(160i) Characterization and Analysis of Dissolvable Microcarrier Byproducts

Microcarriers enable efficient cell scale-up in controlled bioreactors for therapeutic applications. Effective cell recovery from microcarriers often requires the use of concentrated enzymes, extended treatment times, and continuous centrifugation/filtration cycles, which can negatively affect cell health and recovery yield. To address these challenges, Corning Life Sciences developed a calcium-crosslinked polygalacturonic acid (PGA) microcarrier that can be dissolved using ethylenediaminetetraacetic acid (EDTA) and pectinase. Here we describe the characterization of the PGA polymer, dissociation reagents, and the byproducts of bead dissolution and evaluate their impact on subsequent cell growth.

Polygalacturonic acid (PGA), also known as pectic acid, is a water-soluble biopolymer of pectin degradation found in ripe fruits and some vegetables^1,2. Microcarriers of PGA polymer are made by dropping a solution of PGA into a calcium chloride bath, which results in gelation of the droplet as a spherical bead. Excess calcium chloride is removed from the microcarriers through a series of wash cycles before the microcarriers are coated with cell attachment-promoting substrates such as Corning® Synthemax™ II or denatured collagen.

PGA microcarriers can be destabilized by removing the calcium crosslinking between polymer chains using EDTA. EDTA is broadly used as a reagent for cell harvest in biological applications and has been used in the pharmaceutical industry to treat lead poisoning, heart and blood vessel diseases, and a wide variety of other health conditions such as cancer, arthritis, diabetes, and psoriasis³. In the food and cosmetics industries, EDTA has been used to improve the stability, color, and texture of products³. PGA polymers can be further degraded using the enzyme pectinase, which hydrolyzes the linkages between the monomeric units, and hence reduces the molecular weight of the PGA polymer and increases its solubility. In general, pectinase is associated with fruit ripening, as it promotes the softening of plant cell walls; as such, pectinase has been issued a Generally Recognized as Safe (GRAS) notification by the FDA for use as a direct human food ingredient⁴.

Byproducts of PGA Digestion

Since dissolvable microcarriers are made from PGA polymer, we first examined the properties of un-crosslinked PGA polymer. We digested the PGA polymer using EDTA and pectinase and analyzed samples using size-exclusion chromatography (SEC) to separate byproducts by their molecular weight. PGA polymer, EDTA, and pectinase were run separately as controls to determine their relative elution profiles and retention times. As shown in Figure 1, we observed strong pectinase and EDTA peaks due to high absorbance at 214 nm. The overlap between the low molecular weight PGA peaks and pectinase peaks could interfere with detection of larger digestion fragments from PGA. Similarly, EDTA had a peak (molecular weight of 292.17 daltons), potentially masking expected PGA monomer and dimer peaks at molecular weights of 194.14 daltons and 370.26 daltons, respectively. After mixing a solution of PGA polymer with pectinase, we observed low molecular weight periodic peaks, likely a result of different lengths of digested galacturonic acid oligomers (Figure 2). As digestion time increased, the low molecular weight peaks became stronger, suggesting that PGA oligomers were continually digested to lower molecular weight oligomers. After an extended time, we observed a decrease in these periodic peaks and an increase in a low molecular weight peak at 4.17 minutes. The retention time suggests that its molecular weight is lower than that of EDTA and is most likely galacturonic acid monomer. In a separate experiment using Electrospray Ionization Mass Spectrometry (ESI-MS), we observed a strong galacturonic acid monomer peak (m/z = 193) with an increase in digestion, supporting the SEC findings that the PGA polymer will completely digest into monomers.

Next, we sought to compare degradation byproducts of the PGA polymer to that of PGA microcarriers. When only EDTA was added, the microcarriers were dissociated into PGA polymer. As expected, we observed a PGA peak around 2.10 minutes and EDTA peaks at 3.91 and 4.03 minutes (Figure 3). When pectinase and EDTA both were added, microcarrier dissociation and PGA polymer digestion occurred at the same time. We observed additional pectinase peaks between 2.2 and 3.0 minutes, multiple periodic peaks after 3.5 minutes which weakened with increased digestion time, and single strong peak at 4.17 minutes corresponding to the PGA monomer. Due to interference from the EDTA peaks, we could not resolve PGA oligomer peaks between 3.85 and 4.10 minutes. Based upon the strong peak at 4.17 minutes, these results agree with the PGA polymer SEC and MS data and demonstrate that PGA microcarriers dissolve into galacturonic acid monomers.

