Protocol for bevacizumab purification using Ac-PHQGQHIGVSK-agarose

Graphical abstract


Method details
Bevacizumab (trade name: Avastin TM ) is an anti-vascular endothelial growth factor (anti-VEGF) that blocks the growth of tumor blood vessels. This monoclonal antibody (mAb), produced in CHO cells, is used for the treatment of many human cancers. Nowadays its purification is achieved by affinity chromatography (AC) using protein A, which is a high expensive ligand. Moreover, harsh elution conditions, that damage both the mAb and the protein ligand, are required to ensure bevacizumab recovery from the protein A affinity column. On the other hand, small peptides consisting of few amino acids represent ideal affinity chromatography ligands because they are much more physically and chemically stable than protein A and can be readily synthesized by standard chemistry in bulk amounts at a lower cost. Considering the bevacizumab binding site on the 85-Pro-His-Gln-Gly-Gln-His-Ile-Gly-92 VEGF segment [1], a peptide ligand for bevacizumab purification by AC was designed [2]. Val-Ser-Lys was introduced as a spacer arm to facilitate bevacizumab interaction with the immobilized ligand. Ligand site-directed immobilization on the agarose chromatographic support was ensured by the e-amino group of the C-terminal Lys and the acetylation of the N-terminus. The short peptide designed, Ac-PHQGQHIGVSK-NH 2 , has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis.
The present protocol describes the synthesis of the peptide Ac-PHQGQHIGVSK-NH 2 , its immobilization on agarose and the use of the Ac-PHQGQHIGVSK-agarose for affinity chromatography purification of bevacizumab from a clarified CHO cell culture.
The peptide ligand shows higher stability and lower cost than protein A. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix. Bevacizumab binding to the peptidyl-agarose is achieved using as adsorption buffer 20 mM sodium phosphate, 1 M (NH 4 ) 2 SO 4 , pH 7.0. Bevacizumab adsorption at high ionic strength suggests that the binding is largely hydrophobic. The elution is performed quantitatively by removing the (NH 4 ) 2 SO 4 from the running buffer, thus weakening the hydrophobic forces that supported the binding of bevacizumab to the chromatographic matrix. The mild elution conditions preserve the integrity of both the peptide ligand and the mAb. Furthermore, Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices.
Note: The amount of resin to use depends on the amount of peptide to be synthesized. Considering an overall yield of 75 %, 1 g of resin is used to obtain 0.5 mol of peptide. The ligand Ac-PHQGQHIGVSK-NH 2 is synthesized in enough amount to prepare the affinity chromatographic media. Usually, the purity of the crude peptide (>90 %) is enough for using it without further purification. In the case of a lower purity, a purification by reverse phase C 18 liquid chromatography is carried out. 2 Wash the resin three times with CH 2 Cl 2 and then with DMF. 3 Incubate with 20 % piperidine in DMF (v/v) (2 Â 5 min) to remove the Fmoc group. 4 Wash the resin with DMF (5 Â 1 min). 5 Weight the Fmoc-Lys(Boc)ÀOH (3 eq) and TBTU (3 eq) into a dry tube and dissolve them in a minimum amount of DMF. 6 Add the solution to the resin. 7 Add DIEA (4 eq) dropwise to the resin and mix. 8 Incubate the resin with agitation for 45 min at room temperature on an orbital shaker. 9 Wash the resin with DMF (2 Â 1 mL) and CH 2 Cl 2 (2 Â 1 mL) by filtration or decantation. 10 To confirm the reaction coupling completion, test a small amount of resin (1À3 mg) with Kaiser test [3]. If positive, wash resin with DMF (2 Â 1 min) and repeat coupling reaction with fresh reagents as indicated in steps 5-9. If negative, remove Fmoc group, wash the resin and couple the next Fmoc protected amino acid as indicated in steps 3-9. 11 After peptide elongation, acetylate terminal proline by adding Ac 2 O (10 eq.) and DIC (10 eq.) in enough CH 2 Cl 2 to make the swollen resin just mobile to agitation. Step 2: affinity matrix synthesis [5] Material Step 3: evaluation of peptide immobilization on agarose

