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European Biopharmaceutical Review

Controlled Environment

In modern pharmaceutical laboratories, the demand for commercial and clinical success is constantly increasing. Product safety and efficient production is ensured by strictly enforced regulations and guidelines from both internal and external bodies – and all lab equipment and instrumentation must perform in ways that demonstrably meet these requirements.

Biopharmaceutical formulators may use micro-organisms, mammalian cell lines or plant cell cultures to produce biological molecules fundamental to product manufacture. These require controlled incubation in specific environments to develop successful, productive colonies. The choice of incubation system is vitally important, with numerous factors to be considered when dealing with these cell lines, which are often highly sensitive to their environment.

This article examines some of the challenges of culturing cells for the biopharma sector. In this situation, sample security and culture efficiency are essential drivers, as is minimising the risk of contamination during drug production. Recent technological developments, now available commercially, deliver safe, stable cell culture environments for these highly regulated applications.

Regulatory Challenges

European regulations define biological medicinal products (such as vaccines, antigens and cytokines) by their method of manufacture, exemplified by the use of microbial and cell cultures, or extractions from biological tissues (1). However, such biological techniques are often more changeable than physico-chemical reactions. As the vital cell lines employed in these applications are susceptible to changing conditions, any problems that occur during incubation may have potentially disastrous consequences for the final product. This places particular importance on the reliability and control of the equipment and methods used.

Due to this sensitivity, Good Laboratory Practice is a major factor for consideration in biopharmaceutical production. These include clear guidelines on what is expected from heating and storage equipment used in labs, including incubators.

The European Medicines Agency (EMA) Guideline on Human Cell-Based Medicinal Products states that “the manufacturing process of cell-based medicinal products should be carefully designed and validated to ensure product consistency” (2). In order to meet these standards, lab equipment must have proven levels of accuracy and control.

Optimum Growth Environment

New technologies are rapidly developing to meet the rising need for improved control and reliability in lab equipment and support the production of safe, stable cell lines for regulated applications. High reliability, including effective decontamination and precise control of carbon dioxide (CO2) and oxygen (O2) concentrations, temperature and humidity levels, are all essential drivers in creating an optimum cell culture environment.

Controlling Temperature

Cell cultures exposed to inconstant temperatures cannot be considered viable in regulated environments. Fluctuations in temperature impact the metabolism of cells and affect various parameters including adhesion, protein expression and proliferation (3).

Modern technologies and intelligent design can play an important role in ensuring temperature reliability. The MCO-19 incubator series, developed by Panasonic Biomedical, has a patented Direct Heat and Air Jacket, which encloses the chamber in high density foam insulation. This system prevents condensation and protects against ambient temperature fluctuations (see Figure 1). Gentle fan circulation maintains uniform heating, delivering regular temperatures to all cultures in the chamber, regardless of their position.

A direct heating system also ensures highly reactive temperature control in response to door openings, which can be responsible for many variations in temperature – especially in busy labs. By incorporating independent heating sources throughout the incubator chamber – in the top and rear wall, and in the base and outer door – regulated temperatures are maintained throughout the chamber. In addition, the outer door heater warms the inner glass door relative to ambient conditions, preventing condensation and promoting temperature uniformity.

CO2 and O2 Levels

When growing cell cultures with rigorously specified environments and growth conditions, close monitoring and control of CO2 and O2 levels within an incubator is crucial. This is of particular importance when using stem cells, which may naturally reside in locations with very low oxygen requirements, such as bone marrow or adipose tissue. When culturing cells from these sources, the duplication of natural growth conditions requires close control of O2 concentration. In this way, users can optimise the quality of the cell culture, not to mention that of any subsequent daughter cells.

One method used to maintain O2 levels in incubators is a long-life zirconia O2 sensor which upholds sub-ambient levels from 1 to 18 per cent. Using proper safety precautions, enriched O2 levels (from 22 to 80 per cent) can also be achieved. Electronic proportional integral derivative (PID) control ensures high accuracy by preserving temperature and gas set points over the entire system range.

When it comes to controlling CO2 levels, a sensor can provide the necessary levels of accuracy through the use of a single beam, dual detector. This permits a CO2 range of 0 to 20 per cent. The ceramic-based sensor measures sample and reference wavelengths for continuous calibration, and is unaffected by moderate changes in temperature and relative humidity.

The sensor is linked to a sophisticated PID microprocessor controller. This reliably maintains temperature and gas set points, and delivers rapid recovery, without overshoot, during periods of frequent door opening.

Humidity Control

Correct humidity levels are essential when cultivating a healthy cell line, especially when using cell culture dishes or plates, which present more opportunity for evaporation of the media than closed vessels. Should evaporation of cell culture media ever occur, it leads to increased concentrations of salt, encouraging the conditions that can go on to cause cell lysis (3).

