How is Cell Culture Used in Bio-Process?

What is Cell Culture?

Single-Use Cell Culture Media Bottle Assemblies

Cell culture is the growth of plant or animal cells in special containers and in controlled conditions. In the bio-pharmaceutical process space, cell culture is ubiquitous and used for a variety of applications, such as developing drugs, vaccines, bio-markers and in personalized medicine where we can study the effects of a particular treatment on a patient at an individual or group level. Cell culture is highly regarded for its reproducibility.

and because living cells can act as catalysts that facilitate reactions and generate molecules that are not possible or practical with synthetic chemistry.

Cell Culture Media and the Cell Culture Process

Controlled conditions are vital in cell culture because the cells need an environment in which they can grow and thrive. In a lab, we can scale up cell counts by using cell culture media that is often composed of essential nutrients, proteins, salts, serums and other components to aid growth. The cell culture media can be combined with the cells in containers such as flasks or single-use media bottle assemblies. To maintain sterile conditions, single-use media bottles may be equipped with a 0.2 micron filter for air flow and TPE tubing for aseptic welding.

Stages of Cell Culture

Lag – A phase which the cells are adjusting to their new media environment and do not readily grow.
Exponential – Cells start multiplying exponentially.
Stationary – Once the rate of death and the rate of growth are at equilibrium, the cells reach the stationary phase. This is typically the point where cells are added to a bio-reactor or transferred to a larger media container to continue growing.
Death – When more cells are dying than growing. Cells in the bio-processing industry rarely get to this rate because they are harvested before it happens.

Cell expansion is commonly scaled up to a bioreactor that can have a volume capacity of thousands of liters.

How is Cell Culture Used in Bio-Process?

In bio-pharmaceutical process, the upstream process encompasses all steps needed to produce the product. Cell culturing is essentially the first step of bio-processing because these cells generate the main compound of interest. Nurturing and growing cells in media enables industries to mass-produce products. After the cells have developed to the stationary phase in a container such as a flask or single-use media bottle assembly, they are moved to bioreactors for fermentation as required to produce the target molecules. A typical bioreactor is equipped with sensors to monitor parameters such as pH, absorbance, and temperature to ensure ideal conditions. After the product is made, it needs to be refined and purified. This next series of steps preceding final filling and formulation is referred as downstream processing.



Invitrogen, GIBCO. (n.d). Cell culture basics – Handbook. Retrieved from

What is Tangential Flow Filtration (TFF) and Where is it Used?


What is Tangential flow filtration?

Tangential Flow Filtration

Tangential flow filtration (TFF), also known as Cross-flow filtration, is a process of separation widely used in bio-pharmaceutical and food industries. It is different from other filtration systems in that the fluid is passed parallel to the filter, rather than being pushed through a membrane perpendicularly which can clog the filter media. This method is preferred for its continuous filtration and reproducible performance. The particles that pass through the membrane, the permeate, are put off to the side, while the rest, the retentate, is recycled back to the feed.

When is TFF used?

Tangential flow filtration is used in concentration and diafiltration processes.

  • Concentration – Increases the concentration of a solution by removing fluids while keeping the solute molecules. This process is done by selecting a filter significantly smaller than the solute molecules to allow for a higher retention of solute molecules.
  • Diafiltration – The separation of small and large particles, leaving the smaller particles behind without altering the overall concentration at the end by washing out the remaining salt with another buffer. This process can also reduce the concentration of a solution by washing out the remaining salts with water.

Specific applications of TFF include, but are not limited to:

  • Pre-filtration component WFI systems to remove endotoxins.
  • Recovering of bacterial cultures and viruses.
  • Removing PVA that is excreted from the excipient from nanoparticles during drug loading.



