IV. Materials & MethodsIV. Materials & Methods

II. IntroductionII. Introduction

I. SummaryI. Summary VV.. ResultsResults && DiscussionDiscussion Automation of desalting of chlorophyll from plant extract has beenpresented below:

Automation of 96 Gel Column Array for SizeAutomation of 96 Gel Column Array for Size--Exclusi on & Desalting of Exclusion & Desalting of Chlorophyll on VERSA 10 WorkstationChlorophyll on VERSA 10 Workstation

Sikander GillSikander Gill 11 PhD, Rajwant GillPhD, Rajwant Gill 11 PhD, PhD, Barry YelenikBarry Yelenik 22 and Dong Liangand Dong Liang 11 PhDPhDAurora Biomed Inc., Vancouver, BC, CanadaAurora Biomed Inc., Vancouver, BC, Canada 11, EMP Biotech, Howell, NJ, USA, EMP Biotech, Howell, NJ, USA 22

Contact: Barry Yelenik e-mail: byelenik@empbiotech. com or Sikander Gill e-mail: gill@aurorabiomed.com

Gel filtration is widely used in diverse downstream bioprocessesincluding DNA size separations, removal of dyes, salts and other lowmolecular mass reagents, purification of >10 bp oligonucleotides, andbuffer exchange in downstream bioprocesses. CentriPure N96, a gelarray was automated on a very compact VERSA 10 Workstation. Theautomation allows desalting of 96 samples in 4 minutes includingsample addition, pre-run and elution. Subsequent washing andequilibration of the columns automatically continues for the user-defined volume or duration. The eluted samples are auto-incubated atthe user-defined temperature in the range of 2-90°C. A total of 104tips (1000 or 200µl) are used.

The results show that the automated operation performed withconsistency, high throughput, and accuracy did not suffer from anydetectable cross-contamination indicating no dripping or hangingdrops from the columns into eluted samples. The resulted 94.4%recovery was found to be equivalent to that of manual process(94.35%).

Gel filtration, a non-adsorptive chromatography technique separatesmolecules on the basis of molecular size exclusion. It is widely usedfor DNA size separations, purification of sensitive biomolecules,

The validation of the CentiPure N96 Gel Filtration (www.empbiotech.com) wasconducted on VERSA 10 Workstation (www.Aurorabiomed.com) with 8-Channelliquid handling, and 6 deck position configuration as follows:

1.Samples: 96 samples of the followings were purified:1. a. Hyladder DNA, and b. Plant extract (DNA, Protein, and chlorophyll)2. Deck layout: The deck was configured as shown in Figure 1.3. Protocol: Automated steps were performed as follows (Figure 2):a. Equilibration of gel columns with distilled water:

1. Addition of 5 ml by repeating cycles of 300µL.2. The flow out waste liquid was collected in the underneath collection module

which was connected with a tubing to a waste bottle kept under the table.b. Sample addition: Sample of plant extract (25 or 50 or 100 µl) were added

to the columns with a user-defined controlled low dispense speed to minimizedisturbance to the sample distribution in the gel

c. Pre-run liquid addition : A calculated volume of distilled water was followed thesample addition so that sample + Pre-run volume = 350 µl.d. Moving of the CentriPure N96 Gel Array: The Gel array was automaticallymoved between wash module and elution module ensuring the followings:

a. No drippingb. No hanging of dropse. Elution: 300 µl elution reagent (distilled water) was added to each

columns.

1. Recovery of chlorophyll from plant extract: The objective of this experiment was to examine whether theworkstation can achieve recovery of eluted molecules comparative to that of manual process. The averagerecovery of both the manual and automated process was comparable (manual process: 94.35% vs automated94.4%. A CV% of the inter-channel (1.3-6.4%, n=12) and intra-channel (2.1-6.2%, n=12) recovery was alsocompared for the automated process (Table 2).

Table 2 Table 3

2. Cross-contamination test: This experiment was designed to test any cross contamination during the automatedprocess due to cross contaminating liquid handling due to wrong X-Y precision movement of the arm, splashingor over flowing of columns or eluted sample plate, or due to dripping or hanging drops from the bottom ends ofcolumns. The OD680 data from the eluted samples in checker-board format indicated no cross contaminationduring the automated process (Table 3).

Table 4 Table 5

VIII. ReferencesVIII. References

III. ObjectivesIII. Objectives

*Correspondence should be addressed to Sikander Gil l e-mail: gill@aurorabiomed.com, Aurora Biomed Inc. , 1001 E. Pender St., Vancouver, BC, Canada, V6A 1W 2, Tel: 1-604-215-8700, Fax: 1-604-215-9700www.auro rabiomed.com

for DNA size separations, purification of sensitive biomolecules,removal of low molecular mass reagents, adjusting of ionic strength ofa protein or DNA solution, removal of dyes, purification of >10 bpoligonucleotides, desalting, and buffer exchange in diversedownstream bioprocesses1-3. However, manual operation of thisprocess faces many challenges as outlined in table 1 and thusrequires automation.

Table 1

Keeping this in view, a 96 gel column array, CentiPure N96 GelFiltration was automated on VERSA 10 Workstation.

1. To validate the separation of the following moleculesa. DNA from blue dyeb. Chlorophyll and salts from plant extractc. DNA from plant extractd. Protein from extract

2. Optimal size of sample3. Optimal recovery4. Any cross-contamination5. Accurate reagent dispensing

columns.4. Cross-contamination This test was designed as a checker-board with alternatingplant extract samples and distilled water.5. Manual process: The manual process was carried after the automation forcomparative evaluation.6. Analysis: The Hyladder DNA, DNA, protein and chlorophyll in plant extract wasscanned (240-900nm) and OD260, OD280, and OD680, respectively was analyzed onEpoch Microplate Spectrophotomete (www.biotek.com)

Figure 1: a. VERSA 10 Workstation b. Deck Layout

Figure 2: Automated steps of the gel filtration process

3. Throughput: The flow down of the samples and reagents through the columns was carried by gravitation flowwhile the system offers options from mild to strong vacuum (negative pressure) or positive pressure. Thegravitation flow was recorded as 4.46 min/ml. The total duration of steps of the process is presented in Table 4.

In addition to the efficient automation of gel filtration, VERSA 10 Workstation is a very compact (L 57cm x W 45cmx H 54) to fit on standard lab tables with economical spacing and offers various additional applications with orwithout add-on modules (Table 5). The system offers a throughput of 96 samples within 21 minutes and a total of104 tips (1000 or 200µl). The results show that the automated operation performing with consistency, highthroughput, and accuracy, handling of 96 well or 384 well formats can bring savings on time, energy and addconfidence in the accuracy of results. This system with high efficiency air filter hood equivalent to a small ultraclean workbench, equipped with optional modules like shaker, cooling-heating incubator(s), and with smoothliquid handling, effectively avoids cross contamination which is highly essential for diagnostic and otherapplications.

VI. Why VERSA 10 Workstation?VI. Why VERSA 10 Workstation?

1. Escriou etal., Triple helix formation on plasmid DNA determined by a size-exclusion chromatographic method.Anal Biochem.1997; 248(1):102-110.

2. Almadaly etal., Desalted and lyophilized bovine seminal plasma delays induction of the acrosome reaction infrozen-thawed bovine spermatozoa in response to calcium ionophore. Theriogenology. 2015; 83(2):175-185.

3. Xiong etal., Glucomannan microspheres for low-cost desalting of protein solution. Carbohydr Polym. 2014;111:56-62