G418 EvoPure^®

Different batches/purities of G418 sulfate obtained from various suppliers and manufacturers were used to
determine ED90 (leads to 90% death) for sensitive and resistant cells. G418 dilutions are prepared from
50mg/ml active G418 stock solutions.

Results are shown in the following table:

Table 1
Selectivity properties of different batches of G418 sulfate ED₉₀* (calculated from G418
killing curves)

Batch	A	B	C	D
NIH3T3 sensitive ED90 G418 Sulfate concentration	460 units/ml	620 units/ml	510 units/ml	410 units/ml
Surviving rate of Resistance NIH3T3	93%	65%	58%	79%

*All batches of G418 sulfate were vacuum dried at 60C for two hours in order to reach the same water content.

Purity as measured by overall purity (determined by HPLC) and specific impurity profiles of these batches are shown in the following table:

Table 2

Batch	A	B	C	D
Gentamicin X	5.5%	7.6%	6.2%	6.3%
Gentamicin A	3.1%	4.2%	6.1%	4.7%
Gentamicin C	0.7%	6.4%	7.7%	2.1%
G418	90.7%	81.8%	80%	86.9%
Water content	11.3%	12%	9.8%	8.3%

From the above tables, it can be seen that although the purity of G418 sulfate varies from 80% to 90%, the survival rate changes even more dramatically.

Prior to EvoPure^® G418 sulfate, such high purity G418 has not been previously available to test its toxicity to different cell lines and at the same time to prepare its impurities to test the selectivity properties. EvoPure^® G418 sulfate has purity greater than 95% and contains less than 0.5% Gentamicin C contaminants.

LD10 concentraions for various cell lines of the resistant cells for different cell lines and impurities is in the following table:

Table 3

LD10 concentrations for different cell lines and different G418-related fractions showing cell line toxicity:

Resistance to G418 cell lines	NIH 373	CHO	HeLa cells	GH3	U87
G418	7200	5210	6100	4300	6200
Gentamicin X2	527	1240	321	940	689
Gentamicin A	831	1310	937	739	492
Gentamicin C1a	110	216	139	153	138
Gentamicin C1b	53	172	281	156	271
Gentamicin C2	128	93	215	173	96

The above table shows that Gentamicin impurities are much more toxic to commonly used cell lines. The toxicity level varies from 4 times (Gentamicin X2 vs G418 - CHO cells ) to about 120 times (Gentamicin C1b vs G418 - NIH 373).

Normal grade of G418 sulfate was chosen to compare to the 99% pure G418 sulfate of the present invention, at different dates at various concentrations. G418 dilutions are prepared from 50mg/ml active G418 stock solutions. The results of this comparison are shown in the following table:

Table 4

Selectivity properties of different batches of commercially available G418 sulfate comparing with the high purity G418 sulfate*

	Day 7	Day 14	Day 21	D28
NIH3T3 (300u/ml) sensitive surviving rate of supplier A	58.7%	13.2%	5.7%	2.2%
Surviving rate of Resistance NIH3T3	98%	92%	83%	74%
NIH3T3 (500u/ml) sensitive surviving rate of supplier A	19.2%	3.1%	1.1%	0%
Surviving rate of Resistance NIH3T3	82%	63%	42%	23%
NIH3T3 (500u/ml) sensitive surviving rate of 99% G418 sulfate	21%	1.2%	0%	0%
Surviving rate of Resistance NIH3T3	99%	98%	97%	96%
NIH3T3 (700u/ml) sensitive surviving rate of 99% G418 sulfate	9%	0%	0%	0%
Surviving rate of Resistance NIH3T3	98%	95%	92%	90%

*All batches of G418 sulfate were vacuum dried at 60C for two hours in order to reach the same water content.

EvoPure^® G418 concentrations can be increased without the loss of resistant cells. An increase in usage of non-EvoPure^® G418 (commercial grade G418 sulfate (including Geneticin)) causes the loss of resistant cells. At low concentrations, sensitive cells can be hard to kill. Assuming the death rate of 10% for the resistant cells is a successful selection, normal G418 sulfate still has around 10% of the sensitive cells surviving. Assuming a 70% resistant cell surviving rate as successful selection, the concentration of high purity G418 sulfate of the present invention can be very high and the selection process can be done within a week as compared to three weeks.

The following table compares Geneticin 97% pure G418 sulfate (Gentamicin X2 1.1%, Gentamicin A 1.7% Gentamicin C 0.2%) at different dates and various concentrations:

Table 5

Selectivity properties of geneticin comparing with the high purity G418 sulfate (97%)* (calculated from G418 killing curves)

	Day 7	Day 14	Day 21	D28
NIH3T3 (300u/ml) sensitive surviving rate of Geneticin	51.7%	9.2%	1.7%	0.5%
Surviving rate of Resistance NIH3T3	98%	95%	91%	89%
NIH3T3 (500u/ml) sensitive surviving rate of Geneticin	18.2%	4.1%	1.1%	0%
Surviving rate of Resistance NIH3T3	95%	91%	86%	81%
NIH3T3 (500u/ml) sensitive surviving rate of 97% G418 sulfate	20%	0.7%	0%	0%
Surviving rate of Resistance NIH3T3	98%	96%	93%	89%
NIH3T3 (700u/ml) sensitive surviving rate of 97% G418 sulfate	11%	0%	0%	0%
Surviving rate of Resistance NIH3T3	98%	95%	91%	87%

*All batches of G418 sulfate were vacuum dried at 60C for two hours in order to reach the same water content.

From the above table it is seen that the concentration of high purity G418 sulfate can be increased without causing the loss of resistant cells, while the increase of Geneticin causes the loss of resistant cells. At low concentrations, sensitive cells take a long time to be killed. Assuming the death rate of 10% for resistant cells is a successful selection, Geneticin needs 28 days to complete the selection, while a 97% G418 sulfate preparation takes only 14 days. This reduction in time to complete the selection is of significant benefit to researchers in industry and academia. Thus, the surprisingly highly 14 purified G418 preparation of the present invention will be of assistance to the progress of research.

Next Section: Working concentrations of G418...