Carole S. Genovesi, May-June 1983, Several Uses for Tangential-Flow Filtration in the Pharmaceutical Industry, Accessed on 28 August 2018. <>

Pall Corporation, n.d., Introduction to Tangential Flow Filtration for Laboratory and Process Development Applications, Accessed on 28 August 2018. <>

Gautam Dalwadi and Vivian Bruce Sunderland, 26 September 2008, Purification of PEGylated Nanoparticle Using Tangential Flow Filtration (TFF), Accessed on 28 August 2018. <>

Novasep, n.d., Industrial cross-flow filtration technology (Picture), Accessed on 28 August 2018. <>

USP Class VI Testing

What is USP Class Testing?

Plastic Sanitary Tri-Clamp Fittings

USP Class testing is one of the most common methods of testing to determine bio-compatibility of materials. There are six classes, VI being the most rigorous. Class VI testing is aimed to certify that there are no harmful reactions or long-term bodily effects caused by chemicals that leach out of plastic materials. USP Class Testing standards are determined by the United States Pharmacopeia and National Formulary (USP-NF), the organization responsible for the quality and safety of medical devices and foods. Class testing is frequently conducted on plastic materials that come in contact with injectable drugs and other fluids found in various steps of the drug manufacturing process.

USP Class VI Testing Methods

USP Class VI testing is conducted by producing an extract of the product with different extraction fluids, such as polyethylene glycol and vegetable oil, and injecting it in specimen (rabbits and mice) in vivo (alive), to observe the biological response to the extract. Testing is commonly done as per USP <88>, which requires three types of testing: systemic injection, intracutaneous, and implantation.

  • Systemic Injection Test (Acute Systemic Toxicity):
    Test specimen are injected with the extract intravenously and observed for 72 hours. The specimen are monitored for any abnormal toxic reactivity. The scientist determines the test as pass/fail.
  • Intracutaneous Test:
    The purpose of this test is to check for any local skin reactions. Test Specimen are injected with the extract intracutaneously and observed for 72 hours. The reactions are scored and averaged.
  • Implantation Test:
    Specimen are implanted with the product material to observe the reaction of the live tissue in direct contact with the product over a span of at least 120 hours (5 days).

Why should products be USP Class VI?

Class testing is often required for manufacturing drugs for its low toxicity compliance and strict bio-compatibility standards. It is important to know that no fluid-contact surfaces will result in harmful chemicals being extracted in to a conveyed fluid. Class VI testing extensively investigates the reaction in the body, skin, and living tissue to ensure safety. USP Class VI is a common standard for pharmaceutical tubing, fittings, single-use systems, and fabricated parts.

Unites States Pharmacopeia, n.d., <88> Biological Reactivity Tests, In Vivo, accessed 21 August 2018, <>.
NAMSA, 14 April 2014, USP Class Testing, accessed 21 August 2018, <>

WFI Basics and Sampling Methods

What is Water For Injection (WFI)?

Water For Injection is a solvent that is used for the manufacture of injection drugs. It can be used as an excipient as well as the final cleaning rinse agent for Bulk Active Pharmaceutical Ingredient (API) or in a Bulk Pharmaceutical Chemical (BPC) preparation.

Types of Water For Injection

  • USP WFI – USP WFI is the material used to make Bacteriostatic and Sterile WFI. It is not the finished product, therefore, it needs to go through extensive validation to ensure that it meets USP parameters.
  • USP Bacteriostatic WFI – Bacteriostatic WFI is sterile water that has 0.9% benzyl alcohol added as a preservative. It prolongs the water vial for up to 28 days, therefore, allowing repeated usage.
  • USP Sterile WFI – Sterile Water For Injection is stored in a single-use vial and used for intravenous administration. After it is opened or heated, it cannot be reused and must be discarded.

How is Water for Injection Made?

  • Distillation – According to USP, European Pharmacopoeia (EP), and Japanese Pharmacopoeia (JP) guidelines, distillation is the preferred method of manufacturing WFI for its ability to remove 100% of all impurities. Pre-treated water is boiled and the clean water vapor is collected, leaving all impurities behind. The collected vapor then undergoes condensation to return to its liquid state.
  • Reverse Osmosis and Ultra-Filtration – Reverse Osmosis (RO) is permitted by USP and JP regulations, however, JP regulations require ultra-filtration if reverse osmosis is utilized. The water first goes through ultra-filters that ‘squeeze’ water and small impurities through a semi-permeable membrane. Next , the ultrafiltered water passes through a semi-permeable RO membrane which further separates the water and impurities by forcing the water through RO membranes leaving pure water.

