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Chromatography Technique for Purification

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Published: Fri, 13 Jul 2018

  • Okeli Stephen Ade

Introduction

Column packing is an integral part of the purification process in the manufacture of biologics. The goal is always to ensure reproducibility with regard to the technique to be used. Manual packing might sometimes involve several attempts to get an optimal packing as this would affect the purification process. The resin to be used in the packing process has to be well defined as it could impact on the flow rate which could lead to a reduced through put. The mobile phase is also important as the rationale behind the choice would be looking for a solvent that can pack the resin more tightly.

In any chromatography technique that has to be utilised whether for the need to capture, purify or polish four integral parameters which includes, resolution, speed, capacity and recovery are always considered. Resolution is the most difficult to achieve especially during the polishing stage were impurities can be construed as having similar properties to the product. The efficiency of the column packing thus has a significant role to play on this basis as it is a good measure how consistently the column can perform.

1.1 Material: As per SOP

1.2 Key Instrument Components

  • Bubble Trap – BT1
  • Filter Housing – F1
  • Inlet / Outlet Valves – V001 & V101
  • UV Sensor – QIR4
  • pH Probe – QIR3
  • Conductivity Meter – QIR2
  • Drain – V102
  • Column top and bottom connections – DN ¼

1.3 Preparation of column for packing: As per SOP TRG-DSP-052.

1.4 Determination of % slurry: Procedure was followed as per instruction manual

Results: Table 1

Parameter

Volume

Volume of centrifuged resin in 50ml centrifuge tube

______33_______ml (a)

Volume of centrifuged resin in 100ml

(a) x 2

________66_____ml (b)

% Slurry in Container (b)

_______66______ % (c)

Results: Table 2

Parameter

Formula

Measure Value

Volume (L)

Packed bed volume (PBV)

πr2h

Where r = radius of column

h= desired packed bed height

r=5cm

h= 15cm

1.178

Volume of gravity settled resin for packing (Vgs)

PBV x CF

CF = compression factor (1.33)

1.178 x 1.33

1.56674

Slurry volume needed from container (SVc)

(To give you the desired amount of gravity settled resin) (c)

Vgs x 100

% slurry in container

= 1.55574 X 100/66

% Slurry in container = 66%

2.37384

Adjusting the slurry to the desired % concentration for packing

Slurry volume required for packing (SVp)

Vgs x 100

% slurry for packing

= 1.56674 X 100/ 70

% Slurry for packing = 70%

2.2382

Volume packing buffer to add

SVp – SVc

= 2.2382- 2.37384

-0.13564

Volume to be added is thus 0.13564 Litre

Calculations:

Number of theoretical = N

Where VR = volume eluted from the start of sample application to the peak maximum

= 8CM

W h = peak width measured as the width of the recorded peak at half of the peak height

= 0.5CM

N = 5.54 X ²

Number of theoretical plates = 1418.24

HETP = L/N

Where L = Bed height (cm)

As we already know N (1418.24)

HETP = 15/ 1418.24

= 0.0105765

Asymmetry factor (AS) = b/a

Where a = 1st half peak width at 10% of peak height (0.5cm)

b = 2nd half peak width at 10% of peak height (0.5cm)

= 0.5/0.

= 1

As rule of the thumb a good HETP value should be at least two to three times the average matrix bead size and normally in the range of 0.0018cm to 0.035cm. Looking at our column our HETP value was approximately 0.0106 and our bead has a pore size of about 40 microns which equates to 0.004cm and this is about 3 times our HETP. Our column can thus be confirmed to be within the acceptable range.

In the event that our column is not within the acceptable range several factors such as the following can be construed as being responsible.

  • Uneven packing of the column or exceeding the optimal packing flow rate
  • The possibility of channelling in the bed
  • Inadequate CIP can also be a factor as this can result in a build-up of contamination in the column thus impacting on flow and other performance determinants of the column. Cleaning is also important to wash the matrix storage solution which is an unwanted entity during packing.
  • Air entrapment prevalence of air bubbles can also affect the HETP values.
  • The possibility of a void being present at the inlet can also be a contributing factor to the value of HETP not being within specification
  • The choice of resin is also very important as the possibility of the solute reacting with the resin can result in an ambiguous HETP value.

Peak asymmetry is an important measure in the determination of column efficiency and in conjunction with the HETP value is always used in the calibration of a new or existing column. The gold standard is the ability to achieve an asymmetry value of 1 although the acceptable range is normally between 0.8 and 1.2. An asymmetry value greater than 1 indicates the prevalence of extensive tailing while an asymmetry value less than 1 indicates extensive fronting.

Taking our packed column into consideration, our asymmetry value from the chromatogram was 1 and one would generally thus expect a high efficiency and resolution.

However, in the event of our column not being within the acceptable asymmetry value the following reason are the possible causes.

  • Extensive tailing which is characterised by an asymmetry value greater than 1 as mentioned earlier can be a reason. This factor is a result of column being packed too loosely and it can be observed from the chromatogram by the peak tailing gradually.
  • Extensive fronting is also a possible cause and it is characterised by an asymmetry factor less than 1 which is normally as a result of the column being packed tightly and would be noticeable on the chromatogram by the peaks developing slowly.

Possible causes of resin/column deterioration and their remedies

  • Temperature – the resins have a temperature range that is normally specified by the manufacturers and a usually high temperature can cause irreversible damage due to loss of functional groups. It is thus important that operation should always within the optimal ranges and bearing in mind the fact that temperature maxima is only for indication.
  • Oxidation – The functional groups are also attached by oxidation and on this basis one has to ensure that oxidants such as hydrochloric acid , nitric acid are not utilised in the cleaning regime as they can accelerate oxidation which damages the polymer crosslink
  • Fouling apart from impacting on performance of the column can also cause irreversible damage to the resin. Fouling can result due to the presence of iron and silica for this reason special attention has to be paid to the type of resin to be used as prevention they say is better than cure.
  • Drying out and cracking of the resin is also an important reason for column deterioration and this can be remedied by ensuring that the column is well equilibrated.
  • High pressure -The build-up can also cause damage to the resin/column and it could be as a result of flow path restriction due to dirty or worn bed support. Manufacturer’s specification should always be adhered to in ensuring an optimal usage of the resin.
  • The life span of the resin/column should also be taken into account and usage should always be as specified by the manufacturer.
  • Harsh elution is another factor that is responsible for irreversible damages to resin/column. Every resin has a pH range that is optimal and this should be adhered to strictly.

Conclusion:

The practical experience was so interesting and brought the protein purification lectures received into perspective. A better understanding of the process was developed and the practical knowledge is quite adaptive to the day to day operation in a typical Biopharmaceutical plant.


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