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UCD School of Chemical &

Bioprocess Engineering

CHEN30210:
Chemical & Bioprocess Engineering Laboratory 2
2017-18

Experiment 15: Protein Purification with Cation


Exchange Chromatography

Name: Nalin Seth


Laboratory Pair: H

Experiment Performed: Week X


February 20th,2018

KEY LEARNING GAINS FROM THIS EXPERIMENT (INCL. REPORT)

1. Seperation of mictures using AKTA system

2. Calculation of concentration of protein after separation

MOST DIFFICULT CONCEPTS (if any):

1.

2.

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TABLE OF CONTENTS

page

ABSTRACT 3

INTRODUCTION 4-5

MATERIALS & METHODS 6-7

RESULTS 8-10

DISCUSSION 11-14

CONCLUSIONS & RECOMMENDATIONS 15

APPENDICES

Appendix A. Experimental Data 16

Appendix B. Sample Calculations 17

Appendix C. Nomenclature 18

Appendix D. References 19

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ABSTRACT

The main objective of this experiment is to investigate the effectiveness of a cation exchange

column for the purpose of protein separation. This experiment was deter mined to be

successful as the cation exchange column separated a mixture of two proteins – Bovine

Serum Albumin (BSA) and Chicken Embryo Lysozyme (CEL). Additionally, the

concentrations of both proteins in the mixture were determined. Based on the isoelectric

points (pI) of each protein, and the pH of the buffer, it was predicted that the BSA would

flow through the column uninhibited, whereas the CEL would interact with the resin in the

cation exchange column, and would need to be washed out. This prediction was confirmed

based on the absorbance of the samples taken, checked with a spectrophotometer .

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INTRODUCTION

The purpose of this experiment is to separate a mixture of two proteins – Bovine

Serum Albumin (BSA) and Chicken Embryo Lysozyme (CEL), in a cation exchange

column. The basis of this separation is due to the relative charge of both proteins in a solution

with pH 7.4, (determined using the isoelectric points of each protein), and how this relative

charge allows one of the proteins to interact with the resin in the column, but allows the other

protein to flow through without interacting with the resin.In the production of

biopharmaceuticals, all processes produce more than the desired protein, and it is required that

these undesirables are completely removed from/ separated from the desired product before

sending the product for finishing. This is because the patient is often treated with the product

via injection. If this medicine is not pure, than there are very serious and potentially fatal

consequences for the sick patient. The primary aim of downstream processing is to

purify the product, but due to the fact that the desired product is often a protein, this

purification must be done based on mechanical separations, as chemical separation

techniques would denature the protein, rendering it useless for the purpose of treating

illnesses such as rheumatoid arthritis or cancers. There are several unit operations used in

the separation of product from undesirables in downstream bioprocessing. These

techniques can be classified as filtration or chromatography. There are several variations

for both techniques used in downstream processing, and this experiment focuses on

liquid chromatography. Typically one of the first steps in downstream processing is a

filtration step to remove any particles (i.e. cells) from the broth. After this has been done the

product (if it is a monoclonal antibody) is often passed through chromatography columns. The

basis of chromatography is the “selective retardation of solute molecules during passage

through a bed of resin particles” (Doran, 1995). If the desired product is a monoclonal

antibody, the first chromatography step is often an affinity chromatography step, where the

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broth is sent through a column packed with a resin called Protein A. The second purification

step often is either a cation or anion exchange liquid chromatography. The basis of

cation exchange involves having a column packed with a resin of strong negative charge.

Then, a solution containing a buffer of known pH will pass through the column. Substances

with a relative negative or neutral charge compared to the buffer will flow through the

column uninhibited, whereas substances with net positive charge compared to the buffer will

interact with the resin, and flow though the column at a slower rate, thus separating from the

main solution. The basis for determining the net charge of the proteins for this experiment is

the isoelectric point, pI. The isoelectric point is defined as the pH at which the protein has no

net charge. Therefore, if the buffer has a pH greater than pI, the protein will have a net

negative charge, and vice-versa. The isoelectric point of BSA is 4.7 (in H2O at 25°C), and the

isoelectric point of CEL is 11.35 (in H2O at 25°C). The buffers used for this experiment are at

a pH of 7.4. So since 4.7 –7.4 = -2.7, the BSA has a net negative charge, and should flow

through the cation exchange column uninhibited, whereas 11.35 – 7.4 = 3.95, so the CEL

has a net positive charge, and should interact with the resin in the cation exchange column.