Regulations and Sampling

As per USP < 1231>, WFI needs to meet these requirements:

Total Organic Carbon (TOC) [µg C/L, ppb]<500
Conductivity at 25°C (µS/cm)<1.3
Bacteria (CFU/100 ml)<10
Endotoxin (EU/ml)0.25


These levels are monitored through test analysis and sampling periodically. Sampling is done through valves that are located throughout the system. Since WFI needs to be completely rid of microbes, the water needs to be in constant movement at 80-90°C so that microbial growth cannot occur. In order to ensure water quality is within specification, sampling is performed frequently at different use-point valves to ensure that there are no contaminants. The samples are collected and taken to a microbiology laboratory for testing per USP <1231> requirements.

Sampling Process

The collection and testing processes are critical procedures that need to be performed by highly trained technicians to avoid false-positive results due to contamination during the collection by improper techniques, poor hygienic habits, or inadequate sterilization methods. Oftentimes, companies sterilize their sampling equipment using autoclave and then create a kit for collection. Before collecting the sample, the valve is inspected for cleanliness and it is flushed for 30 seconds or more at a rate of 8 ft/s or more in order to remove bio-film structures.

The pictures below depict the sampling procedure in progress (left) and it shows an example of a use-point valve (right) from where water is collected for sampling.

During collection, technicians have to manually place a gasket onto the flow path before clamping the sample device into the use-point valve. Placing the gasket poses a tremendous risk of contamination because the technician touches parts where the sample will flow, compromising the integrity of the results. Whenever a result is positive, costly measures are taken to investigate the source of contamination and the problem is often traced back to the collection procedure, not the system itself. This problem can be easily solved through single-use systems like our aSURE™ WFI Sampling Kit or aSURE™ Tri-Clamp Fittings. These sanitary connections come with a fused gasket, which allows the technician to successfully collect a sample without coming into contact with the fitting, removing an immense contamination risk. Our kits are gamma-radiated for microbial control, double bagged in dual laminate medical-grade pouches with easy-peel seals.

Example of a WFI Filtration and Storage System:



Mettler Toledo, Pharmaceutical Waters Guide, accessed 16 August 2018, <>.

U.S. Food & Drug Administration, 7/93, High Purity Water System, accessed on 16 August 2018, <>.

U.S. Food & Drug Administration, 12/31/86, Water For Pharmaceutical Use, accessed on 17 August 2018,

Pure Flow Inc., Spring 2015, How to Properly Sample Water Systems, accessed 17 August 2018, <>.

United States Phamacopeia, <1231> WATER FOR PHARMACEUTICAL PURPOSES, accessed on 17 August 2018, <>.

Critical Process Filtration Inc., (n.d.), Filters in USP Water System [Diagram], accessed 17 August 2018, <>.

Additional Photo Credit: MQA Laboratories

Differences Between Cleanroom Classes

What is a Cleanroom?

A cleanroom is a space in which the number of contaminants in the air per unit of volume, such as dust and other airborne microbes sized between 0.1µm and 5µm, are controlled to decrease chances of contamination. These particles are controlled by a high efficiency particulate air (HEPA), which filters the air before entering the cleanroom, and it is changed multiple times per hour, according to the class of the cleanroom, as established by the International Standard Organization (ISO) 14644-1. Cleanrooms with the intention of keeping a product without contaminants are kept at a positive pressure so that the particles flow out, from the cleanest area to the least clean one. However, there are cleanrooms kept at a negative pressure in order to not let anything escape the cleanroom, such as quarantine stations and chemical testing facilities.

