The Next Level of Quality for Cell Therapy Media – Beyond Xeno-Free

By on January 18, 2018

A Guest Blog by Randall Alfano, Ph.D., Vice President, Product Development, InVitria

What Does InVitria’s “Blood-Free” Really Mean?

Since the advent of in vitro biology, mammalian cell culture and cell culture-based assays have served as an indispensable tool in the elucidation of biological processes. This article will discuss the current landscape of FBS and human serum use and their limitations, as well as introduce a powerful alternative: InVitria’s blood-free cell culture media components.

Fetal Bovine Serum

Fetal bovine serum (FBS) has been a central component in the establishment of in vitro cell culture as successful expansion of cells ex vivo is dependent on cell adhesion promotion via extracellular matrix proteins, stimulation of cellular proliferation by the presence of growth factors, and nutritional support through the presence of sugars, amino acids, and other undefined components provided by FBS [1].

Although the use of FBS has broadly been adopted, its removal from cell culture media has been recognized as an urgent priority amongst cell culturists for several reasons:

  • Primarily, the addition of FBS introduces an unacceptable degree of unpredictability in any in vitro Studies have confirmed that different lots of serum can fluctuate in a concentration of known components in some cases more than 10-fold in addition to the inconsistent presence of other undefined components [2]. These noted differences in FBS composition can have profound effects on the outcome in cell culture systems [3].
  • Further, due to the large demand for FBS and the absence of exact annual production volumes, adulteration and fraud have been well noted in the serum industry. 30,000 liters of FBS were documented to be sourced from New Zealand in 1994. However, the country itself only reported 15,000 liters annual production [4]. Another prominent adulteration case was identified in 2013 with the dilution of FBS with bovine albumin, water, and other growth-promoting substances by a major global supplier [5]. Subsequent investigations conducted by the FDA determined that approximately 280,000 liters of FBS were affected between 2008-2013 [5].

In the spirit of innovation and a desire to better define the culture environment of cells in vitro, attempts to remove FBS enabled the formulation of media that was able to function without FBS as long as specific components purified from bovine serum, most notably bovine albumin, and transferrin, were included at specified concentrations [6, 7].

The natural progression of these formulations eventually prompted the substitution of the bovine serum-derived components for those purified from human serum. These serum-free media formulations that incorporate human serum sourced albumin and transferrin, known as xeno free, perform well in supporting cellular proliferation ex vivo and exhibit a higher degree of media definition than FBS-containing media. However, these human serum protein-containing serum-free media still possess the issues of supply reliability and variability exhibited by FBS-containing media.

A Limited Supply Chain

In terms of security of supply, albumin can be examined in greater detail although the same supply limitations that can affect serum transferrin. Currently, the global demand for human serum albumin is approximately 663,000 kg and is increasing due to the application of human serum albumin in a multitude of therapeutic and emerging biotech applications [8, 9]. Human serum albumin is typically sourced from plasmapheresis, although both time-expired blood and placental material have also been utilized [8, 10]. Other plasma-derived components, such as Alpha-1 antitrypsin, immunoglobulin fractions, and clotting factors, are also utilized and thus reduces the amounts of available donor plasma for albumin purification.

Overall, it is projected that 40 million liters of plasma will be recoverable in 2018 from the United States and Europe, estimating the supply of serum albumin between 1.4 and 2.0 million kg from western countries prior to any purification or consideration of other plasma-derived product manufacturing [9]. Indeed, 130 plasma donations are required to manufacture enough intravenous immune globulin therapy for a single patient with primary immunodeficiency for one year [11].

Product Variability

The amount of plasma available for serum component manufacture is one aspect to take into consideration. The dynamic nature of these serum proteins in circulation in health and disease and diverse population pools used to source plasma that could potentially drive final product variability is another.

Variable oxidation states in human serum albumin lots intended for clinical use have been described when compared to fresh isolations of healthy volunteers [12]. Further, other significant posttranslational differences in commercial human serum albumin preparations, which may also adversely affect protein function, were also found to variable degrees [12]. Given the central functional role of these proteins combined with the high concentrations in media, inconsistencies in these components can greatly affect the overall media performance.