This can be checked using the spectrophotometer at the end of each run.

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MATERIALS & METHODS

The materials and methods used were as described in the CHEN30210 Chemical &

Bioprocess Engineering Laboratory 2 Laboratory Manual. (Kieran, 2018). The AKTA

system had been set up and calibrated beforehand by the laboratory demonstrator, who went

through the set up at the beginning of the lab. Additionally, all three buffers (A, B and C) and

a sample solution of BSA and CEL had been prepared pre-lab by the laboratory demonstrator.

Before conducting the experiment it was pointed out that L, the light path length for

the spectrophotometer was 1cm, and that the value of 2mm written in the laboratory manual

was for the light path length for the flow cell incorporated in the AKTA Start Fast Protein

Liquid Chromatography (FPLC) instrument.

Experiment 15 Flowsheet

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UCD SCHOOL OF CHEMICAL & BIOPROCESS ENGINEERING
CHEN30210 SAFETY SUMMARY SHEET

Experiment No.: 15 Title: Protein Seperation


Academic/Technical
Ghayah Alsaleem
Staff Responsible:
Sheet prepared by: Nalin Seth Date: 29th April 18

MATERIAL HAZARDS & ASSOCIATED PREVENTIVE ACTIONS


Material Hazard(s)* Preventive Action(s)
Sodium Chloride Skin and eye irritant Wear PPE
Sodium Phosphate Skin and eye irritant Wear PPE
Dibasic Anhydrous
Sodium Phosphate Skin and eye irritant Wear PPE
Monobasic
Monohydrate
Bovine Serum Skin and eye irritant Wear PPE
Albumin
*The globally harmonised system (GHS) hazard symbol is shown first, followed by the pre-2011 European hazard symbol.

OPERATING HAZARDS & ASSOCIATED PREVENTIVE ACTIONS


Technique/Procedure Hazard(s) Preventive Action(s)
Handling of buffers and Chemicals Minimise Exposure
chemicals

Handling of Glassware Broken Glass Tidy up Carefully

WASTE DISPOSAL INSTRUCTIONS


Waste Disposal Instruction(s)
No Waste is generated N/A
during this experiment

RECOMMENDATIONS FOR AMENDMENTS TO EXISTING RISK ASSESSMENT AND/OR MSDS


Document Section/Page Recommendation & Justification

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RESULTS

Figure 1: Experimental Run 1 Conductivity and Absorbance v/s time


Two graphs were generated with the results obtained, one for Run 1 containing Buffer A & B,
and the other Run 2 containing Buffer A & C. In first case there was no first peak this might
be due to an error reading the absorbance values. Also, in the second case two distinct peaks
can be seen with the second peak being shorter and wider each peak represents a protein that
has been separated. The conductivity which represents the ionic nature of the sample- in both
cases increases linearly from approximately 7 and 8 minutes onwards respectively. Reaching
A maximum of 46 mS/cm for Run 1 and 78.9 mS/cm for Run 2. This as expected as the
concentration of NaCl in Buffer C is twice that of Buffer B.

Figure 2: Experimental Run Absorbance and Concentration v/s time

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Figure 3: Plot of Absorbance vs time from AKTA for run 1 – Buffer A + Buffer B.

The first peak was determined to be BEL and the second peak was determined to be CEL
based on the isoelectric points of the protein. The CEL peak occurs approximately 4 minutes
earlier in Run 2 than in Run 1 and approximately 1.5 times wider in Run 1. The peak relating
to BSA occurs over the same time frame in both the runs, however The BSA peak in Run 1 is
1.3 times taller than in run 2. The average absorbance of all fours peaks was calculated using
equation 2. The average absorbance of peak ranges from 25.2 mAU to 49.2 mAU. The
concentration of each protein was calculated using equation 1 to give values ranging from
3.23 to 5.65 the highest concentration was found to be in CEL in BUFFER A+C. This data
along with absorbance and concentration of pooled fractions is summarised below.

