Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #1200: Handling of Novozymes GroPep LONG®R³IGF-I for use in Cell Culture

Introduction:

LONG®R³IGF-I may be effective in promoting growth of any cell line which bears the Type I IGF receptor. The effectiveness of this peptide as a growth promoting agent varies from cell line to cell line and therefore must be tested in every new application. The concentration range at which the peptide is effective is 10 - 100 ng/ml.

The recommended procedure for the preparation of LONG®R³IGF-I for addition to cell culture media is described. Small peptides and proteins such as LONG®R³IGF-I can bind non-specifically to plastic, glass, filter membranes or other surfaces, resulting in the irreversible loss of peptide. This document includes the correct handling of LONG®R³IGF-I in order to minimise or eliminate this problem.

Handling Procedure:

The procedure involves the reconstitution of the peptide, filter-sterilization of the peptide solution and then the final addition of the peptide to the cell culture media. Note that it is important to add the solution of peptide after the media has been filter-sterilized, thus reducing the possibility of peptide binding non-specifically to the filter membrane.

Step I. Reconstitution of the peptide:

LONG®R³IGF-I is supplied lyophilized in a glass vial in an atmosphere of nitrogen at a slight vacuum (-25kPa). Care should be taken to equilibrate this slight negative pressure when opening the vial and reconstituting the peptide to avoid losses. An air-filled syringe may be introduced through the bung to equalize the pressure before opening. Peptides in the lyophilized state should be stored at 2-4°C and are stable for at least 3 years.

I.1. Remove the metal cap from the glass vial and intoduce an air-filled 2 ml syringe through the bung to equalize the pressure.

I.2. Add sufficient 100 mM acetic acid solution to the peptide vial to achieve a peptide concentration of 1 mg/ml.

Note that if the peptide vial purchased contains less than 1 mg, reconstitute the peptide in 100 mM acetic acid to achieve a stock concentration of 0.1 mg/ml. Addition of a carrier protein such as bovine serum albumin at a concentration of 0.1 - 1 mg/ml should be added to prevent the loss of peptide activity due to non-specific absorption to glass and other surfaces.

I.3. Mix thoroughly to ensure all peptide is dissolved in solution.

IMPORTANT: Irrespective of size, always reconstitute the entire vial in situ and then aliquot. Each vial contains the net amount of peptide indicated on the label but ~15 - 20 % more by mass due to the residual moisture retained through the freeze drying process. DO NOT estimate peptide content by weight or gravimetric means as this will underestimate the actual peptide content by ~15 - 20 %.

I.4. The resulting stock solution can be stored for at least 3 months at -20°C or -80°C. Longer term stability tests are continuing and extended stability is expected to be demonstrated.

LONG is a trade mark owned by Novozymes GroPep Limited

LONG®R³IGF-I is covered by the following patents assigned to Novozymes GroPep:

US patent 5,330,971; European patent 429,586; Japanese patent 2,682,738; Australian patent 633,099; Canadian patent 2,033,176;


Step II. Filter-sterilization of the peptide solution:

Filter-sterilize the peptide solution using a membrane with low protein binding. An example of a low protein binding membrane would be the Millipore Millex-GV Cat. No. SLGV025LS.

Step III. Addition of peptide solution to cell culture media:

Add the desired quantity of sterilized peptide solution to the pre-sterilized cell culture media.

The recommended final concentration range of LONG®R³IGF-I for use as a growth factor supplement in cell culture medium is 10 - 50 ng/ml. Given that the optimum concentration varies depending upon the cell line and the concentration of fetal bovine serum present, it is recommended that a titration of LONG®R³IGF-I be performed for each cell line.

LONG®R³IGF-I has been demonstrated to be stable in culture medium at the working concentration of 10-50 ng/ml in the presence of 0.01-1 mg/ml carrier protein (bovine serum albumin) for up to eight days at 37°C. Losses in biological activity will occur in the absence of carrier protein, presumably due to absorption of peptide to glass or plastic surfaces. We recommend that a carrier protein be included in working solutions of LONG®R³IGF-I if the peptide is to be used in serum-free media.

Trial Filter Sterilization of LONG®R³IGF-I undertaken at Novozymes GroPep:

5 mls of a 0.1 mg/ml solution of LONG®R³IGF-I was sterilized using a Milli-GV filter unit (membrane area 4 cm²). Analysis by HPLC did not indicate any detectable loss of LONG®R³IGF-I from the solution. However, when the peptide concentration was dropped to 0.01 mg/ml protein and 5 mls filtered-sterilized, HPLC analysis revealed that approximately 50% of peptide was lost by non-specific binding.

Important recommendations:

*Do not add the peptide to low protein or protein-free cell culture media prior to filter sterilization.

* Use a filter membrane with low protein binding characteristics

* Filter sterilize the LONG®R³IGF-I separately at a concentration of 0.1 mg/ml or greater.


Further reading:

Thomas, J.N. & Fung, V. "Comparison of LONG®R³IGF-I with insulin in the support of cell growth and recombinant protein expression in CHO cells" in Animal Cell Technology. Products of Today, Prospects for Tomorrow. European Society for Animal Cell Technology. The 12th Meeting. Ed. Spier, R.E., Griffiths, J.B. & Berthold, W. Butterworth Heinemann, 1994.

Francis, G.L., Ross, M., Ballard, F.J., Milner, S.J., Senn, C., McNeil, K.M., Wallace, J.C., King, R., & Wells, J.R.E. (1992) "Novel recombinant fusion-protein analogues of insulin-like growth factor (IGF) -I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency." J. Mol. Endocrinol. 8: 213-223.

* NOT FOR USE IN HUMANS


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