of aseptic conditions during sampling
• Sterile disconnection and transfer after sampling

Cobetter Lifecube? SA aseptic sampling system is a kind of flexible aseptic fluid transfer unit, which consists of sampling holder, sampling bag or bottle with needle or needle-free port. It can be flexibly designed as single sampling or multiple sampling based on the volume and numbers of sampling each time. The sampling base and needle come pre-assembled for direct connection with stainless steel reactor and online steam sterilization (SIP). 

Following sterilization, aseptic sampling is performed using pre-sterilized single or multi-port sampling bags/bottles, effectively preventing cross-contamination during the sampling process. The sterile disconnection after sampling can be achieved by metallic sleeve+crimping tool or TPE tubing. The entire sampling process is fully closed and easy to operate, guaranteeing sample sterility from the point of collection to the analysis.

2. Aseptic Sampling Design in Mixing Process

In biopharmaceutical manufacturing processes, sampling is required during media and buffer preparation to verify critical quality indicators including mixing homogeneity, solution purity, concentration, pH, impurities, microbial load. Similarly, the temporary storage of intermediate products and bulk drug substances also requires sampling to test their stability and pre-release testing (e.g., purity, potency). The sampling design for single-use systems must not only ensure sterility and operational efficiency, but also be tailored to specific container configurations and application requirements.

2.1 Sampling Design for Different Storage and Mixing Containers

In the preparation and storage of solutions, there are different mixing containers and storage containers on the market. For sampling design of mixing bags and storage bags, the sampling methods need to be flexibly adapted according to the specific port locations of the containers. A common sampling solution is directly integrating sampling line at designated ports on the corresponding containers. However, in cases where the container ports are limited or restricted, the sampling line design requires special customization. 

2.2 Sampling Design for Drug Substance Frozen Storage

Prior to and following cryopreservation, the bulk drug substance must be tested for activity, purity, concentration and physicochemical properties.

• Sampling line prior to cryopreservation is usually designed in the final storage stage before aliquoting
• Sampling line or sterile connectors during or after cryopreservation can be integrated on freeze-thaw bags

The aseptic connector design enables multiple sampling when integrated with multi-port sampling bags, ensuring zero cross-contamination during the sampling process. In contrast, dedicated sampling lines offer superior operational convenience and minimized dead volume.

3. Sampling Design in Final Filling

Formulation filling is the final and high-risk step in biopharmaceutical manufacturing, which imposes more stringent requirements on the design of single-use systems and sampling protocols. For sterile-process formulations, during the sterile filtration stage, samples must be taken to test the product’s critical quality attributes, and final filling can only be proceed after confirming compliance with quality standards. During the filling process, filled products are typically selected as samples to verify the uniformity of the filling process, sterility, and the products’ critical quality attributes.

With full customization capability and an end-to-end single-use system supply chain, Cobetter delivers tailored sampling solutions without compromising quality or timelines. This enables biopharmaceutical partners to refine process control, mitigate contamination, and optimize productivity.

Advantages of Cobetter Customized Solutions

• Wide Range of Self-produced Single-Use Accessories (500+)
• 13,000+ filtration process validations for pharmaceutical products, and 35,000+ process validation reports
• Manufacturing in the ISO Class 7 clean zones
• Stable and sustainable supply chain assurance