Infectious Agents

Finally, the aspect of adventitious agent contamination from blood is of constant concern. Several safety measures have been implemented in the manufacture of human serum-derived components, such as prescreening voluntary donors, pooling large numbers of donors for contaminate virus dilution, and viral inactivation steps during purification and final packaging [13].

However, the risk of transmission of infectious agents cannot be eliminated as viral titer and virulence reduction steps can be escaped [13]. Hepatitis virus has been shown to be resistant to chemical and physical inactivation as serum contaminated with Hepatitis B virus was shown to be infectious in lab chimpanzees post-pasteurization for 10 hours at 60⁰C. However, high initial titers would be required to retain infectivity in the final product which lowers the probability of actual occurrence.

To support this, there is no known case of virus transmission from purified serum albumin in man although cases of hepatitis B transmission have been identified in plasma protein fractions [13]. Nevertheless, the utilization of human plasma-derived products represents a known risk of virus transmission due to the constant threat of new outbreaks and emergence of novel pathogens.

Blood-Free: InVitria’s Innovative Solution

To circumvent some of the issues with the use of native derived proteins, that same innovative spirit that first prompted the removal of FBS from cell culture media has again struck and InVitria has taken the desire to chemically define cell culture media to the next level.

Recombinant protein expression technology has advanced at impressive rates and expression methods of non-mammalian origin are now being utilized to manufacture human serum proteins at large scale [14]. As a result, recombinant human albumin and transferrin are now readily available and have been proven functional in cell culture media that is completely void of any human or animal blood-derived components. These animal-free components, when incorporated into media, are known as mammalian blood component-free, or simply blood-free, and have demonstrated excellent ability in supporting cell growth of phenotypically correct and functional cells compared to both FBS-containing and xeno free media in multiple cell types.

Although important, functional characteristics of these blood-free recombinant proteins are only a single consideration that must be accompanied by other quality requirements, such as security of supply chain, manufacturing standards, and origin of these recombinant proteins. InVitria retains control of the entire manufacturing supply chain of these recombinant proteins, from genetic construct to final product and does not outsource any aspect of the manufacturing process. Further, these products are manufactured in the United States in an ISO9001-certified animal component free facility.

Thus, InVitria has raised the bar in that the blood-free classification encompasses a new standard of performance and quality in cell culture media components that to date ensures the highest degree of chemical definition in cell culture systems. Responsible and sustainable manufacturing methods solely based in the United States produce blood-free recombinant proteins at commercially and economically viable scales. Incorporation of these blood-free proteins ensures better chemical definition in cell culture media. This enhanced definition drives reproducibility, a deeper understanding of factors that drive desirable outcomes in cell culture and fuels additional R&D, and ultimately ensures a safer therapeutic for the patients who depend on them.

If you are interested in learning more about blood-free recombinant proteins and media design and optimization that incorporates these components, be sure to catch us at Cell and Gene Therapy in Miami, Florida, or get in touch with one of our experts here.


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[2] Baker M, “Reproducibility: Respect your cells!,” Nature, pp. 433-5, 2016.
[3] Baker M, “1,500 scientists lift the lid on reproducibility,” Nature, pp. 533, 452-454, 2016.
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[8] Raoufinia R, et al., “Overview of albumin and its purification methods,” Adv Pharm Bull, vol. 6, no. 4, pp. 495-507, 2016.
[9] Marketing Research Bureau, “Product Forecast for Serum Albumin,” 2013.
[10] Matejtschuk P, et al., “Production of human albumin solution: a continually developing colloid,” British Journal of Anaesthesia, vol. 85, no. 6, pp. 887-95, 2000.
[11] Baxter, “Baxter,” Baxter , September 2012. [Online]. Available: [Accessed 8 January 2018].
[12] Bar-Or D, et al., “Heterogeneity and oxidation status of commercial human albumin preparations in clinical use,” Crit Care Med, vol. 33, no. 7, pp. 1638-1641, 2005.
[13] Erstad B, “Viral infectivity of albumin and plasma protein fraction,” Pharmacotherapy, vol. 16, no. 6, pp. 996-1001, 1996.
[14] Zhang D, et al., “Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.).,” Protein Expr Purif. , pp. 74(1):69-79, 2010.


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