It is therefore recommended that cell culture incubators are maintained at a relative humidity of approximately 95 per cent, in order to avoid cell desiccation and the potential loss of whole cell lines. These high levels of humidity can be maintained through natural evaporation, together with gentle fan circulation.

However, it must be remembered that humidity pans can become a source of contaminants, such as mould, bacteria and yeast, so frequent cleaning is essential.

Continuous Decontamination

In research labs that work routinely with cell cultures, mycoplasma is a common bacterial contaminant that can cause genuine problems. These bacteria occasionally prompt infections, resulting in cellular changes such as alterations in metabolism and cell growth, as well as chromosome aberrations (4). Severe mycoplasma infection can destroy a cell line completely; thus, some form of continuous contamination control is necessary.

Incorporating a ultraviolet (UV) lamp into the design of incubators is a proven method of providing contamination control. Figure 2 shows the summary of four tests on separate strains of mycoplasma using MCO-19 incubators.

It can be seen that the lamp in the UV range (located at the base of the chamber) has a much greater decontamination effect than lamps emitting at visible wavelengths.

By ensuring that the isolated, narrow bandwidth, ozone-free UV lamp switches on for a specified period after each door opening, any airborne contaminants that may enter the chamber are efficiently eliminated. Water-borne organisms in the humidity water reservoir can also be eradicated in the same way, ensuring the security of any active cell cultures.

Surface protection is equally important. Constructing incubators from intelligent material – such as InCu saFe® copper-enriched, stainless steel alloy – provides continuous germicidal protection and prevents the growth of moulds, fungi and bacteria within the chamber. Contamination sources are consequently eliminated and the effects of common airborne contaminants reduced.

H2O2 Decontamination

As mentioned, effective decontamination of equipment is essential when maintaining the integrity of the cell lines in biopharmaceutical applications. While traditional direct heat decontamination systems will effectively remove the majority of contaminants from incubators, it may take as much as one full day to complete a single decontamination cycle. With labs under increasing pressure to complete processes in shorter time frames, this can cause unnecessary delays.

Because of this, low temperature hydrogen peroxide (H2O2) in biological safety cabinets is now becoming a widely used alternative to direct heat sterilisation, especially in the biopharma industry. The H2O2 vapour decontamination process permits quick turnaround of the cell culture incubator, even when a complete, validated decontamination is required. This means that equipment downtime is substantially less than similar direct heat decontamination systems.

The H2O2 decontamination process follows several steps. Once the cycle is activated, the door is automatically locked and H2O2 is vapourised. An airflow system circulates interior air to ensure all surfaces come into contact with the H2O2 vapour. After this, a UV lamp switches on for 90 minutes, causing the H2O2 vapour to decompose into water vapour and oxygen. Once the cycle is complete, the door automatically unlocks.

When H2O2 decontamination is engaged, all the incubator’s interior components, such as shelves, shelf brackets and the humidity tray, are decontaminated in situ. This eliminates the need for any additional cleaning. Furthermore, critical parts such as the CO2 sensor do not need to be removed during the process or recalibrated afterwards, contributing to the reliable operation of the system.

Top Quality Cells

In biopharma applications, the culturing of healthy, viable cell cultures is a top priority. By developing the very best quality cells, scientists are able to lay a strong foundation for further development. In an environment where every phase is tightly managed and monitored, the reliability and control of lab equipment is essential, and contributes greatly to ensuring the success of a process and end product.

These innovative technologies enable safe, regulated incubation of biological samples in ways that meet with today’s stringent regulations. The combination of technologies delivers outstanding control over environmental factors, including CO2 and O2 concentrations, temperature, humidity levels and contamination. With this range of control systems and options now available in a single system, environmental reliability is easily achievable in all labs.

1. Supplementary guidelines to the EC-GMP Guide with specific requirements for the manufacture of biological medicinal products for use in humans, Annex 2: Manufacture of Biological Medicinal Products for Human Use
2. Guideline on Human Cell-Based Medicinal Products, 4.2.2. Manufacturing Process, EMA, 2008
3. In vivo-like cell culture conditions, ibidi GmbH. Visit: applications/live-cell-imaging/in-vivo-like- cell-culture-conditions
4. Ryan J, Understanding and managing cell culture contamination, Technical bulletin, p24, 2008

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Jacqueline van der Zijden-Anusic has been Product Manager Europe at Panasonic Biomedical Sales Europe BV since 2012.With an extensive background in marketing and sales, and a focus on product management, Jacqueline completed her Bachelor’s degree in International Business and Languages at the Inholland Business School of Rotterdam in 2001, for which she developed a strategic marketing plan for the non-paint product group. She has also gained work experience at Braun GmbH in Kronberg, Germany, in the business management communication department.
Jacqueline van der Zijden-Anusic
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