Technical Brief

Citric acid or acetic acid?

Understanding the impact of elution buffer
on mAb purification processes

Introduction Experimental Methods

Successful affinity chromatography relies on a
specific interaction between the target molecule
and the chromatography resin, as well as the ability to
separate the product from the resin using an elution
buffer. In Protein A affinity chromatography, process
developers need to choose from a range of available
elution buffers and conditions to implement at
Varying concentrations of two elution buffers (citric acid
and acetic acid) were used in a standard protein A
chromatography methodology. The elution buffer
concentration were as follows:
Concentration (mM) pH

production scale. Elution buffer choice can significantly Citric acid buffers
impact the characteristics of the elution pool and the 100 3.0
subsequent downstream steps. 100 3.5
100 3.7
This study explores the impact of a range of elution 50 3.0
buffer conditions, including molarity (0.01 M-0.1 M), 50 3.5
pH (3.0-3.7) and buffer type (citric acid and acetic 50 3.7
acid), on two different commercial Protein A resins 20 3.0
(Eshmuno® A and ProSep® Ultra Plus affinity resins). 20 3.5
Measured outcomes included antibody yield, 20 3.7
chromatographic profile, pool pH, and volume of 10 3.0
the elution pool. The effects of elution buffer 10 3.5
concentration and pH on these parameters were 10 3.7
evaluated. Many factors need to be considered when Acetic acid buffers
selecting an elution buffer and this work demonstrates
100 3.0
the impact that elution buffer choice can have on the
100 3.5
efficiency and process robustness of mAb purification.
100 3.7
50 3.0
50 3.5
50 3.7
20 3.0
20 3.5
20 3.7

Merck Millipore is a business of

 Process steps for Protein A chromatography
Step Buffer CV RT (min.)
Strip Same as Elution 3 3
Equilibration 50 mM Tris 25 mM NaCl 5 mM EDTA pH 7 7 3
Load 4 mg/mL Polyclonal IgG in EQ buffer Load to 32 mg/mL 3
Wash 50 mM Tris 25 mM NaCl 5 mM EDTA pH 7 4 3
Intermediate Wash 0.1 M Citric Acid pH 5.5 4 3
Wash 50 mM Tris 25 mM NaCl 5 mM EDTA pH 7 3 3
Elution Varies 8 6
Wash 50 mM Tris 25 mM NaCl 5 mM EDTA pH 7 4 3
Strip 6M Guanidine HCl 3 3
Equilibration 50 mM Tris 25 mM NaCl 5 mM EDTA pH 7 5 3

Experiments used either ProSep® Ultra Plus or Eshmuno® A affinity resins packed into Omnifit® columns with
1 cm i.d X 5 cm bed height. Both resins were tested under all of the above experimental conditions, with one resin
duplicated for each condition. The feed was human polyclonal IgG. Elution peaks were collected between 100 mAU
and 200 mAU at UV 280 nm and analyzed for yield, conductivity, pH, and volume.

Results and Discussion Elution pool pH

Citric acid and acetic acid were used separately as elution A second set of data identified the optimal pH range for
buffers with two different Protein A resins, ProSep® Ultra each elution buffer. For citric acid (Figure 2A), the pH
Plus and Eshmuno® A affinity resins. The results were of the elution pool decreased as elution buffer molarity
then compared side-by-side to determine the optimal increased. ProSep® Ultra Plus affinity resin appeared to
buffer and pH for affinity chromatography. Product maintain a lower elution pH than Eshmuno® A affinity
yields (not shown) were consistently within the resin over the range of conditions tested. But, the general
acceptable range for all conditions explored. trend for the two resins was consistent. For acetic acid,
the elution pool pH was relatively high at lower acetic
acid molarity for both resins. The actual elution pool pH
Elution pool volume for Eshmuno® A affinity resin does not follow the same
general trend as that of citric acid. Acetic acid’s volatility
The first criterion for comparison was elution pool
and low buffering capability are likely the cause of the
volume. As seen in Figure 1, citric acid buffers resulted
observed inconsistency (Figure 2B).
in consistent elution pool volumes (less than 1 CV
variation) across a range of pH and salt concentration
values for both ProSep® Ultra Plus and Eshmuno® A
resins. In contrast, acetic acid buffers resulted in greater
variation (>1 CV) of the elution pool volumes for both
affinity resins. This is likely due to the lower buffering
capability of acetic acid.