Material
Absorption UV/VIS spectrophotometer 10 mm silica UV cell Procedure Peptide attachment is measured indirectly by quantifying the NHS released as a result of peptide immobilization.
1 Measure the absorbance at 260 nm of the filtrate saved in step 2 (item 5). 2 Calculate the NHS concentration, whose molar extinction coefficient (e) is 9600 M À1 cm À1 at 260 nm [6].
Step 4: Adsorption isotherms for bevacizumab binding to Ac-PHQGQHIGVSK-agarose Note: To add the 50 mL of the chromatographic matrix to each tube, prepare a 1:1 suspension of the chromatographic matrix in adsorption buffer and while agitating measure 100 mL of the suspension with an automatic pipette with the tip cut at the end in order to increase its diameter. 3 Prepare another set of labeled tubes with the same volume of protein stock solution and buffer but without matrix. 4 Gently shake the tubes overnight at 25 C in Thermomixer to enable the system to reach its equilibrium. 5 Separate the resin by filtration using labeled polypropylene columns fitted with a polyethylene porous disk. 6 Measure the protein concentration in the filtrate with Bradford reagent. 7 Determine free protein concentration at equilibrium (c*) with the first set of tubes and the total protein concentration at the beginning of the experiment (c t ) with the second set of tubes. 8 Calculate the amount of bevacizumab bound to the immobilized peptide at equilibrium, per unit of total chromatographic matrix volume (q*), as: q* = (c t -c*)1000/50 (1) 9 To determine the maximum adsorption capacity for bevacizumab per volume of chromatographic matrix (q m ) and the dissociation constant (K d ), non-lineal curve regression of the q* = f (c*) graph is performed with Sigma Plot software, using a one-to-one Langmuir binding mode [7]: q* = q m Ác*/(K d + c*) Step 5: bevacizumab purification by peptide Ac-PHQGQHIGVSK-agarose affinity chromatography from the CHO cell culture Note: the amount of bevacizumab loaded to the column must be lower than the maximum capacity of the chromatographic matrix synthesized. 5 Wash the column with adsorption buffer until the absorbance at 280 nm achieves the baseline value. 6 Elute the bound protein by adding 20 mM sodium phosphate, pH 7.0. 7 Measure total protein concentration with Bradford reagent using pure bevacizumab as the protein standard [8]. 8 Measure bevacizumab concentration by HPLC using a protein A analytical column as per Zou et al. [9]. 9 Perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12.5 % under reductive conditions) as described by Laemmli [10] and stain gels with Coomassie Blue following the standard procedure.

Method validation
Peptide Ac-PHQGQHIGVSK-NH 2 is easy to obtain with high purity by solid phase peptide synthesis.
Ligand site-directed immobilization on the NHS activated agarose is ensured by the e-amino group of the C-terminal Lys and the acetylation of the N-terminus. The reaction is performed in organic solvent to prevent NHS ester hydrolysis, thus promoting higher immobilization yields [5]. When using Dry Pierce TM NHS-Activated Agarose, 17 mmol of peptide per mL of matrix was obtained with a maximum capacity of 38 mg of bevacizumab per mL of matrix. Similar results may be obtained using other NHS activated matrices such as NHS-activated Sepharose from GE Healthcare. Fig. 1 shows the profile, together with the corresponding SDS-PAGE of the chromatographic fractions, using 20 mM sodium phosphate, pH 7.0, 1 M (NH 4 ) 2 SO 4 as adsorption buffer and 20 mM sodium phosphate, pH 7.0 as elution buffer. The peptidyl-agarose adsorbs bevacizumab from the CHO cell culture filtrate while most contaminants pass through. Although the pass-through peak of the chromatogram is much higher than the elution peak, the protein concentration in both fractions are comparable. That is due to the contribution of the culture medium to the absorbance at 280 nm of the pass-through peak. Table 1 shows the purification obtained using buffers 20 mM sodium phosphate, 1 M (NH 4 ) 2 SO 4 , pH 7.0, and 20 mM sodium phosphate, pH 7.0, as adsorption and elution buffer respectively. The yield was 94 % and the purity 98 %.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.