#2C Chromatography Systems

8/10/2019 #2C Chromatography Systems

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Chromatography systemsBy Assoc. Prof. Dr. Maizirwan Mel

© Azmir 2008



Contents

• Engineering aspects of scale-up

•Process considerations

•Scale up of adsorption process

•Scale of Partition Chromatography

•Equipment•General requirements

•Chromatography columns

•Pumps

•Detectors

•Valves, Piping, and Vessels

• Automated Chromatography Systems

•Design: Liquid Handling Unit

•Design: Data Handling Unit

•Operation

•Installation, Commissioning, and Validation



Introduction

The purity requirements for recombinant proteins intended for human

therapeutic applications are extremely high

Modern analytical methods have the sensitivity to detect impurities present at

a concentration of only a few parts per million (ppm)

Chromatography is one of the few techniques that can achieve both the high

resolution and the high capacity needed to generate large quantities of highly

purified product

However even high resolution chromatography is unable to achieve the

required purity in a single operation, and therefore several chromatographicsteps in succession are usually required

Each step utilizing different aspects of the product’s chemistry (for example,

molecular charge, size, or hydrophobicity) to meet the purity required for

pharmaceutical applications



Engineering aspects of scale up

Process Considerations:

The ideal purification process for economic scale up involves only a small number of

integrated chromatography steps that utilize aqueous buffers and do not require organic

solvents

The process should be designed to operate with step changes of buffer composition

instead of continuous concentration gradients and should not require the collection of

numerous fractions across a product elution peak

The buffer used should not support bacterial growth and the chromatographic matrices

should be compatible with clean in place (CIP) solutions to allow efficient sanitization and

decontamination

The matrices should be able to withstand high flow rates and pressures of several bar in

order to reduce purification cycle times

In addition, the purification process as a whole, including buffer makeup, column packing

and testing, in process sampling, etc. should ideally fit comfortably into five eight-hour

shifts



Process Considerations (contd.)

Affinity chromatography is among the easiest of chromatographic procedures to

scale up and automate

It has very high specificity and relative insensitivity to operating conditions such

as flow rate and product concentration

For more difficult purifications, (for example, where product and impurities have

very similar chemical properties), high pressure or high performance liquid

chromatography (HPLC) is used



HPLC is generally used to describe chromatography on matrices composed

of rigid, small particles (5-25 µm diameter) having a narrow particle size

distribution

Advantage of HPLC:

It can achieve extremely high resolution, enabling chemically similar molecules to be separated from each other in a single unit operation

Disadvantage of HPLC:

a. It require expensive chromatography equipment

b. It require expensive matrices that need specialized column packingprocedures

c. When HPLC used with reverse phase column, it will require organic solvents

that can irreversibly denature many proteins and with expensive handling

procedures



Process Considerations (contd.)



Engineering aspects of scale up

Scale up of Adsorption Processes

The overall aim of scaling up a purification procedure is to increase the rate of production in

the most economical manner

The column sizes required in the manufacturing operation are generally set by the scale of

upstream operations and by any subdivision of one batch of product into successivepurification styles

The stability of the product at the intermediate stages of purification process affects any

subdivision of one batch into cycles and therefore influences column sizes. Thus in practice

the first chromatography step in a purification sequence is often performed as quickly as

possible in one cycle on a relatively large column so that any proteolytic enzymes and other

deleterious impurities and contaminants are rapidly removed

The guidelines that are suggested for scaling up chromatographic column dimensions are

different for adsorption chromatography and partition chromatography



Scale up of Adsorption Processes (contd.)

Suggested Guidelines for Scale-Up:

• Use exactly the same grade of chemicals and the same gel

• Keep the ratio of sample volume to column volume constant

• Maintained the same linear flow rate

• Keep the bed height constant, and vary the bed diameter only

• For gradient elution, maintain the same ratio of total gradient volume tocolumn volume




The shape or aspect ratio of the column is an important factor in process scale chromatography. By increasing the bed diameter and not the bed height when scaling up, it is possible to keep both the

cycle time and the pressure drop across the bed constant.

