Cell Culture Media: Balancing Resources with Results

Why does medium matter?

It all starts with media. Without an appropriate growth medium, no biopharmaceutical process would exist. The proper medium allows for cells to grow and generate product. Going beyond the fact that cell culture processes would simply not exist without cell culture media, the medium (and associated feeds for fed-batch processes) represents a large portion of the cost of a biopharmaceutical process. Cell culture media are complex entities that encompass large quantities of raw materials as well as a high level of technical expertise, driving up the cost per litre.

What are the advantages of an optimized medium formulation compared to an off the shelf product? 

Many people will choose to start with standard off the shelf products for their cell culture processes. While there are a wide variety of products available on the market, there are several important reasons to consider using an optimized medium formulation instead. The first is to tailor a medium formulation specifically for your own cell line. Every cell line will respond differently to standard products, and optimizing a medium formulation is the best way to ensure that you use a medium that works best for your application and requirements. Additionally, as you generate different cell lines with varying genetic constructs for new products, your cells may begin to respond differently. Having an optimized medium formulation allows you to adapt to these changes while maintaining growth and productivity. Using a standard product also means you are bound to a single vendor with no access to proprietary formulations. With your own optimized medium, you have full access to the formulation, with the ability to manufacture it with a vendor of your choice.

What factors should I consider when developing an optimized medium?

As discussed previously, optimizing a medium formulation to get you the best growth and productivity is the main driving factor, and allows you to minimize the amount of medium used and therefore your overall COGS. It is important to also consider what the criteria are for your optimized medium. For many industries, having a fully chemically defined, non-animal origin medium is critical. This minimizes lot to lot variability and is highly desirable from a regulatory standpoint. Ease of use is also important to consider. This encompasses storage aspects, such as shelf life, and shipping and handling conditions. Ease of use also involves simplifying the process for the operator. As media and feeds become increasingly complex, how difficult does it become to formulate? Will operators have to perform risky pH adjustments with large volumes of corrosive reagents? Will high temperatures be needed to dissolve certain components, and can these high temperatures be achieved as the industry moves towards more single-use technologies? These and other factors must be considered when developing an optimized medium formulation.

What resources are required to develop an optimized medium?

The actual raw materials that form the bulk of a cell culture medium are generally inexpensive. The resources required for an optimized medium formulation that drive up the ultimate cost per liter are time, personnel, equipment, and expertise. With the increasing use of multivariate approaches (Design of Experiments, Principal Component Analysis), the use of high throughput systems become highly advantageous, reducing personnel requirements. Finally, expertise in media development is something that comes only with years of experience in the industry and cannot be bought with any amount of money.

So how do I develop an optimized medium formulation?

There are several approaches to media optimization. Some methods include titration, reverse engineering of other media, and metabolomics. While some of these methods are effective, when implemented on their own, they are time consuming, labor intensive, and heavily resource-dependent. A modern, DoE based approach to media development for CHO based processes is the CHOptimizer® Media Builder. CHOptimizer® consists of three distinct phases. The package is designed to be integrated into our ambr™15 system for automation and ease of use. In the first phase, four chemically defined, non-animal origin based media are blended in different ratios, according to a mixtures design DoE approach. Subsequent phases incorporate spent media analysis and fractional factorial DoE approaches to develop an optimized medium formulation, feed formulation, and corresponding feeding strategy. CHOptimizer® base media are developed using the expertise of Lonza Biologics, and combines a modern, high throughput approach with traditional ideas to deliver an optimized medium in a short timeline, with field based support. It also offers full access to the formulation for ultimate flexibility in the future.

To learn more about CHO media optimization join Sartorius Stedim Biotech’s workshop, chaired by Dr. Michael Gillmeister, Lonza at the IBC Cell Line Development & Engineering Conference, San Francisco, 13th June at 11.45am.

About the author:

Dr. Michael Gillmeister received his Ph.D. in Chemical and Biomolecular Engineering from the Johns Hopkins University in collaboration with the University of Maryland School of Medicine specializing in glycosylation, transient protein production, and neurobiology. In 2009, Mike joined the Gibco® research and development group and was responsible for next-generation media and sera projects. He then led media and process development projects to modulate product quality and maximize titer for PD-Direct® Custom Media Services using high-throughput and bioreactor technologies. Currently, Mike leads Protein Expression Media R&D and the CHOptimizer™ media optimization service for Lonza Walkersville.