At TBL, we are equipped with an ISO Class 7 & Class 5 Cleanrooms. Our cleanrooms feature UV lights designed to kill most germs. These cleanrooms are utilized for assembling single-use systems packaging and injection molding.

Clean-room assembly of single-use tubing kits and manifolds. Gamma irradiation available

Cleanroom Regulations

The first cleanroom standard was the US Federal Standard 209E. This regulation separated cleanrooms into classes, Class 1 having the lowest count of particulates per cubic meter, while Class 100,000 had the highest. As cleanrooms became more prominent throughout the world, an international standard became a necessity. The International Standard Organization assembled a committee and developed the ISO 14644-1. It was based on the Federal Standard 209E, but it had two more classes before Class 1, and one more after Class 100,000 to represent room air. The ISO 14644-1 superseded the FS209E in 2015.

Current Cleanroom Classes

The table below shows how the classes are divided by the number particles allowed per cubic meter along with the range of air changes per hour in each class.

Number of Particles per Cubic Meter by Micrometer Size
Class0.1 micron0.2 micron0.3 micron0.5 micron1 micron5 micronsAir Changes per Hour
ISO 11021.020.350.0830.0029360-600
ISO 210023.710.
ISO 31,000237102358.30.29360-540
ISO 410,0002,3701,020352832.93300-540
ISO 5100,00023,70010,2003,52083229.3240-480
ISO 61,000,000237,000102,00035,2008,320293150-240
ISO 7352,00083,2002,93060-90
ISO 83,520,000832,00029,3005-48
ISO 935,200,0008,320,000293,000

How to Determine Tubing Burst Pressure and Working Pressure


How to determine the pressure rating of tubing?

To ensure that your desired tubing is fit for your needs, one must understand how pressure ratings affect different materials. If the material is chosen for inappropriate use, it can burst at the most inconvenient time; however, this can be prevented by using Barlow’s formula, which shows the theoretical maximum operating pressure of a tube by using its tensile strength at yield (maximum pressure at which there is no permanent damage) and its interior and exterior diameters. This formula can be used for all of your tubing needs, as it is based on a 4:1 safety factor, which is appropriate for compressed air and gases, as well as fluids with working pressure greater than 1MPa.

Barlow’s Formula

barlow's formula for calculating tubing pressure rating

Burst Pressure in MPa or psi
Tensile Strength @ Yield in MPa or psi
X= OD/2 in inches
Y= ID/2 in inches

Tensile Strength Values

Click on a product to be redirected to its page.

MaterialProductTensile Strength @ Yield (psi)Tensile Strength @ Yield (MPa)
Phathalate Free VynilClearGreen®190013.1
Silicone (Non-Braided)Platinum Cured Silicone12008.3
Peroxide Cured Silicone159511
TPEPharm-A-Line™ I972 (At Break)6.7
Pharm-A-Line™ VI210014.5
Weldable TPECellGyn®8706
PFAFluor-A-Pure™ PFA420028.9
FEPFluor-A-Pure™ FEP400027.5
PTFEFluor-A-Pure™ PTFE350024.1


Note: These are theoretical values. Material properties may be affected greatly by temperature, operating pressure, chemical concentration, the presence of other chemicals, and other factors. Ultimately, users should determine the compatibility of any product through field testing under their particular process conditions.

How do we Achieve Exacting Tolerances with Plastic Tubing? Lasers.

For many critical applications, it is essential that plastic tubing is produced within strict dimensional tolerance limits. Spatial measurements of a tube, such as inside diameter (ID), outside diameter (OD), wall thickness, concentricity, and overall length all play separate roles in ensuring a tube performs up to intended standards. For example, Peristaltic-pump tubing is especially susceptible to performance flaws when tubing is manufactured out of specification.

The following are three common instruments employed for measuring tubing dimensions: pin gauges, comparators, and ultrasound/ laser systems.




