Abstract
BackgroundStandardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined.MethodsSupernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools.ResultsGrowth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)–CV); VEGF: 950–81.4; FGF-b: 27–74.6; PDGF-AA: 7883–28.8; PDGF-AB: 107834–32.5; PDGF-BB: 11142–48.4; Endostatin: 305034–16.2; Angiostatin: 197284–32.9; TGF-β1: 68382–53.7; IGF-I: 70876–38.3; EGF: 2411–30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential.DiscussionResults deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.
Highlights
Standardized animal-free components are required for manufacturing cell-based medicinal products
We have applied our statistical algorithm to analyze the data matrix of growth factor concentrations measured in single donor supernatant rich in growth factors (SRGF)
The algorithm was programmed to create in silico 500 groups of random data and to repeat this operation increasing group statistical sample size step by step from n = 2 to n = 20
Summary
Standardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Ex vivo cell expansion is often required to obtain advanced therapy medicinal products (ATMPs) for clinical use. Fetal bovine serum is the most common source of growth factors, and it is routinely used in research protocols as culture medium additive to promote cell expansion. Replacement of fetal bovine serum use is strongly recommended throughout Europe and United States [7, 8] for cell product manufacturing under GMP guidelines. To comply with GMP guidelines [1], separate batches of medium additives must be characterized by consistent concentrations of growth factors in order to consistently promote ex vivo cell expansion. Pooling together several single donor preparations can minimize the final product variability [13, 14]; otherwise, the lowest number of single donor SRGF, needed for production of pool products characterized by sufficient consistency between batches, would still need to be assessed
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