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Striving to find new development methods for chemically defined media

In a recent article at European Business Review, Francesc Gòdia took an in-depth look at how to scale up the production of cell culture media in a safe way.  Reliability of supply, variability in performance and risk of viral infection has caused manufacturers to find new ways to develop these cell cultures.  The race is on and three alternatives to traditional methods are insulin, trasferrin, and albumin.  To examine the opportunities, Gòdia designed an experiment that screened for optimal compounds and another that optimized their concentration.  Read the results on page 36 of the European Biopharmaceutical Review.

This March, Gaurav Chauhan, M.S., Associate Scientist II, Cell Culture and Fermentation Sciences, MedImmune, will be on hand to share the presentation A Mathematical Model: Predict How HTST Impacts Media Treatment and Resolve Scale-Up Issues at Biopharmaceutical Development and Production Week.  For more information on this session and the rest of the program, download the agenda.  If you'd like to join us March 30-April 2, 2015 in Huntington Beach, California, as a reader of this blog when you register to join us and mention code BDP15JPLA2, you can receive the current lowest rate available to the event!

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How is the growing use of chemically-defined media increasing our understanding of the complexity cell culture manufacturing?

This week, we turn our sights to look at raw materials and supply chain management at Biopharmaceutical Development and Production Week.  Speaker David Kolwyk, M.S., MBA, Principle Scientist, Material Science, Amgen Inc. recently sat down to join us for an interview and previewed many of the topics he plans on look at during his presentation Strategies for Managing Impurities in Upstream Raw Materials.

Today, he looks at:
How is the growing use of chemically-defined media increasing our understanding of the complexity cell culture manufacturing?

When you look at the development of cell culture media, originally when we used, for instance, basal media plus fetal bovine serum – and fetal bovine serum was thousands, if not millions, of components in a supplement for the basal media that varied from year-to-year due to its biological origin. Or even when we went to hydrolysate and protein extracts. Once again, maybe it was just mostly protein, but it was thousands of different peptides, some vitamins, trace metals. It was very hard at that point to discern what was influencing or what components individually were influencing the performance of the cell culture process. You might find some markers. You might find some ratios of amino acids, but ultimately it was still fairly difficult. Whereas when you remove the chemically-defined media, at this point now you have a media, it has a defined list components – probably 50 to 70 different components. And that now makes it possible that you can really start to focus in and figure out which components in that media are actually critical to the performance of your manufacturing. And because they are chemically-defined components in the media, you can actually start to discern what attributes of those components are critical to your process performance. From doing that, you can then start to learn, “Okay if these attributes are important in this cell line, can I apply it to other processes? Can I further improve and increase the robustness of my media in such a way to minimize the impact of the variability of this component that is inherent to the manufacturing process?”

So, by moving to chemically-defined media, I think we’ve been able to build our understanding of how individual components in the cell culture process can impact our process performance and better develop strategies to be able to modulate and ensure that our processes are more robust and less susceptible to inherent variability in those cell culture components.

Download David's full podcast here.

David will be presenting Strategies for Managing Impurities in Upstream Raw Materials on Wednesday, March 26 in San Diego.  For more information on her session and the rest or the program, download the agenda.  If you'd like to join us for Biopharmaceutical Development and Production Week, as a reader of this blog, when you register to join us and mention code BDP14BLOG, you can save 20% off the standard rate.

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Cell Culture Media Supplements – The Role of Recombinant Proteins in Biopharmaceutical Manufacturing

Author: Brandy Sargent, Editor, Cell Culture Dish, www.CellCultureDish.com.  Brandy will be joining us this year at BioProcess International as a guest blogger on September 16-19, 2013 in Boston, MA.  If you'd like to join us at BioProcess International, as a reader of this blog, register to join us and mention code BLOG13JP to save 20% off the standard rate!

Defined supplements that improve growth, productivity and consistency are highly attractive for cell culture optimization projects. And, recombinant proteins are increasingly being used as defined supplements in biopharmaceutical, vaccine and stem cell manufacturing. The use of recombinant proteins is an important step in moving toward defined manufacturing for production cell lines. Recombinant proteins allow for the replacement of proteins that in the past have been only available through animal sourcing or from fractionation of donor blood.

This product review will cover six of the most commonly used proteins and their role in manufacturing.

Insulin
Insulin is an essential media component for most cell lines when removing serum from culture. In mammalian cells, insulin regulates metabolism and is responsible for cell signaling that controls glucose and amino acid uptake. Insulin is a key growth factor in cell culture media and has also been shown to promote cell survival and reduce apoptosis.

