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Hygromycin B EvoPure®

Technical characteristics

EvoPure® Grade Hygromycin B can lead to faster and more accurate transfection results.
Several experiments have verified this result.

Table 2

LD 50 Hygromycin B Concentration (µg/mL) for Sensitive Cells from Different
Commercial Scources (calculated from hygromycin B killing curves)

Sensitive Cells Batch
  S128 I-A10 R-01 T-94 T-99
Tobacco Calli cells 4.3 4.5 4.1 4.7 5.2
293 cell 8.8 8.3 9.2 8.5 8.1
Fungi cell 13.2 14.6 13.7 14.2 13.8
Escherichia Coli 8.2 8.1 8.4 8.2 8.6

From the above table (Table 2), it can be seen that the Lethal Dose 50 (LD 50) Hygromycin B concentration for sensitive cells is about the same for these suppliers of Hygromycin B.

Table 3

LD 50 Hygromycin B Concentration (µg/mL) for Resistant Cells from Different Vendors

Resistant Cells Hygromycin B Batches
S128 I-A10 R01 T94 T99
Tobacco Calli cells transformed
with the Hygromycin B
phosphotransferase gene
72 220 81 490 720
293 cells transformed
with the Hygromycin B phosphotransferase gene
51 136 69 394 570

From Table 3, it can be seen that T-94 (94%) and T-99 (99%) Hygromycin B have lower toxicity to resistant cells relative to S128, I-A10 and R01. It is possible that the toxicity to the resistant cells is mainly due to the 78.1 minute HPLC retention time impurity (impurity DF).

LD 50 comparison for resistant cells for different cell lines:

The impurities of commercial Hygromycin B are collected during the preparation of the high purity Hygromycin B. Each impurity is individually isolated by preparative HPLC with C18 as the stationary phase for preparation of Hygromycin B with HPLC purity greater than 99%. The impurities are then used to add to the media of the following cell line to test the toxicity of the individual impurities to cell lines carrying the resistant gene of Hygromycin B.

Table 4

LD 50 for different impurities (µg/mL) for resistant cells transfected with a hygromycin resistant gene.

Resistant Cells Impurities
  A C D E F
Tobacco Calli cells transformed
with the Hygromycin B
phosphotransferase gene
193 2.9 0.5 4.2 16
293 cells transformed
with the Hygromycin B
phosphotransferase gene
240 7.2 1.7 3.2 11

From Table 4, it can be seen that the toxicity of different impurities varies for different cell lines and the toxicity is much higher than the purified EvoPure® Hygromycin B. The impurities may not share the same resistant gene as Hygromycin B itself. In order to make low cell toxicity Hygromycin B, all the impurities need to be controlled. In particular, the concentrations of impurities C, D and E are relatively more important to the toxicity to the resistant cells.

Table 5 is a killing curve comparison between EvoPure® Hygromycin B and another well-known vendor's Hygromycin B (I-A10).

Table 5

Death Ratio of different resistant cells at several Hygromycin B concentrations of T-99 I-A10 Batches

Hygromycin B concentration T-99
+
Tobacco cells
T-99
+
293 cells
I-A10
+
Tobacco cells
I-A10
+
293 cells
0.1 mg/ml 100 100 100 100
1 mg/ml 99.7 100 99.2 98.6
10 mg/ml 100 100 83.6 81.2
100 mg/ml 97.3 90.4 65.2 51.5
1000 mg/ml 43.6 39.5 23.7 16.3
3000 mg/ml 11.7 50.7 1.8 0.0
5000 mg/ml 6.2 1.5 0.0 0.0
10000 mg/ml 2.2 0 0.0 0.0

Table 5 shows that batch "T-99" aka EvoPure® Hygromycin B has much lower toxicity to the resistant cell lines tested.

The high Hygromycin B concentration of high purity Hygromycin B of EvoPure® grade antimicrobials kill sensitive cells much more quickly than the lower concentration of commercial grade Hygromycin B, and the resistant cell survival rate is much higher. Thus, the selection process is done more quickly with high purity Hygromycin than with currently available commercial grade Hygromycin B.

This reduction in time to complete cell selection that is provided by the present invention is of significant benefit to researchers in industry and academia. Thus, the surprisingly high purified Hygromycin B preparation is of assistance to the progress of research and medical developments.