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Figure 4: Plot of Absorbance vs time from AKTA for run 1 – Buffer A + Buffer B.

Also, the fact that the spectrophotometer used in this experiment does not worked as
expected. Values repeatedly changed and does not settle for a single absorbance value, so the
middle of the range observed was taken. This indicated that the results obtained from this
apparatus is unreliable and should therefore be discussed with caution.

Average Concentration Absorbance of Concentration Absorbance


Absorbance (𝜇M) pooled of pooled of mixture
(mAU) fraction(mAU) fractions(𝜇M) (mAU)
BEL (A+B) 40.3 4.59 95 2.2
system
CEL (A+B) 25.2 3.23 45 1.2
System
BEL (A+C) 49.2 5.61 90 2.1
System
CEL (A+C) 44.0 5.65 90 2.3
System
300

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DISCUSSION

Conductivity is seen to increase linearly in both runs from approximately 7-8 mins onwards.
The AKTA system was set up to introduce buffers B and C alongside buffer A in 10%
augmentations, beginning with 100% of buffer A. Since buffer A contained no NaC1, there is
no conductivity in the samples. Buffers B and C contain NaC1 and therefore exhibit a
conductivity when they are introduced, 10% at a time, since the ions in the solution can
transfer current. The increase is linear as the second buffer (B or C depending on the run) is
introduced at a constant rate. The maximum conductivity reached in Run 1 is 46.0 mS/cm and
in Run 2 is 78.9 mS/cm. This aligns with the reasoning as the NaC1 is twice as concentrated
in buffer C when BSA is the first protein to elute. The isoelectric point of BSA is 4.7, much
lower than compared with buffer B. that of CEL — 11.35 (Kieran P.M., 2018). The pH of the
buffers is 7.4, meaning BSA is above its isoelectric point and CEL is below its isoelectric
point in the buffer solution. Therefore, BSA exhibits a net negative charge in the buffer and
elutes almost immediately as there is no interaction with the column packing, which is
anionic. On the other hand, CEL exhibits a net positive charge and interacts with the anionic
column packing, thus, preventing it from eluting (Kieran P.M. 2018). When the second buffer
is introduced, the sodium ions interact with the column packing more strongly than the CEL
protein, causing it to be displaced and collected in the samples after elution. This mechanism
explains the longer retention time of CEL over BSA. In Run 2, buffer C is used in place of
buffer B. Buffer C contains 1M NaC1 compared with 0.5M NaCl in buffer B.

The higher concentration of NaC1 means there are more sodium ions which can compete with
CEL to interact with the column packing. For this reason, when using the higher
concentration buffer (buffer C) CEL elutes over a shorter period, thus, the peak is narrower. If
the same amount of CEL elutes in a shorter period, this implies that the concentration of CEL
is higher. And since concentration is proportional to absorbance, the peak is taller due to
greater absorbance (Kieran P.M. 2018). It is seen in the results presented, that elution of CEL
does not begin until conductivity reaches approximately 10 mS/cm. This may be considered a
threshold over which there are enough sodium ions to begin completely displacing CEL from
the column. This is a slight difference in concentration and may be due to a slight error made
at the beginning of the run by the operator — having the line in the incorrect test tube for an
abbreviated period. In theory both values should be quite close as BSA does not interact with
the column packing and y, hence elutes immediately, unaffected by the presence of buffers B

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or C. The absorbance of the mixture was found to be 300 mAU compared with 95 mAU and
90 mAU for the pooled fractions of BSA and 45 mAU and 9Or1AU for the pooled fractions
of CEL. The values for the pooled fractions are significantly lower than those of the mixture.

This is because the pooled fractions comprise buffer solution, meaning they are more dilute
than the mixture. And since absorbance is directly proportional to the concentration, the lower
concentration of the pooled fractions harvests a lower absorbance than the mixture. Based on
the findings of this experiment, the use of a 1M NaCl buffer is more favourable than that of a
0.5M NaCl buffer for the elution of BSA from CEL in the shortest time possible. This is
based on the theories projected in this report as the result that both proteins eluted 2-3 mins
earlier when the more concentrated buffer was used. In more general terms, the use of a more
concentrated salt solution is more favourable when carrying out cation exchange
chromatography with BSA and CEL.