In the past, insulin has been sourced from animals, primarily pigs. Porcine insulin is the closest to human insulin with only a single amino acid variation, which made it a common choice for cell culture. In the 1980’s recombinant insulin was developed and approved to treat diabetes in humans. As production of recombinant insulin became more widespread, scientists began using the product in cell culture to replace animal-derived insulin.

Due to the length of time that recombinant insulin has been available, its use is well established in cell culture. There are two types of recombinant insulin available: recombinant insulin and animal-free recombinant insulin available. In recombinant insulin that is not animal-free, animal-derived enzymes are used in the manufacturing process, the product is manufactured in facilities where animal-products are used, or manufacturing uses serum during banking. In animal-free recombinant insulin, all products used in the manufacturing process are animal-free and it is manufactured in a dedicated animal-free environment.

Albumin
Albumin is the most abundant protein in serum and has many functions in cell culture. It increases overall cell health and productivity by carrying nutrients (lipids) binding and sequestering toxins and stabilizing hormones and peptides in media. Albumin also binds free radicals that can damage cells and cause apoptosis. As a result of these many benefits, the addition of albumin to serum-free cell culture media often provides an increase in growth and productivity. Albumin is also used in other areas of manufacturing and is often used in formulation to stabilize viral and protein based vaccines.

In many cell lines, scientists have been able to remove serum from media, but have needed to supplement with either bovine derived albumin (BSA) or human blood donor derived serum albumin (pHSA) to maintain healthy cells. Until recently, there were no recombinant albumin options available and this made removing serum from some cell lines nearly impossible without suffering an enormous performance hit. Now that recombinant albumin is available, it is reasonable that adoption of this protein will continue to increase, particularly in applications where completely animal-free manufacturing is required.

Transferrin
Transferrin is also found in serum and in culture is responsible for delivering iron to the cells. Transferrin is a universal iron carrier designed to deliver the appropriate amount of iron to cells in a receptor-mediated transfer of iron from transferrin to the cell. Specific transferrin receptors on the surface of cells up regulate or down regulate iron-bound to transferrin based on their iron need. Transferrin is uniquely able to deliver only the amount of iron needed by the cells. This is important because it eliminates the problem of cells taking in too much iron and causing cell damage, which can happen with other iron sources.

In serum-free systems, transferrin either needs to be replaced by bovine transferrin, human blood donor derived transferrin or other iron sources. Due to the absence of animal-free alternatives, some cell lines were adapted to grow with the use of other iron sources. However, many cell types do not grow as well with iron compounds as they do with transferrin. Now with the availability of recombinant transferrin, there are more animal-free options available for use in media formulation.

Trypsin
Trypsin is a serine protease and its main function is to hydrolyse proteins. For cell culture, it is frequently purified from animal sources, primarily porcine or bovine. It is an important tool used in cell culture to suspend adherent cells either during harvest or moving to a new plate. Trypsin cleaves the proteins that bind the cells to the plate or substrate. These properties make trypsin a key ingredient in vaccine manufacturing, some E. coli based manufacturing (including recombinant insulin production) and for harvesting stem cells.

Any process that uses trypsin to detach cells can’t be called animal-free unless the trypsin is recombinant and animal-free itself. Some sources for recombinant trypsin include the following: Roche carries a recombinant trypsin, Life Technologies carries a trypsin-like protease product called TripLE, and Sigma carries a recombinant bovine trypsin, Trypzean.

Aprotinin
Aprotinin inhibits trypsin and other proteolytic enzymes and prevents protein degradation of cells. The use of aprotinin is an important step in the manufacturing of recombinant proteins produced in E. coli. During cell lysis, enzymes are needed to break the cell walls and release the protein produced in the cells. While these enzymes are necessary to release the recombinant protein from the cells, they can also denature the protein itself and lower overall yield. Aprotinin added at the right stage in the process can inhibit enzyme action and prevent degradation of the recombinant proteins.

Aprotinin is most commonly purified from bovine lung tissue and thus prevents animal-free manufacturing if it is used. There are a few recombinant aprotinin options available; Sigma, Millipore and Cell Sciences all offer a form of recombinant aprotinin.

