40 CHROMATOGRAPHY
Tackling the COMPLEX AREA OF PEPTIDES
The challenges of peptide analysis and purification. By Cecilia Mazza & Fredrik Limé
A
s industry and academia continue to expand their work into the promising
Chromatographic results for a challenging peptide mixture
area of peptides, with the focus of bringing more effective therapeutic drugs to the market, there is an increased need for the analysis and purification of peptide mixtures. Traditionally, peptide molecules are separated and purified using reversed phase chromatography with silica based stationary phases at low pH conditions. However, as the studies of peptide structures develop and some of those innovative structures become more basic in nature, not all peptide mixtures can be isolated to satisfaction with the standard conditions of low pH, representing a big challenge to the scientific community. Furthermore, while many of the therapeutic peptides are initially analysed and purified at a small scale within drug discovery departments, there is the expectation that some of
the peptides will exhibit positive results to the point that scale-up and manufacturing will occur. As many of these substances are expensive to produce and their impurities need to be removed to very low concentrations, HPLC is the ideal technique to use in the purification of these drugs also at a large scale. Tere is therefore a need to determine successful preparative methods early on that can be transferred from discovery to development and then to production. At the same time as the field of therapeutic peptides grows rapidly, state-of-the-art stationary phases are being developed based on organosilane enforced silica to expand the range of pH scientists can operate at, maintaining the chemical stability of traditional silicas but at high pH. Tese innovative materials provide a new tool to separate and purify substances, including peptides. Te example discussed
here is one of a challenging peptide mixture where the main
substance is a small peptide that needs to be purified. Te chromatographic results are depicted in the figure. As shown, the purification at pH 8 is carried out in the same manner as with a classic silica material, meanwhile the purification at pH 9.5 is normally used with a reinforced silica material. Te purity and yield results are also shown, indicating that the work carried out at higher pH is more effective. In fact the results at higher pH are significant because under overloaded conditions, the main compound and its impurities travel at a different pace through the column, producing purer fractions. When these types of results are found at the discovery phase, there is an aspiration to continue using the same stationary phase, mobile phase and run conditions in the scale up to production because of the time savings this represents to a given project where companies are striving to be first to market. Te example shown here is
relevant as results are impressive and the purification is already carried out with a 10µm material considered to be a traditional particle size in larger scale chromatography, where the scientist is already future- proofing the development steps if the substance is deemed of value for the drug company. Consequently, there is more
stress on using stationary phase materials that can be scaled with the needs of the pharmaceutical or biotechnology company.
For more information ✔ at
www.scientistlive.com/eurolab
Cecilia Mazza & Fredrik Limé are with AkzoNobel.
www.kromasil.com
www.scientistlive.com
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100