CAPTURING OF A scFv FROM E.COLI USING TOYOPEARL® AF-rPROTEIN L-650F

Abstract

The optimization of the capture of single chain variable fragments on TOYOPEARL® Protein L resin using a robotic parallel chromatography system leads to significant purity improvement. More specifically, post-load washing with chaotropic agents can significantly reduce HCP content. It may further support on-column refolding of misfolded and aggregated product.

scFv CAPTURING Single chain variable fragments (scFv) are antibody derived molecules. The  variable  part  of  the  light  chain  and  the heavy  chain  of  a  mAb  are  connected  via  a  peptide  linker. The  molecular  weight  of  the  whole  construct  is  approximately 30 kDa. ScFvs are apt for being produced in E.coli, since  they  lack  the  glycosylation  site  of  a  full-length  IgG. Other  advantages  over  a  full-length  IgG  include  rapid  target access and good tissue penetration. However, renal clearance delimits serum half-life to hours instead of days, compared  to  typical  serum  half-lives  of  mAbs.  In  some  therapeutic  strategies,  this  may  be  an  advantage.  Hence, straight-forward and efficient capturing solutions similar to Protein A for mAbs may pave the way for the future success of this class of molecules. Herein we describe general conditions for capturing of an exemplary  scFv  from  E.coli  with  TOYOPEARL  AF-rProtein L-650F.  Further,  host  cell  protein  (HCP)  removal  was  optimized in a parallel chromatographic approach.

LAB-SCALE EXPERIMENT The κ light chain of mAbs, Fabs and scFv binds to Protein L at  neutral  pH  and  physiological  conductivity.  HCPs  flow  through  the  column  at  the  applied  conditions.  The  bound  product can then be recovered at pH 2.0-3.5, depending on the particular format and molecule. As a rule of thumb, larger molecules can be eluted at comparatively higher pH. Most methods can be set-up based on these general guidelines. In  detail,  the  herein  presented  experiments  use  100  mM  sodium phosphate buffer, pH 6.5 for column equilibration. 15 column volumes (CV) of a periplasmic scFv feedstream were loaded onto the 6.6 mm ID x 6.7 cm L column packed with  TOYOPEARL  AF-rProtein  L-650F.  The  column  was  washed  with  equilibration  buffer  and  the  scFv  was  recovered  in  100  mM  glycine/HCl,  pH  2.0.  The  method  was  conducted  at  300  cm/h  except  for  column  loading,  which  was conducted at 3 min residence time.

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