Cell-based therapies like CAR-T cell therapy have transformed modern medicine in the fight against cancers. Autologous CAR-T therapies involve isolation of primary T cells via leukapheresis. This is followed by an initial ex vivo activation and expansion phase before transduction of the CAR transgene. Final steps include a secondary cell expansion phase before cryopreservation and eventual infusion into the patient.

 

 

One of the major challenges, particularly for autologous cell therapies, is limited expansion or scale-up potential of isolated cells. Therefore, successful delivery of the final cell product is a function of and directly dependent on ancillary materials being used during the ex vivo bioprocessing of the isolated cells of interest.

 

Described in USP <1043> and Ph.Eu. 5.2.12 guidelines, ancillary materials for cell therapy manufacturing workflows may include cell culture media, supplements like sera or serum substitutes, antibodies for expansion and activating functions, and recombinant cytokines and growth factors. Thus, by extension, the quality of ancillary materials, including recombinant cytokines and growth factors, is critical to the success of the cell therapy.

 

In this blog, we discuss how purity, safety, and consistency must be key considerations when selecting recombinant cytokines and growth factors for use in manufacturing of cell therapeutics.

 

Purity

 

Purity of a recombinant protein can be defined as the quantitative measurement of the extent to which a protein of interest has been purified or isolated from other non-specific proteins, DNA, and miscellaneous impurities.

 

By definition, recombinant biotherapeutics are manufactured using recombinant technology in a variety of expression systems including bacteria, yeast, mammalian, insect, and plant. During the expression and purification process, inadvertent, and perhaps unavoidable, introduction of impurities originating from the host cell might become a part of the final purified recombinant protein. Because recombinant proteins can be used in ex vivo cell manufacturing workflows for cell therapy, the presence of unquantified impurities or microbial contamination could enter the production stream and affect product quality, safety, and efficacy. Therefore, determining purity of a recombinant protein is an important specification when considering use of a recombinant protein for cell bioprocessing workflows. Typically, SDS-PAGE is an industry-wide accepted method of purity quantification for recombinant proteins. While purity as close to 100% as possible is desired, > 95% purity is generally an industry-wide accepted purity level.

 

Safety

 

As discussed previously, in the context of cell therapy workflows, impurities or contamination directly affect usability, performance, and controllability of recombinant proteins. There are three critical safety specifications that need to be met when selecting recombinant proteins:

• Endotoxin test by LAL assay described according to USP <85> guidelines¹. Endotoxins are components in the cell walls of Gram-negative bacteria that are released during bacterial lysis and can be ubiquitous in the laboratory. Ensuring ultra-low levels of endotoxins in the final purified recombinants helps ameliorate potential severe anaphylactic responses.
• Bioburden/sterility test for bacteria and fungi described in USP <71> guidelines².
• Mycoplasma test described in USP <63> guidelines³. Mycoplasma, a group of bacteria without cell walls, are common and can cause serious contamination in cell cultures.

 

Additional safety specifications that should be evaluated are Host Cell Protein (HCP) content as described in USP <1132>⁴ and Host Cell DNA (HCD) content as described in USP <509>⁵. During the expression and purification process of recombinant proteins, certain amounts of host-cell derived residual proteins and DNA get introduced into the final product. HCP test facilitates quantifying these non-specific host proteins by ELISA while HCD can be determined by qPCR.

 

Finally, N-terminal sequencing for recombinant proteins is another safety specification that is evaluated while considering the cell therapy workflows. Denoted as an analytical specification, N-terminal sequencing provides information on sequence, purity, and correct translation of recombinant proteins. Having the correct sequence influences protein function, including its subcellular distribution, degradation, and turnover rate. Described in USP <1052> guidelines⁶, typical N-terminal sequencing provides sequence information on the first 10 – 15 amino acids determined by either Edman Degradation or Mass Spectrometry.

 

Consistency

 

Conclusion

 

In conclusion, purity, safety, and analytical characteristics must be key considerations when selecting recombinant cytokines and growth factors to manufacture final cell therapy products. Therefore, robust ex vivo bioprocessing of cells, using cell culture media supplements such as cytokines and growth factors to signal cell differentiation and expansion, is critical to obtain a successful final product.

 

References

1. <85> Bacterial Endotoxins
2. USP <71> Sterility
3. USP <63> Mycoplasma Tests: A New Regulation for Mycoplasma Testing
4. Host Cell Proteins: Detect and characterize problematic host cell proteins to improve overall drug quality
5. USP Reference Standards for measurement of residual host cell DNA.
6. <1052> Biotechnology-Derived Articles— Amino Acid Analysis