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QUESTIONS
1: Why does the conductivity increase during the chromatographic runs? Why does it
increase linearly?
The conductivity increases linearly during each run, as the AKTA machine linearly increases
the volume of buffer B (run 1) and buffer C (run 2) flowing through the machine,
whilst decreasing the volume of buffer A (ensuring that the capacity of the column (1mL)) is
not exceeded. So initially each run starts with pure buffer A flowing through the CEX, and by
the end of the run, only buffer B/C is being used.
2: Which protein elutes first? Discuss your answer based on the principles of CEX
chromatography and the properties of each protein that has been separated.
It is predicted that the BSA will elute first, as it will not interact with the resin in the cation
exchange column. This is because the isoelectric point of the BSA is 4.7. In a buffer with a
pH of 7.4, this means the net charge on the BSA is -2.7. Since the net charge is negative, the
BSA protein will not interact with the negatively charged resin, flowing through uninhibited.
On the other hand, the isoelectric point of the CEL is 11.35, and thus the CEL has a net
positive charge of 3.95, and will interact with the resin in the CEX, only flowing through
when the net positive charge of buffers B and C are greater than the net charge of the protein.
3: Which protein would elute first if anion exchange chromatography were used
to separate BSA and CEL using the same buffer system?
If the same buffer system was used, then the BSA would still have a net negative charge, and
CEL would have a net positive charge. Then, the CEL will pass through the column
uninhibited, while the BSA will interact with anion resin, and will need to be washed out by a
negatively charged buffer.
4: Why is the second peak narrower and taller in the A+C experiment?
The peak indicates the elution of the CEL flowing out of the CEX. Since the conductivity of
Buffer C is greater than the conductivity of Buffer B (due to the greater concentration
of Sodium Chloride – 1M compared to 0.5M) than the CEL is washed out at a quicker rate.
Since the same volume of CEL is washed out, but in a shorter time period (a narrower
peak), t he peak will need to be taller to have t he same area under t he curve.
5: Does the increase in conductivity correlate with the elution time for each peak? If so,
why?
The increase in conductivity correlates with the elution time for the second peak, but
doesn’t have an effect on the elution time or rate for the first peak. This can be seen
in figure 2. However, the increase in conductivity decreases the time it takes for the CEL

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(peak 2) to elute. This is because, the greater the conductivity, the more it breaks the
interaction between the CEL and the resin, allowing the CEL to flow through the column.
The reason that it doesn’t affect the BSA is due to the net negative charge of the BSA, which
allows it to flow through the cation column exchanger uninhibited, negating the effect of the
conductivity in the column.
6: Compare the absorbance values obtained for the sum of pooled fractions with
the absorbance obtained from the protein mixture (Results, step 8 (Kieran, 2017)).
Which is higher? Why?
The pooled fraction values for run 1 and run 2 are (2.2/1.2) and (2.1/2.3) respectively. This
shows that the pooled fraction for run 2 is higher. Combining them gives a value of , which is
less than the 0.2 from run 2, but greater than 0.1 from run 1.
7: Based on your answer for question 7, briefly discuss why a concentration step, such as
ultrafiltration or dialysis, is employed immediately after chromatographic steps in
an industrial setting?
From the calculations, it can be seen that the concentration of the proteins after separation is
extremely low – in the order of mM, so these must be concentrated before heading to a fill
finishing facility, otherwise the medicine would be too diluted to have any significant effect
on the patients health.
8: Considering your results, what separation conditions would you adjust to reduce the
time it takes to separate BSA and CEL? Justify your answer.
Since the BSA doesn’t interact with the resin, it flows through uninhibited. So to decrease the
time it takes to separate the BSA and CEL, the CEL must be eluted more quickly after the
BSA is eluted. One possible way of doing this would be to increase the concentration of the
Sodium Chloride in the second buffer. This will increase the conductivity in a shorter period
of time, thus eluting the CEL in a shorter time period, and at a quicker rate.