Pin Gauges

Pin gauges are metal pins of strictly calibrated diameter. When measuring a tube with pin gauges, the pin with the lower limit diameter should fit effortlessly through the inside diameter of the tubing, while that of the upper limit should not. Pin gauges are inferior as a sole means of dimensional analysis as they can not be used for continuous in-process control nor do they imply any information about a tubes outside diameter or concentricity. Samples need to be taken, and statistical methods are employed to determine the likelihood that the ID of a particular length of tubing will be within specification. Pin gauges are better utilized as a tool for post-manufacturing quality control.


Optical comparators display an enlarged shadow image of the tubing on a display screen. The tubing is placed in a fixed position in the comparator, illuminated with light sources, and the image projected onto a display. Computer software is used to convert the coordinates of the tubing on the screen to measurements of the internal and external diameter, etc.. Comparators are not normally used on the factory floor, but are useful instruments for examining samples in the QC laboratory. Again, statistical methods are needed to determine the probability that a particular length of tubing will be within specification..

Ultrasound/ Laser Measurement Systems

A laser , such as the technology utilized by TBL Performance Plastics, allows for continuous “real time” measurement of extruded tubing as it is produced. The instrument will continuously measure and record the ID, OD, wall thickness, and concentricity, with high accuracy to ensure all critical dimensions remain within specification. In practice, the sensor output can be fed directly to the servo drive motors in the extruder, so any deviations can be immediately corrected.
Typically, a laser system consists of a controller linked to the following devices:

  • “UltraScan” Gauge – This gauge uses ultrasound to determine the wall thickness and concentricity of the extruded tubing. The ultrasound is reflected from the inner and outer tube surfaces.
  • “LaserSpeed” Detector: This is a non-contact unit, which measures the speed of the extruded tubing using lasers, from which the length produced can be calculated.

Data from the whole of the production run can be collected, and retained for subsequent audit if necessary.


Continuous laser measurement devices are superior to pin gauges or comparators as an in-process measurement device. However, pin gauges and comparators are valuable quality-control tools for carrying out a final check of the tubing dimensions, and to analyze which materials have a tendency to expand or shrink after they are allowed to set over time. A robust quality control system will utilize several of such methods to ensure that tubing is manufactured consistently and within specification.

So, which is better, Platinum or Peroxide?

Silicone tubing has many beneficial properties and has been utilized in medical and pharmaceutical applications for over 50 years. It is made from silicone polymers that are extruded then crosslinked and “cured” in to solid form using an assortment of curing methods. The two most common of these methods are platinum-catalyzed addition polymerization (platinum curing) [see Figure A] or peroxide-initiated free-radical polymerization (peroxide curing) [see Figure B]. Platinum-cured silicone tubing is widely accepted in applications where purity is a concern, where peroxide-cured silicone tubing typically exhibits enhanced mechanical strength.


It is important to note platinum curing has no byproducts. Peroxide curing does result in byproducts, which tend to be volatile organic acids [1]. Although a high-heat post-curing method can be employed to drive out many of these impurities, they are a major reason why platinum-cured silicone is often preferred for medical and FDA applications. In addition to its purity, it is sometimes favored for its inherent optical clarity, as peroxide-cured varieties tend to be a bit hazier in appearance, interfering with a user’s ability to visually inspect the contents of the tubing. The tear strength of platinum-cured silicone is usually higher due to the nature of its crosslinks.

A benefit to peroxide-cured silicones is that they typically have superior mechanical properties. In addition, they are generally less expensive. However, their use in the life-sciences industries are limited because of potential liability due to toxicity. To meet the mechanical-performance characteristics of peroxide-cured products, some manufacturers are using platinum-cured low hysteresis silicone, resulting in pump life similar to that seen with peroxide-cured tubing and high-accuracy dosing for peristaltic-pump applications.

Effect of Sterilization Methods on Mechanical Properties

There are four methods most commonly employed for sterilizing non-reinforced silicone tubing. They are electron beam (e-beam) irradiation, gamma irradiation, autoclave (steam sterilization), and treatment with ethylene oxide (EtO) gas.


