Stem cell therapies are some of the most cutting-edge and sophisticated therapeutic developments. They offer an¬†attractive alternative approach to more¬†widely used treatments for conditions¬†such as multiple sclerosis, metabolic¬†diseases, cardiovascular disease, liver¬†disease, and cancer. But developers still¬†face challenges, some of which can be¬†addressed by the use of recombinant¬†human albumin. As a long-established
ingredient of cell culture media, albumin¬†is well recognized for its ability to¬†facilitate growth of many cell types. The¬†industry is expanding its use of high-quality, fully recombinant, current good¬†manufacturing practice (CGMP)¬†excipients in cryopreservation and¬†formulation of stem cell therapies. Herein¬†we describe the manufacturing,¬†formulating, and handling challenges¬†associated with the development of cell¬†therapies and assess how the use of fully¬†recombinant human albumin in the¬†culture, expansion/differentiation,¬†cryopreservation, and formulation of¬†stem cells compares with alternative¬†approaches.
Cell Therapy and Albumin
Cell heterogeneity is a main concern in¬†cell therapy development and¬†formulation. It is a function of the entire¬†value chain design ‚ÄĒ from harvesting,¬†upstream culture, and cell preservation to¬†therapeutic administration. Consequently,¬†biomanufacturers strive to understand¬†and control all process steps to optimize¬†cell viability and variability.
Serum and human serum albumin¬†(HSA) are useful stabilizers in cell culture¬†and preservation media. In stem cell¬†culture development, albumin contributes¬†different functions. Present at amounts of¬†about 40 g/L, human albumin is the most
ubiquitous protein in blood. It serves as a¬†buffer or reservoir for smaller entities such
as metals, hormones, fatty acids, and¬†toxins, and it shuttles such molecules¬†from areas of high concentration to sites¬†of low concentration. Albumin constitutes¬†about 75% of the colloidal oncotic¬†(colloidal osmotic) pressure of blood, and¬†its single free cysteine makes up most of¬†the reducing equivalents in blood. These¬†properties make albumin a useful¬†component in development of stem cell¬†therapies.
Regulatory concerns over blood-borne¬†contaminants (e.g., mycoplasma, viruses,¬†and prions), potential issues with¬†reliability of supply, and the performance¬†variability of HSA and undefined serum¬†all have led to an increased industry¬†demand for more well-defined, well-characterized, and easily controlled¬†media. Recombinant human albumin is a¬†serum-free, CGMP raw material of high¬†consistency and purity that enables¬†complex culture media to be chemically¬†defined. Regulatory agencies prefer the¬†use of chemically defined media because comprehensive quality information must¬†be available for their constituents.
Recombinant Albumin in Cell Therapy
Cell therapy developers can encounter a¬†number of challenges when optimizing¬†cell performance and controlling¬†variability. From a processing¬†perspective, aggregation, shear, surface¬†interaction growth rate, and¬†reproducibility all can pose serious issues
for manufacturing. Efforts to maintain¬†cell identity and multipotency and to¬†ensure cell survival during transformation¬†also can lead to problems during¬†development. Formulation, quality¬†control of final products, storage and¬†transportation, apoptosis, loss of identity,¬†multipotency, and safety all can be¬†sources of risk to product development.
Recombinant human albumin¬†enhances cell therapy applications, thus¬†making it one of the best ways for cell¬†therapy developers to improve product¬†quality. Albumin readily coats the surface¬†of cells, thus eliminating unwanted¬†interaction between cell and surface. This¬†protein also can prevent aggregation of¬†cells in formulation, protect cells from¬†shear stress during processing, and¬†stabilize cells during freeze and thaw.¬†Most important, albumin does not¬†contain blood-derived impurities that¬†otherwise could activate unwanted cell¬†pathways. And recombinant albumin can¬†help maintain the safety and efficacy of¬†final drug products during storage.
Recombinant Albumin in Stem Cell Cryopreservation
For development of commercial stem cell¬†therapies, cryopreservation is a vital and
often necessary step because it¬†eliminates the requirement for a¬†continuous culture for maintaining¬†viability, which increases flexibility and¬†logistical advantages. Cryopreservation¬†ideally takes place at two stages in a¬†development process leading to a¬†differentiated stem cell therapy dose. The¬†first stage generally is at an upstream¬†point, typically as close to harvest or¬†stem cell generation as possible. The¬†second stage takes place as far¬†downstream in a process as possible,¬†ideally after production of a
differentiated stem cell therapy dose.¬†
The first step in cryopreservation can¬†contribute economies of scale because¬†multiple batches can be produced either¬†in parallel or in combination, depending¬†on the nature of a therapy. The second¬†step allows for flexibility in distribution¬†and dose administration. Such¬†advantages make cryopreservation¬†highly useful in product development.¬†
During cryopreservation, stem cells¬†usually are removed from their growth¬†medium (which supports the cells‚Äô¬†biological need for growth), and they are¬†placed in a cryopreserving medium. That¬†environment generally contains only salts¬†and buffers (and no other small-molecule¬†nutrients) to maintain pH and isotonic¬†conditions and different cryopreservation¬†agents such as dimethyl sulfoxide¬†(DMSO) and albumin. After the¬†formulation step, stem cells are quickly¬†frozen. Once thawed, the cells are either
returned to a growth medium supporting¬†further processing or introduced to a¬†final formulation solution, depending on¬†whether they are to be further processed¬†or administered.