2
 A. ProSep® Ultra Plus resin A. Eshmuno® A resin
Contour plot of elution volume (CV) Contour plot of elution volume (CV)
vs. concentration (mM), pH vs. concentration (mM), pH
100 100
Elution volume(CV)
90 90
Figure 1.
< 1.50
Concentration (mM)

Concentration (mM)
80 80 1.50 –Elution
1.75 pool volume using
70 70 1.75 –citric
2.00 acid (A) and acetic
2.00 –acid
2.25 (B) as the elution
60 60 2.25 –buffers
2.50 on ProSep® Ultra
50 50 2.50 –Plus
2.75 (left) and Eshmuno® A
2.75 –(right)
3.00 affinity resins.
40 40 3.00 – 3.25
ProSep® Ultra Plus resin Eshmuno® A resin 3.25 – 3.50
30 30
Contour plot of elution volume (CV) Contour plot of elution volume (CV)
vs. concentration (mM), pH vs. concentration (mM), pH 3.50 – 3.75
20 20 3.75 – 4.00
100 100
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 > 4.00Elution volume(CV)
90 pH 90 pH
< 1.50
Concentration (mM)

Concentration (mM)
80 80
B. ProSep® Ultra Plus resin B. Eshmuno® A resin 1.50 – 1.75
Contour
70 plot of elution volume (CV) Contour
70 plot of elution volume (CV) 1.75 – 2.00
vs. concentration (mM), pH vs. concentration (mM), pH 2.00 – 2.25
100 60 100 60 2.25 – 2.50
90 50 90 50 2.50 – 2.75
2.75 – 3.00
Concentration (mM)

Concentration (mM)

80 40 80 40 3.00 – 3.25
70 30 70 30 3.25 – 3.50
3.50 – 3.75
60 20 60 20 3.75 – 4.00
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 > 4.00
50 50
pH pH
40 40

30
ProSep® Ultra Plus resin 30
Eshmuno® A resin
Contour plot of elution volume (CV) Contour plot of elution volume (CV)
20 vs. concentration (mM), pH 20 vs. concentration (mM), pH
100 100
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7
90 pH 90 pH
Concentration (mM)

Concentration (mM)

80 80
A. 70 ProSep® Ultra Plus resin A. 70 Eshmuno® A resin
Contour plot of elution pool pH vs concentration (mM), pH Contour plot of elution pool pH vs concentration (mM), pH
60 60
100 100
50 50 Elution pool pH
90 90 Figure 2.
40 40 < 1.50
Concentration (mM)

Concentration (mM)

80 80 1.50 – Elution
1.75 pool pH using citric
30 30
1.75 – acid
2.00 (A) and acetic acid (B)
70 70
20 20 2.00 – as the elution buffers on
2.25
60 3.0 3.1 3.2 3.3 3.4 3.5 3.6 603.7 3.0 3.1 3.2 3.3 3.4 3.5 3.6 2.25
3.7 – ProSep®
2.50 Ultra Plus (left)
pH pH 2.50 – and
2.75 Eshmuno® A (right)
50 50
2.75 – affinity
3.00 resins.
40 40
ProSep® Ultra Plus resin Eshmuno® A resin 3.00 – 3.25
30 Contour plot of elution pool pH vs concentration (mM),
30 pH Contour plot of elution pool pH vs concentration (mM),3.25
pH – 3.50
3.50 – 3.75
20 100 20 100
3.75 – 4.00
3.0 3.1 90 3.2 3.3 3.4 3.5 3.6 3.7 3.0 3.1 90 3.2 3.3 3.4 3.5 3.6 3.7 Elution pool pH
> 4.00
pH pH < 1.50
Concentration (mM)

Concentration (mM)

80 80 1.50 – 1.75
B. ProSep® Ultra Plus resin
B. Eshmuno® A resin
70 70 1.75 – 2.00
Contour plot of elution pool pH vs concentration (mM), pH Contour plot of elution pool pH vs concentration (mM), pH 2.00 – 2.25
60 60 2.25 – 2.50
100 100
50 50 2.50 – 2.75
90 90
2.75 – 3.00
40 40
Concentration (mM)

Concentration (mM)

80 80 3.00 – 3.25
30 30 3.25 – 3.50
70 70
3.50 – 3.75
20 20 3.75 – 4.00
60 60
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 > 4.00
50 pH 50 pH
40 40
ProSep® Ultra Plus resin Eshmuno® A resin
30 Contour plot of elution pool pH vs concentration (mM),
30 pH Contour plot of elution pool pH vs concentration (mM), pH

20 100 20 100
3.0 3.1 90 3.2 3.3 3.4 3.5 3.6 3.7 3.0 3.1 90 3.2 3.3 3.4 3.5 3.6 3.7
pH pH
Concentration (mM)

Concentration (mM)

80 80

70 70

60 60

50 50 3
40 40
 Elution peak profile
A third set of experiments compared the elution peak profiles for citric and acetic acids.
Citric acid produced a relatively stable elution profile for both resins tested, at citric acid
concentrations of 0.02 M and above (Figure 3A). A broadening of the peak was observed
at the lower concentration of 0.01 M on both resins (Figure 3A). By contrast, the elution
profile on either resin was more scattered when acetic acid was employed as the elution
buffer in comparison to that from citric acid (Figure 3B). Peak broadening and shoulders
were observed at lower acetic acid concentrations or higher pH. Elution peaks were
most consistent when acetic acid was at 0.1 M.