However there is clearly a limit to how wide a column can be before it is necessary to increase the bed

height so as to prevent channeling through the column and breakthrough of product prior to elution.

The recommended ratio of diameter to height of adsorption chromatography columns for process scale

work is approximately 2.0-4.0




Table5.3



Scale up of Partition Chromatography

Gel filtration chromatography is a inherently a low capacity purification technique that is notwell suited to industrial scale use

Many matrices produced for gel filtration are mechanically weak and can compress when

packed in large columns

However, gel filtration is often used as a final stage in many purification schemes to remove

trace amounts of impurities and self aggregates of the product, and to buffer exchange the

sample into the final formulation buffer

In gel filtration, resolution is a function of: The column length approximately one meter when using modern gels that have some rigidity

The ratio of the sample volume to the column volume typically around 5% when gel filtration

is used on a process scale for protein purifications The linear flow velocity lower than adsorption chromatography and lead to much longer

cycle times

The sample concentration protein conc. is usually is keep as high as possible to provide

high throughput typically 10-50 g/L



Scale up of Partition Chromatography (contd.)

•The guidelines for scaling up gel filtration unit operations are broadly similar

to those for adsorption chromatography, but careful attention to detail is

required since a large number of factors can affect the separation obtained

•Process optimization studies need to be done to determine the highest

protein concentration, linear flow velocities, and ratios of sample volume tocolumn volume that can be used without a detrimental effect on the required

solution when the process is operated with bed heights of approximately 60-

100 cm.



Equipment

General requirements:There are four fundamental requirements that are common to all items of equipment:1. The materials of construction must be compatible with the proposed buffers, solvents and cleaning solutions.

Gaskets, plastics, and so forth should not leach any plasticizers or other chemical into the product stream, and

stainless steels need to be of appropriate grade if chaotropic chemicals, high conc. of salt, or solutions of low pH

are used in the cleaning or running buffers

2. The equipments must be designed to allow hygienic operation. Internal surfaces should be smooth and highlypolished to minimize bacterial adhesion and corrosion. Wherever possible, threaded connections should be

avoided, since bacteria and other deposits can accumulate in the threads

3. The equipment must function reproducibly in order to produce a quality product with no batch-to-batch variability.

Pumps, for example, should be able to maintain their set flow rate irrespective of back pressure, and monitors

should have stable outputs without drift of the signal. Where chromatography gels are reused, product qualityand safety should be independent of gel cycle number within the known limits

4. Reliability is of central importance because, with high value compounds, a single batch of product can have a

greater value than the equipment used for its purification. All instrumentation must be serviced and calibrated

regularly; all electronic equipment should be immune from electrical noise, and should be able to tolerate high

levels of condensation. Critical pieces of equipment such as pumps and UV absorption detectors also need to

have extensive alarm functions to safely shut down the system if a failure occurs



Chromatography columns

The column itself must be able to withstand the operating pressures without flexing. The end plates need to becarefully designed and constructed to produce uniform laminar flow across the entire width of the gel bed (Figure

5.2).

The volume of the flow distribution chamber within the end plate should be as small as possible in order to

reduce zone spreading.

If the end plate incorporates multiple inlets, then these need to be of a sanitary design and must prevent the

formation of air locks.

The end connections need to be robust and one should avoid the use of plastic compression-type connections .

The seals should contain no potentially contaminating substances such as oils or hydraulic fluids.

For low pressure chromatography, glass or acrylic columns are frequently specified.

In high pressure applications, columns are generally constructed of 316L stainless steel.

Chromatography gel should be able to withstand high flow rates without compression, and should not swell or

shrink with changes in buffer pH, ionic strength, and chemical composition.



Chromatography columns (contd.)