Lysozyme
Lysozyme is an enzyme that lyses bacterial cell walls. It is an important component of recombinant protein manufacturing in E. coli, as it is often used to extract recombinant proteins or DNA during product harvest. It is preferred over mechanical processes such as sonication and homogenization when shear forces of these processes result in degradation or denaturing of the product. Compared with detergents, Lysozyme does not denature the desired product. Some scientists use Lysozyme in combination with mechanical or detergent-based lysis. In these cases, they reduce potential loss of the target protein by reducing the intensity of sonication or homogenization, or reduce the concentration of detergents used. Recombinant lysozyme is offered by Merck KgA’s EMD Millipore division.

Defined Supplements and Recombinant Advantages
While each protein discussed above provides its own unique function to cell culture, there are some advantages that the recombinant versions offer. When animal-free manufacturing is required, recombinant proteins can offer a viable alternative to animal sourced products. In addition, they can provide more consistency and can also be easier to source with fewer regulatory restrictions than their animal-derived counterparts. While recombinant versions can be more expensive at first glance, it is important to factor in the advantages. After weighing the benefits, recombinant proteins in manufacturing can be quite cost-effective.

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A short history of cell culture media and the use of insulin

Today, we feature this article from our partners at BPI Magaine.

A short history of cell culture media and the use of insulin

The ancestors of cell culture media were the balanced salt solutions (BSS) devised by early workers interested in studying isolated organs ex vivo.

Such solutions provided irrigation and supply of water, bulk inorganic anions essential for normal cell metabolism, osmotic balance (isotonicity), a carbohydrate such as glucose as energy source, and a buffering system to maintain the medium within the physiological pH range (7.2 - 7.6) usually monitored by the addition of phenol red (1).

The first BSS was composed in 1885 by Sydney Ringer (1836-1910), a British clinician and pharmacologist from University College London wh
o worked on the isolated frog heart (2).

A version containing lactate is still used for intravenous perfusion. Ringer's solution was modified by Maurice V. Tyrode (1878-1930), an American physiologist born in Besançon to study isolated kidneys. Another popular balanced salt solution was devised by John H. Hanks (1906-1990), an American microbiologist, in order to attempt to cultivate the leprosy bacillusin vitro (he never succeeded) (3). Renato Dulbecco, an Italian born (1914) American virologist (who won the Nobel Prize in Physiology and Medicine in 1975 with Howard Temin and David Baltimore for their discovery of reverse transcriptase), described the much used Phosphate Buffered Saline (PBS) (4).

However it became clear that in order to get cells to actually proliferate in vitro, additional nutrients had to be added to culture media.

The first successful attempt at tissue culture is attributed to Ross Harrison (1870-1959) at Johns Hopkins University, who explanted in 1907 tadpole tissues in clotted frog lymph as nutritional and growth support, using a hanging drop on a glass slide. He was successful in observing neurite expansions (5,6).

Building on Harrison's success, the major early figure in tissue culture was Alexis Carrel (1873-1944), a French surgeon. He moved to the University of Chicago in 1903 and obtained the Nobel Prize in Physiology and Medicine in 1912 for inventing a method to suture blood vessels. In 1906 he moved to the Rockefeller Institute for Medical Reseach and started his work on tissue culture, in which he did pioneering work (124 articles, 7,8) and dominated the field, for better or for worse, until his death. He adopted Harrison's hanging drop technique but replaced the frog lymph by a plasma clot and fed the explants serum, a salt solution and chick embryo extract, which he called "plasmatic medium", which became a standard until the 1950's. His most famous experiment was the maintenance for over 34 years  of an embryonic chicken heart explant, finally discarded two years after Carrel's death. This nurtured Carrel's firm belief that primary cells in culture were immortal given the appropriate nutrition. Definitive evidence that this is wrong was provided by Leonard Hayflick (born 1928), an American gerontologist and professor of anatomy at UCSF, who demonstrated in 1965 that normal human diploid fibroblasts do not survive in vitro for more than approximately 50 divisions ("Hayflick's limit") (9).  The survival of Carrel's culture was likely due to the daily feeding of new embryonic cells from the embryonic extract.

The first continuous culture of a rodent cell line (L929) from a single cell was generated by Wilton R. Earle at the National Cancer Institute in 1943 (10). In 1951, George O. Gey (1899 -1970) at Johns Hopkins University developed the first continuous human cancer cell line from Henrietta Lacks's cervical carcinoma, the celebrated HeLa cell line whose eagerness to grow has resulted in crosscontamination of many cell cultures…

To read the rest of the white paper, visit BPI Magaine.

Follow the jump to see the References.

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