38
August / September 2019
ADVERTORIAL
Development of a novel combined IEX-RP chromatographic process for the purification of bivalirudin
By Alessandra Basso, Purolite Ltd, UK , Benjamin D. Summers*, Purolite Ltd, UK, Christopher Bresner, Purolite Ltd, UK, Simona Serban, Purolite Ltd, UK * Corresponding author email
benjamin.summers@
purolite.com
A novel coupled ion exchange and reverse phase chromatographic process was investigated in the purification of bivalirudin from a semi-crude mixture using Purolite Chromalite®
resins. The highly hydrophobic backbone of the ion exchange resin Chromalite® PCG1200FS contributes to the
purification by means of a pseudo-mixed mode purification. The peptide bivalirudin was purified from an initial 80-85 % sample to >99 % purity. The process also shows excellent recovery of the peptide samples, with >99 % yield obtained.
Introduction
Peptides represent a highly valuable section of the pharmaceutical market, making up 13% of newly FDA-approved drugs in 2017 [1]. However, the highly regulated nature [2] of pharmaceuticals requires the careful and thorough purification of these therapeutic agents. Additionally, as these drugs are not orally bioavailable [3], they must be directly injected - again mandating a highly pure compound. The biological aspect of peptides is responsible both for a high level of specificity and activity in treating conditions, however these also result in strong off-target effects if insufficiently purified or conjugated in the case of peptide conjugates [3, 4].
Separation of therapeutic peptides often relies heavily on reversed-phased HPLC (RP-HPLC), which is the most commonly used method in both the development and production of these species [5]. This method can be inefficient, however, as similarities between peptides produced industrially and their synthetic by-products result in challenging purifications [6]. The small differences in affinity for the stationary phase between the target peptide and by-products, as well as long retention times [7], and a significant effect of small changes in the mobile phase composition on the retention, further complicate purification [8].
A common solution to increase the separating power of RP-HPLC is to make use of an ion-pairing agent, such as trifluoroacetic acid, and conduct the separation at low pH; this coupled basic amino acids with the ion pairing agent, results in a net zero charge - increasing the
affinity of the peptides to the stationary phase [6]. Currently, ion exchange purification is generally avoided due to the difficulty in removing polar non-peptide impurities [9] as the elution of peptides will generally occur alongside that of some quantity of these impurities. Additionally, it can be challenging to ensure the removal of peptide by-products due to the close relation in charge between these molecules [10].
The use of a highly hydrophobic cation ion exchange stationary phase offers the possibility to carry out the ion-pairing process in reverse - the basic amino acids in the peptide pair with the acidic groups on the resin, while the hydrophobic backbone stabilises the interaction and exploits the ‘reverse-phase’-like attraction between the non-polar areas of the peptides and the resin to increase separation. This approach also facilitates the removal of upstream impurities, including host cell proteins and DNA/RNA, as well as offering the possibility for virus clearance due to the low pH of the process [5].
Experimental Resin Data
Chromalite® PCG1200FS 15AD2
Particle Size 20 - 50 µm 12 - 18 µm Pore Diameter 300 - 500 Å 200 - 300 Å Surface Area >600 m2
/g >500 m2
Functional Group
Volume Capacity
Backbone Basis Styrene/DVB Polymer Sulfonic Acid None 0.69 eq/L N/A
Styrene/ DVB
/g
Cation Exchange Chromatographic Purification
Described here is the full purification procedure following all development work: A GE Tricorn 5/200 column was packed with Purolite Chromalite®
PCG1200FS resin
according to standard practice, with an asymmetry of 0.8-1.8 as acceptable. The column was equilibrated with 5 column volumes (CV) of 20 mM citrate, 150 mM NaCl, pH 2.5 then 4 mL of a 10 mg/mL solution of crude bivalirudin applied to the column. The column was then washed with 3 CV of 20 mM citrate, 150 mM NaCl, pH 2.5 then a step elution carried out with 10 CV of 20 mM citrate, 1 M NaCl, pH 5 followed by a further elution with 10 CV dH2O. The process was carried out at a linear velocity of 300 cm/hr throughout.
Reverse Phase Chromatographic Purification
A GE Tricorn 5/200 column was packed with Purolite Chromalite®
15AD2 resin according
to standard practice, with an asymmetry of 0.8-1.8 as acceptable. The column was equilibrated with 5 column volumes (CV) of 0.1% trifluoroacetic acid (TFA) then 4 mL of the purified solution of bivalirudin applied to the column. The column was then washed with 3 CV of 0.1% TFA followed by a gradient elution carried out over 10 CV to a final concentration of 0.1% TFA in 60% acetonitrile. A final hold was carried out with 3 CV 0.1% TFA in 60 % acetonitrile. The process was carried out at a linear velocity of 300 cm/hr throughout.
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