This document summarizes experiments to optimize the chromatography process for purifying GFP protein. It finds that purified GFP has a higher binding capacity than clarified lysate. For anion exchange chromatography (AEX), gradient elution provided higher purity than step elution at similar recovery levels. Hydrophobic interaction chromatography (HIC) was also tested as an intermediate purification step, with gradient elution again providing better purity and recovery than step elution. However, HIC only achieved about 80% purity. Overall, gradient elution produced fractions with fewer impurities than step elution for both AEX and HIC.

1. Process Development Studies on Maximizing Recovery and Purity of GFP from Chromatography
Fractions
Kathryn Howard
BEC 436, Golden LEAF Biomanufacturing Training and Education Center, North Carolina State
University
September 28, 2016
Introduction
Chromatography exploits the target protein’s
inherent properties to purify the solution by
allowing the protein to bind to a stationary resin
while impurities pass through the column. In
biopharmaceutical applications, it is common to
employ different types of chromatography in
succession for optimal recovery and purity.
The purpose of the experiments within the scope
of this lab report was to determine an
appropriate chromatography process for
capturing and intermediately polishing GFP
from a Q Sepharose FF Eluate stream. An
effective chromatography process is necessary
in order to maximize the amount of target
protein recovered while sufficiently purifying
the target protein. Establishing optimal
chromatography parameters was accomplished
by evaluating purity and recovery results from
both gradient and step elution.
Materials and Methods
Using a 5 mL HiTrap™ Q Sepharose FF
column, a breakthrough run was executed to
determine the dynamic binding capacity (DBC)
of Q Sepharose FF for GFP in clarified lysate.
The column was first equilibrated with 50mM
Tris buffer pH 8.0 before loading the GFP in
clarified lysate to allow the column to become
fully saturated. The column was then washed
with the same buffer and eluted using 50 mM
Tris pH 8.0 and 1M NaCl, collecting fractions
throughout the process. A breakthrough curve of
GFP concentrations in the fractions collected
and the fractions’ corresponding volumes was
created, for which the breakthrough volume,
defined as the point where 10% of the column
was saturated with GFP. Using this value, the
DBC was calculated using Equation 1. The
maximum volume of clarified GFP lysate that
could be loaded to the Pall LRC column for
capture chromatography was then calculated
using Equation 2.
Equation 1. DBC = (Co∙Vbthru)/Vbed,1
Equation 2. Vload = (0.5∙DBC∙Vbed,2)/Cload
The Pall LRC column was then equilibrated
using 50mM Tris buffer pH 8.0, and the Vload
from Equation 2 was fed to the column in order
to perform anion exchange chromatography
(AEX). The column was washed with the same
buffer prior to elution. For the step elution, 10
column volumes (CV) of 1M NaCl were fed to
the column, while gradient elution utilized 10-
20% 1M NaCl over 4 CVs. Fractions of 12mL
were collected for concentration and
fluorescence analysis, and appropriate fractions
were then pooled together for best purification
and recovery results.
For intermediate purification, Hydrophobic
Interaction Chromatography (HIC) was
employed. The column was equilibrated with
2M ammonium sulfate + 50mM Tris prior to
loading the column with a 1:1 mixture of
fraction pool + ammonium sulfate. The column
was then washed with the same buffer, and GFP
was eluted with 50mM Tris in a similar manner
as the previous step and gradient elution.
Results and Discussion
The first analysis completed involved
determining whether clarified GFP lysate or
purified GFP possessed a better binding
capacity. As shown in Table 1, purified GFP had
a significantly higher binding capacity due to

2. having less contaminants competing for column
binding.
GFP Sample DBC (mg/mL)
Clarified GFP Lysate 25.265
Purified GFP 83.1
Table 1. Comparison of DBC values for purified
GFP and clarified GFP lysate.
Figure 1 depicts the comparison between
recovery and purity of GFP in gradient elution.
Fraction pools that contribute high GFP
recovery while also highly purifying the protein
are ideal. While other fraction pools were
considered, the pool of samples A3-A12
provided the best compromise of percent of GFP
recovered as compared to an acceptable percent
of GFP purified. In all cases, purity was
calculated by dividing the total amount of GFP
by the amount of total protein in each fraction or
fraction pool.
Figure 1. Plot of % GFP recovery vs. % GFP
purity in AEX for gradient elution.
The results from step elution were not as
expected, since the calculated total GFP (in mg)
of some fraction pools exceeded the total GFP in
the feed, inhibiting comparison of the two
elution methods. However, as compared to step
elution, it is expected that gradient elution would
have a higher purity at a similar recovery due to
gradient allowing for impurities to unbind from
the column and elute prior to the strong salt
concentration eluting the GFP. While some
more tightly-bound impurities will still be
present, this method of eluting impurities earlier
in the capture step causes a higher purity of GFP
in the later fractions obtained, thereby reducing
any further purification necessary.
HIC was used for intermediate purification of
GFP, as the buffer used exposes the hydrophobic
regions of GFP and results in a conformational
change of the protein. This allows for increased
GFP binding to the column as compared to
impurities, resulting in a more pure product.
Figure 3 demonstrates the results obtained from
HIC for both step and gradient elution. The
same fraction pooling process as earlier was
used in order to determine the fraction pool that
delivered the best compromise of GFP recovered
as compared to percent of GFP purified; the
optimal fraction pools for each elution method
have been marked. As shown, gradient elution
provides a better GFP recovery for similar
purification. However, HIC caused a sizeable
reduction in the amount of GFP recovered after
this intermediate purification and did not offer
the purity desired after only reaching
approximately 80% purity at its highest.
Figure 2. Plot of % GFP recovery vs. % GFP
purity in HIC for gradient and step elution.
Conclusions
Utilizing gradient elution for HIC allowed for
GFP fraction volumes to contain less impurities
as compared to fraction volumes from step
elution. While an effective comparison could not
be deduced from the data collected, it is
expected that the same analysis would be true
for the previous AEX step due to the timing of
impurity elution. However, both elution methods
would cause a decrease in percent of GFP purity
as percent GFP recovery increases since a larger
fraction pool will inherently allow for greater
possibility of impurities.
References

3. Introduction to Downstream Processing - BEC
436/536 Course Pack. North Carolina State
University: Golden LEAF Biomanufacturing
Training and Education Center, Fall 2016.
Print.