Calcium and EDTA Concentration

Calcium content measured by ICP-MS was 90-110 mg/L, or approximately 2.25-2.75 mM calcium ions in the dissolvable microcarrier digested solution. SEC data showed an approximate 60% reduction in the EDTA peak at 3.91 minutes in a digestion solution containing 4 mM EDTA, which agrees well with the ICP-MS results of the amount of calcium released. This data suggests that most of the calcium released from dissolved microcarriers binds to EDTA, and EDTA is in excess at 4 mM.

Removal of Soluble Digestion Components from Recovered Cells

Since dissolvable microcarrier digestion products include small PGA oligomers (monomers), EDTA, calcium, and pectinase which may have negative impacts on subsequent cell growth. To determine if these components can be easily separated from cells, post-recovery, we completed a series of wash and centrifugation cycles. The results suggested that the final concentration of residuals could be reduced to 0.4% of the original value after 4 wash/centrifugation cycles. Based on these results and considering the byproducts are proteins (pectinase) and small water-soluble molecules (PGA oligomers, EDTA), it is reasonable to expect that tangential flow filtration or other commonly-used cell purification methods will be able to remove these byproducts.

Digestion Components Impact on Cell Growth

To better understand if digestion byproducts and reagents have a negative impact on subsequent cell growth, Vero cells cultured on dissolvable microcarriers were harvested then reseeded into T-75 flasks in fresh culture media containing a dilution of the collected digestion solution (1:5 to 1:100). Vero cell growth was significantly impacted by the presence of the harvest solution at low dilutions (1:5 to 1:10), and modest inhibition of growth was observed up to 1:40 dilution. These results suggest that there are components in the digestion solution that inhibit cell growth. To further investigate the impact of pectinase and EDTA on reseeding Vero cells on dissolvable microcarriers, pectinase (10 to 100 U/mL final concentration) or EDTA (1 to 5 mM final concentration) was spiked into disposable spinner flasks containing dissolvable microcarriers. Result showed that the presence of pectinase in the cell culture medium at concentrations ≥40 U/mL resulted in decreased cell yield, and the cell concentration was minimally impacted by pectinase concentrations up to 30 U/mL (less than 10% reduction). The results suggest that the digestion solution should be diluted greater than 1:3 to minimize inhibition of cell growth due to pectinase. Similarly, EDTA had a significant impact on cell growth, and concentrations ≥2 mM had an impact on dissolvable microcarriers integrity which resulted in dissolvable microcarriers dissolution. To minimize the impact of EDTA, the final concentration should be less than 1 mM. Further, the addition of calcium chloride during cell reseeding was unable to neutralize EDTA and pre-equilibrating calcium chloride with EDTA-containing cell suspensions prior to cell seeding had a negligible effect. Based upon these results, we recommend removal of the microcarrier digestion solution from cells via centrifugation, filtration, or perfusion prior to reseeding or optimize microcarrier dissolution at lower concentrations of pectinase and EDTA (e.g., 30 U/mL pectinase, 1 mM EDTA).

Conclusions

• Polygalacturonic acid (PGA), pectinase, and EDTA use in pharmaceutical and food industries has been well documented.
• PGA microcarrier digestion using EDTA and pectinase results in small soluble monomers, as measured by SEC and MS.
• SEC and ICP-MS analysis shows that the calcium ions released during bead dissolution are captured by an excess of available EDTA.
• Soluble components resulting from bead dissolution include PGA monomers/oligomers, calcium, EDTA, pectinase, and surface coatings, and these can be reduced or removed from recovered cells through a series of wash/centrifugation cycles.
• Beyond a threshold concentration, residual pectinase and EDTA used for bead digestion have a negative impact on subsequent cell growth. These components should be removed or significantly reduced via centrifugation, filtration, or perfusion prior to cell passage or long-term storage.

References

1. Minzanova ST, et al. Biological activity and pharmacological application of pectic polysaccharides: A Review. Polymers (Basel). 2018 Dec 19;10(12). pii: E1407. doi: 10.3390/polym10121407
2. Pornsak Sriamornsak, Chemistry of pectin and its pharmaceutical uses: A Review. Silpakorn University International Journal 01/2003; 3:206-228.
3. WebMD for EDTA https://www.webmd.com/vitamins/ai/ingredientmono-1032/edta
4. Pectinase GRAS notification. https://www.fda.gov/food/generally-recognized-safe-gras/enzyme-preparations-used-food-partial-list