Figure 3.
Elution peak profile using citric
acid (A) and acetic acid (B) as
the elution buffers on ProSep®
Ultra Plus (left) and Eshmuno®
A (right) affinity resins.
A. ProSep® Ultra Plus resin elution peak profiles A. Eshmuno® A resin elution peak profiles
tion peak profiles with different citric acid elution conditions with different citric acid elution conditions
dtion peakconditions
elution profiles
d elution conditions
2,000 2,000 0.1 M
0.1 M pH 3 0.1 M
0.1 M
0.1 M pH
pH 3.5
3
mAU (300 nm)

mAU (300 nm)

1,500 0.1 M
0.1 M
M pH
pH 3.7
3.5 1,500
0.1 0.05 M
0.1 MMpH
0.05 pH3.7
3 0.05 M
0.05 M
0.05 M pH
pH 3.5
3 1,000 1,000 0.05 M
0.05 M pH 3.5
0.05 M pH 3.7 0.02 M
0.05 M
0.02 M pH
pH 33.7
500 500 0.02 M
0.02 M
0.02 M pH
pH 3.5
3
0.02 M
0.02 M
0.02 M pH
pH 3.7
3.5
0 0 0.01 M
0.02 M
0.01 M pH
pH 33.7
36 37 38 39 40 41 42 43 36 37 38 39 40 41 42 43 0.01 M
0.01
0.01 M
M pH
pH 33.5
0 41 42 43 CV CV
0 41 42 43 0.01 M pH 3.5

B. ProSep® Ultra Plus resin elution peak profiles B. Eshmuno® A resin elution peak profiles with
peak profiles with with different acetic acid elution conditions different acetic acid elution conditions
peak profiles with
tion conditions
tion conditions 2,000
0.1 M
0.1 M pH 3 2,000
0.1 M
0.1
0.1 M
M pH
pH 33.5 1,500
0.1 M
0.1
0.1 M
M pH
pH 3.5
3.7
mAU (300 nm)

mAU (300 nm)

1,500 0.05 M
0.1
0.05MMpH
pH3.7
3 0.05 M
0.05
0.05 M pH 33.5
M pH 1,000
0.05 M
0.05 1,000
0.05 M pH 3.5
M pH 3.7 0.02 M
0.05 M
0.02 M pH
pH 33.7
500 0.02 M
0.02 M pH 3
0.02 M pH 3.5 500 0.02 M
0.02 M
0.02 M pH
pH 3.7
3.5
0.02 M pH 3.7
0 0
36 37 38 39 40 41 42 43 36 37 38 39 40 41 42 43
40 41 42 43 CV CV
40 41 42 43

Summary
Main effects plots were prepared using the results from all experiments in this study
(Figure 4). The data indicated that the elution volume was impacted by each variable
studied. As elution conditions become stronger or the buffering capacity was increased,
the elution volume decreased (Figure 4A). Further, the resulting elution pool pH was
strongly impacted by the molarity of the elution buffer and type of elution buffer, and
was not impacted by resin choice (Figure 4B). The increase in elution pool pH and
elution buffer pH is not linear, and this might be attributed to other interactions,
such as elution pool dilution and a buffering effect from the IgG itself.

4
 A. Main effects plot for elution volume (CV)
Data Means
Elution buffer molarity Elution buffer pH
2.7 Figure 4.
2.4 Main effects plots for this
study, comparing elution
Elution Volume (CV)

2.1 volume CV (A) and elution

1.8 pool pH (B).
1.5
20 50 100 3.00 3.50 3.70
Concentration (mM) Elution Buffer pH

Elution buffer Resin

2.7
2.4
2.1
1.8
1.5
Citric Acid Acetic Acid Eshmuno® A resin ProSep® Ultra Plus resin

B. Main effects plot for elution pool pH

Data Means
Elution buffer molarity Elution buffer pH
4.2

4.0
Elution Pool pH

3.8
20 50 100 3.00 3.50 3.70
Concentration (mM) Elution Buffer pH

Elution buffer Resin

4.2

4.0

3.8
Citric Acid Acetic Acid Eshmuno® A resin ProSep® Ultra Plus resin

Conclusions and Recommendations

In this study, citric acid elution buffer provided a more consistent elution pool than
acetic acid at comparable elution buffer pH for the resins tested. Citric acid’s higher
buffering capacity and lower volatility likely contributes to this phenomenon.

Although acetic acid of the same molarity typically generates lower conductivity elution
pools, its higher volatility and lower buffering capacity should be considered when
choosing a Protein A elution buffer for production. Many factors need to be considered
when selecting an elution buffer and this work demonstrates the impact that elution
buffer choice can have on the efficiency and process robustness of mAb purification.

5
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