Using albumin can provide several¬†unique advantages to a cryopreservation¬†process. Albumin can help stem cells¬†endure media change and other¬†transitions because it covers cell surfaces¬†and enhances the buffering capacity and¬†stability in solution. The purity and¬†source of albumin (plasma-derived or¬†recombinant) play significant roles in
preventing human mesenchymal stem¬†cells (hMSCs) from progressing to late-state apoptosis (Figure 1). That ensures a¬†higher postthaw viability of stem cells
cryopreserved with recombinant human¬†albumin and a longer shelf life than is
achievable with other techniques.
Figure 1 shows the postthaw viability¬†of hMSCs, comparing Albumedix
recombinant albumin (AlbIX) with HSA.¬†With HSA, viability drops significantly below the release criteria of 70%,¬†whereas the AlbIX albumin maintained¬†significantly better cell viability after¬†thawing than HSA. The product met the¬†acceptance criteria after 72 hours,
whereas products conditioned with HSA¬†were out of specification after 24 hours.
Recombinant Albumin in Stem Cell Formulation
The right formulation is critical to¬†reproducibly delivering a functional cell¬†therapy. Typically, formulation takes place¬†immediately after generation or thawing¬†of differentiated cells. Following¬†formulation, several time-consuming¬†release assays must be performed before¬†a product is administered to patients. As¬†a result, the longer that cells can be¬†maintained in a stable state, the greater¬†the applicability and flexibility of a cell¬†therapy.
Preparing stem cell formulations with¬†controlled media generally can make the¬†release of a therapy go smoothly by¬†enabling analysts to focus on the therapy¬†itself rather than the potential effects and¬†impurities from its medium. A controlled¬†formulation should reduce variation in¬†the background of biological assays.¬†However, because of carryover between¬†process steps, it is not enough to use¬†controlled media to generate a final
formulation. If a controlled final¬†formulation is needed, it must be¬†designed into a process sufficiently¬†upstream to ensure that enough dilutions¬†and exchanges have taken place to¬†mitigate risks from uncontrolled¬†substances. So substances such as
recombinant human albumin should be¬†considered and introduced in advance of
final formulation to minimize additional¬†regulatory scrutiny.
Albumedix conducted an in-house¬†study showing that reduced variability of¬†AlbIX recombinant albumin created the¬†same effect across multiple batches¬†(Figure 2). The product also supported¬†growth and outperformed both HSA and¬†another recombinant albumin.
In many cases, cell therapies are modified¬†to alter their function, to activate in the¬†case of immune cells, or to express¬†certain proteins or antigens on their¬†surface. When such modifications are¬†made using viral-vectored gene therapies¬†to deliver recombinant DNA, chimeric¬†antigen receptors, or CRISPR-Cas9¬†modifications, recombinant albumin may¬†be able to improve the efficiency of such¬†activities. An article by researchers at the¬†University of North Carolina (1) showed¬†that adding human serum albumin¬†before cryopreservation or during¬†formulation improved adenoassociated¬†virus (AAV) vector transduction five- to¬†seven-fold, resulting in a concomitant¬†increase in expressed and active protein.
Those researchers also conducted¬†mechanism studies and suggest that¬†‚Äúhuman albumin increased AAV vector¬†binding to the target cell surface and¬†resulted in faster blood clearance after¬†systemic administration but did not¬†impact AAV infection pathway.‚ÄĚ The¬†implication is that albumin can act as a¬†‚Äúchaperone‚ÄĚ to assist in the interactions
between AAV vectors and cells. It is¬†interesting to note that AAVs enter cells¬†by means of their endosomes, which also¬†is the pathway by which albumin is¬†recycled. Thus, whether such vectors are¬†used to modify cell therapies or to serve¬†as standalone therapeutics, adding¬†albumin can significantly improve the¬†performance of modified cells. It is not¬†clear whether the use of albumin similarly¬†would improve other viral vectors, so¬†further exploration would be warranted.¬†For that effort, fully recombinant albumin¬†would be preferable to HSA in such¬†cutting-edge technologies.
A Safe Alternative
In both upstream and downstream cell¬†suspensions, recombinant human¬†albumin contributes many useful traits in¬†the development of stem cell therapies. It¬†can act as a nutrient carrier to cells to¬†ensure optimal growth conditions, as a¬†stabilizer for cell membranes and¬†coformulated proteins, as a scavenger of¬†free radicals, and as a viscosity moderator.¬†Recombinant human albumin offers a¬†safe solution for optimized cell¬†performance. Its use not only improves¬†the regulatory pathway of stem cell
therapies, but also can considerably¬†improve the cell viability and morphology¬†while controlling batch-to-batch¬†variability through higher tolerance to¬†stress. Such attributes call for cell therapy¬†and stem cell media developers to begin¬†looking at recombinant albumin in a new¬†way and consider its potential for clinical¬†development of cell therapies. It aids in¬†cryopreservation and expansion¬†improvements and has been a critical¬†tool for enhancing the functional¬†performance of cutting-edge stem cell
therapies. Cell therapy manufacturers can¬†benefit from greater intrinsic safety and
regulatory advantages provided by using¬†recombinant albumin.
We acknowledge our collaborators Xcelia, a¬†biotechnology company located at Banc de¬†Sang I Teixits, the UAB (University Aut√≥noma¬†Barcelona), and Eva J√łrgensen for her input¬†and expertise in creating this article.
1 Wang M, et al. Direct Interaction of¬†Human Serum Proteins with AAV Virions to
Enhance AAV Transduction: Immediate Impact¬†on Clinical Applications. Gene Ther. 24(1) 2017:¬†49‚Äď59; doi:10.1038/gt.2016.75.
Phil Morton is science director of bioprocess characterization, and Dr. Daniel Shelly is business development director at Albumedix; firstname.lastname@example.org; 44-1606-889-194.