Incorporating recombinant proteins to produce blood-free media formulations for therapeutic cell types


Inclusion of human plasma or plasma-derived proteins, such as albumin and transferrin, in cell culture systems produce media with acceptable performance at small scale. However, scale up to clinical manufacturing with these components proves to be more difficult. Most often plasma or plasma-derived components need to be screened prior inclusion in manufacturing processes as these raw materials often induce unacceptable variability in the final cell product. These variabilities often manifest in unexpected cell or cell product yield or unwanted drifts in the expected phenotype or function of cell or cell-based products. Further, the inclusion of human plasma or plasma-derived components introduces the risk of contamination from adventitious agents.

Advances in recombinant protein expression has produced recombinant versions of serum albumin and transferrin that are both biologically active as well as economically viable. Incorporation of these proteins can produce media formulations that may simplify research and development through the higher degree of chemical definition as well as to facilitate the regulatory approval process for clinical products due the absence of blood-derived components. However, successful incorporation of these recombinant proteins to produce fully animal component free formulations requires further formulation work to replace undefined biologically active compounds that are present in native protein preparations. Haphazard substitutions of these recombinant proteins into formulations that are designed for serum-derived proteins may produce less active media formulations, thereby often resulting in a quick revert. This technical challenge in incorporating these recombinant proteins is only further exacerbated by the ease of use of native derived proteins.

This week InVitria presents Randall Alfano, Ph.D., Vice President of Product Development, as an industry expert in the application of recombinant albumin and transferrin for the expansion of primary, stem, and biomanufacturing cell types. InVitria has been successful in helping clinical development phase vaccine, stem, and immunotherapy groups to remove all blood-derived components from the manufacturing processes by formulating custom blood-free media formulations that have been designed around recombinant albumin and transferrin.




Question 1

For which primary cell lines have you been able to create a blood-free media? Do you find that different cell lines require varying levels of albumin/transferrin?

The levels of incorporation of both recombinant albumin and recombinant transferrin will vary depending on the cell line. I would invite you to visit and download the cellastim-s  and optiferrin product bundles. These bundles contain suggested incorporation levels of both recombinant albumin and transferrin for an array of different primary, stem, and biomanufacturing cell types.

Question 2

How has the transition from xeno-free or FBS supplemented media to your formulations been? Is it a difficult transition for the cells?

Typically transition from xeno-free or FBS -supplemented media is straight forward. With direct adaptation, cells usually adapt within a passage and do well in the blood-free media in subsequent passages. However, adaptation is cell-type specific and some cell types require a step-wise gradual transition from xeno-free media to blood-free.

Question 3

Are these supplements or are these formulated into the media. Do your proteins need to be added during culture or just in the beginning?

InVitria offers recombinant versions of critical components that are often sourced from human serum, albumin and transferrin. These components, known as Cellastim S (albumin) and Optiferrin (transferrin) are incorporated into complete media formulations that also include recombinant insulin found in ITSE and animal free cytokines that are specific to the cell type of interest. These components would be present for the entire time span of the cell culture you are working with.

Question 4

Have you tested the inclusion of your proteins in cryopreservation media? Any impact on thaw viability?

Cellastim S has been evaluated in cryopreservation media. The data suggests that Cellastim S can in fact replace human serum-derived albumin in cryopreservation media.

Question 5

Can these be used with feeder-free cultures?

Yes. Blood-free media formulations are typically complete enough to be used without feeder cells.

Question 6

Do you include any other proteins or growth factors in your media besides the albumin and transferrin?

Yes. Most cell types require supplementation with cytokines. There are several different sources of cytokines that are considered to be animal free and produced in E. coli. The choice of which cytokines that should be used will be dependent on the cell type that is trying to be expanded. For example, T cells require IL-2 where as VERO expansion is dependent on EGF.

Question 7

Is there any evidence that these supplements could help cell recovery post thaw?

Cells that are thawed directly into blood-free media typically recover well from cryo-preservation. Addition of the individual components, such as Cellastim S, can facilitate different processes in cell culture such as single cell cloning. For example, addition of Cellastim S, Optiferrin, and ITSE to commercial CHO media can reduce timelines in CHO cell line selection from single cell cultures to having adequate number of cells to analyze growth and expression potential of clonal populations.

Question 8

Has there been any impact on karyotype of the cells with the recombinant versions of the proteins?

We routinely look at cellular phenotype via expression of phenotypic markers specific for the cell type of interest as well as cellular function and differentiation (if relevant). We have found that, in terms of phenotype and growth kinetics, that cells cultured in the presence of these recombinant proteins are normal and display the expected growth kinetics. Further, in specific applications, we find that cellular function is actually better in blood-free media versus human serum component-containing media through the deletion of bioactive serum-derived contaminates. Though karyotype is an interesting, we currently have not looked at this quality parameter.

Question 9

Have you discovered any significant differences in cell health and growth between use of recombinant and native supplements?

Overall, we have not seen significant differences in cell health and growth between recombinant and native supplements. However, there are particular instances where we have seen better growth kinetics in the blood-free media versus xeno-free SFM and serum-containing media. This is due to the presence of undefined components in native supplements that are actually having adverse effects on cell growth.

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