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CONCLUSIONS & RECOMMENDATIONS

This experiment was successful in separating a mixture of two proteins – Bovine Serum

Albumin and Chicken Embryo Lysozyme. It was predicted that the BSA would elute from

the Cation Exchange Column first, and the CEL would elute when the conductivity of the

system (i.e. when a sufficient concentration of Buffer B/C was in the flow) was enough to

break the interaction between the resin in the CEX and the CEL. This was confirmed by

testing the absorbance of the samples with protein, checking which protein had eluted first.

One possible recommendation to further enhance this experiment would be (if there was

enough time) to do a third run, but with a greater concentration of Sodium Chloride (e.g.

2M), to further investigate the effect of increasing the conductivity on the elution time and

rate of elution on the system (particularly the elution rate of CEL).

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Appendix A. Experimental Data

Chromatographic Run (Buffer A +Buffer B) Chromatographic Run (Buffer A+Buffer C)


Time Absorbance Conductivity Time Abrorbance Conductivity
0 0 3 0 0 3
0.5 17.6 3 0.5 11.7 3
1 163.9 3 1 156.9 3
1.5 210.1 3 1.5 156.9 3
2 78.3 3 2 45.9 3
2.5 18.6 3 2.5 9.8 3
3 5.9 3 3 3.6 3
3.5 2.5 3 3.5 1.3 3
4 1.4 3 4 0.6 3
4.5 0.9 3 4.5 0.4 3
5 0.6 3 5 0.2 3
5.5 0.5 3 5.5 0.1 3
6 0.4 3 6 0.2 3
6.5 0 3 6.5 0.2 3
7 0 3.1 7 0.3 3
7.5 0 3.7 7.5 0.2 3
8 0 4.6 8 0.2 3
8.5 0 5.1 8.5 0.1 3.9
9 0 6 9 0.1 5.8
9.5 0 6.9 9.5 0.1 7
10 0 8.1 10 0.2 8.5
10.5 0 9.3 10.5 0.1 10.2
11 0 10.6 11 0 12.6
11.5 0 11.9 11.5 0 15
12 0 13.1 12 0.2 17.4
12.5 0 15.5 12.5 1.3 19.7
13 0 16.8 13 9.7 22
13.5 0 18 13.5 65.6 24.7
14 0 19.2 14 124.3 26.4
14.5 0 20 14.5 130.9 28.9
15 0.4 21.5 15 69.3 30.5
15.5 3.4 22.6 15.5 26.6 32.6
16 13.1 23.8 16 7.1 34.7
16.5 36.4 24.9 16.5 1.6 36.9
17 72.1 26.3 17 0 38.6
17.5 99.7 27.3 17.5 0 41
18 91.5 27.9 18 0 42.8
18.5 63.8 28.3 18.5 0 44.8
19 52.4 29.6 19 0 46.6
19.5 32.7 30.6 19.5 0 48.6
20 11.1 31.6 20 0 50.3
20.5 4.1 32.7 20.5 0 52
21 1 33.7 21 0 54.1
21.5 0 34.7 21.5 0 55.9
22 0 35.7 22 0 57.7
22.5 0 36.7 22.5 0 59.3
23 0 37.7 23 0 61
23.5 0 38.7 23.5 0 62.8
24 0 39.7 24 0 64.7
24.5 0 40.8 24.5 0 66.2
25 0 41.7 25 0 67.7
25.5 0 42.5 25.5 0 69.2
26 0 43.4 26 0 71.3
26.5 0 44.4 26.5 0 72.9
27 0 45.1 27 0 74.4
27.5 0 45.7 27.5 0 75.8
28 0 46 28 0 77.1
28.5 0 46 28.5 0 78.2
29 0 46 29 0 78.7
0 29.5 0 78.8
0 30 0 78.9
30.5 0 78.9
Experimental data recorded for Run 1 and Run 2 (Buffer (A+B) & Buffer (A+C))

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Appendix B. Sample Calculations

Figure 5: Sample Calculations

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Appendix C. Nomenclature

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Appendix D. References

Kieran, P. 2018. CHEN30210 Chemical & Bioprocess Engineering Laboratory 2 Laboratory


Manual. Dublin: University College Dublin.

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