With respect to e-beam and gamma irradiation, a study done by Adamchuk et. Al. [2] showed peroxide-cured silicone, in particular, exhibited a very significant drop in tensile strength and increase in hardness and tensile modulus (resulting in decreased flexibility), while there was a relatively small change in these values for the platinum-cured sample. Tear strength decreased significantly for platinum and peroxide cured samples, but much more so for the peroxide cured sample. According to the Second Edition of Effect of Steriliaztion Methods on Plastics and Elastomers, some grades of platinum-cured silicone can withstand up to 9 megarads radiation and not experience a significant change in mechanical properties, while peroxide-cured silicone is usually limited to less than 5 megarads [3].

Ethylene Oxide

In the same study, when treated with EtO gas, both samples actually showed an increase in tensile strength and negligible changes in tensile modulus, hardness, and tear strength.


In a separate study, three platinum-cured silicone samples were autoclaved 25 times using three different methods. The methods: flash autoclave (10 minutes at 132°C, at 30psi), standard gravity autoclave (30 minutes at 121°C, at 15psi), and pre-vacuum high-temperature autoclave (30-35 minutes at 121°C). No significant change in physical properties was noted [3].


For applications where high purity, critical dosing, or repeated sterilization is required, platinum-cured silicone is often the material of choice. Peroxide-cured silicone is a common choice for less demanding (in terms of purity) applications. It is commonly, but not universally, a less expensive alternative to platinum-cured silicone, and often exhibits longer pump life in peristaltic-pump applications.


Figure A: Platinum Curing of a Silicone Polymer

platinum-cured silicone

Figure B: Peroxide Curing of a Silicone Polymer












Single-Use Pressure Sensor – New Alternative!


single-use pressure sensor gauge

Our team at TBL is extremely proud to launch our aSURE™ instrument fitting, which was developed to provide a sterile barrier where disposable manifolds and tubing assemblies are used on hybrid single-use process equipment.

Fixed or tethered pressure-monitoring devices provide extremely high accuracy and are often hard-wired into a central control panel. The aSURE™ instrument fitting provides a practical means of providing a sterile barrier on a complex manifold set, and provides a barrier without the need to have a gauge present during the sterilization process.

When comparing our aSURE™ fitting system to single-use pressure sensor technology, single-use sensors have many important drawbacks.

Drawbacks of Single-Use Pressure Sensors

  • Proprietary equipment required to relay the proper signal in to existing control systems
  • Generation of waste electrical and electronic equipment (WEEE)
  • Unable to use preferred pressure sensor in process

Benefits of the New aSURE™ Instrument Fitting

  • Use the pressure gauge of your choice on a single-use system
  • No need for gauge to be installed during sterilization process
  • No generation of waste electrical and electronic equipment (WEEE)
  • No proprietary monitor or transmitter/ line conditioning required
  • Integrate gauges from Anderson-Negele, WIKA, REOTEMP, Emerson Instruments, & Endress+Hauser

Designed for Pharmaceutical/ Bio-Pharmaceutical Manufacturing

Representative samples of each fluid contact material have been tested and have meet the following regulatory standards:

  • USP Class VI
  • Animal Derived Component Free (ADCF)
  • ISO 10993
  • California Proposition 65


Oina Peristalitc Pumps

TBL Plastics is proud to announce our partnership with Oina, an industry leading manufacturer and developer of peristaltic pumps. As the master distributor for our tubing products in Northern Europe, Oina has validated all of our pump-grade tubing in their pumps. With their technical knowledge and experience in the medical, diagnostic, industrial, and OEM markets. Oina is ideally suited to represent our products in these markets.

A Quote from Oina CEO, Anders Lovas:

“We have during the last year conducted TBL tube tests in multiple pump configurations and applications for several different tube dimensions with very satisfactory results. We have started selling and distributing TBL Pharm-A-Line tubes in analytical instruments, bio-reactors, process industries and pharmaceutical applications.”

Visit or Contact Us for application assistance.

Oina Peristaltic Pumps