Figure 5.2



Pumps

Pumps should be of the positive-displacement type, such as diaphragm pumps

The key requirements are:

The set flow rate should be maintained irrespective of back pressure

The handling of the protein solution should be gentle, so as not to shear or

denature the protein; and The pulsations should be minimized, to avoid disturbance of the chromatographic

bed

Major disadvantage of peristaltic pumps are the likelihood of breakage of the

flexible tubing used in the pump head

However when extremely high levels of containment are required peristaltic

pumps are frequently used Gear pumps or cog-wheel-type pumps are not recommended for sensitive

proteins

HPLC pumps must be able to deliver low flow rates at high pressures



Detectors

Three types of detectors are used to monitor and control the progress of purificationprocedures:

1. pH detector

2. conductivity detector

3. UV absorption detector

• The requirements for all detectors are that they should not cause significant back

pressures, should not give rise to dilution or back mixing of the product stream,

and should not be the source contaminants

• Due to high protein concentrations that are frequently encountered in process-

scale operations, UV detectors need to have short optical path lengths so as toensure the detector is operating in its linear range



Valves, Piping, and Vessels

Piping The piping used to connect the individual components should ideally be of

stainless steel, although flexible piping is a cheaper alternative.

The piping should be seamless, to give gentler fluid handling, and ideally should

be able to withstand temperatures up to 150ºC, to allow either steam sterilization

or autoclaving.

The piping should be polished internally after welding, to eliminate all crevices. The dimension of the piping should be wide enough not to give rise to a significant

pressure drop, but narrow enough to produce slight or moderate turbulent flow to

reduce zone spreading.

Table 5.4



Valves, Piping, and Vessels (contd.)

Valves

Valves used to direct the process fluids must completely seal to prevent contamination and

must be easy to clean.

A feedback mechanism with an alarm function should be incorporated, to relay information

of the actual valve position to the controller.

Ball valves should be avoided if possible, as the hollow balls cannot be flushed clean when

is closed, because the trapped fluid is not accessible to the cleaning medium.

Where the operating pressures allow, weir type diaphragm valves should be used, as they

are relatively easy to clean.

In high pressure systems, diaphragm vales are not suitable because can cause leakage

Ball pumps is the best alternative but must paid special attention on cleaning regimes



Valves, Piping, and Vessels (contd.)

Vessels

Variety of vessels are required in order to operate a chromatography system, such as buffer

makeup tanks, supply tanks, and receiver tanks

For small volumes (less than 20L), use polypropylene vessels which are convenient,

compatible with most process streams, and relatively easy to clean and sterilize

For larger volumes, stainless steel tanks are used (example: 316L) : relatively resistant to

corrosion

Reflective finishes with 200 grit or finer are expensive to produce but reduce the risk of

contamination, easier cleaning and sterilization

For liquids that are not compatible with 316L, other alloys or fluoroplastic or glass lined

tanks are required



Automated Purification Systems

Design: Liquid Handling Unit The simples chromatographic system consist of a pump, a column, a detector, vessels, and

interconnecting tubing.

These components form the core of any purification system, but complete large-scale

system differ in at least two important respects:

a. Additional components and mechanisms are incorporated to improve product safety

and ensure fail-safe operation; and

b. Automation is used to increase reproducibility and decrease the need for manual

intervention

Automation not only reduces labor costs and increase throughput by allowing 24-h

operation but also enables quality to be built into the product by reducing process variability

The additional components incorporated into production-scale systems are used to ensure

that air does not get into the system (air sensors and bubble traps), that pressure does not

build up in the system (pressure detectors), and that it operates in as sterile manner as

possible

More additional components: programmable controller, automatic valves, CIP facilities



Design: Liquid Handling Unit (contd.)

Gambar 5.4




Gambar 5.4




Gambar 5.5 and 5.6




Figure 5.4 shows a range of systems of increasing complexity

Process scale systems have a number of buffer tanks and collection tanks

connected to the purification systems via either two or three way valves

Automatic solenoid or pneumatic valves allow application of various buffers in

sequence without manual intervention

Air sensor is used on the sample inlet line so as to allow loading of the entire

sample volume onto the column

A bubble trap located immediately prior to the column serves not only to

eliminate air that may have entered the system but also as a pulse dampener

When designing layout of piping, engineer should minimize (or preferably)

completely eliminate dead legs, to ensure that he system can be fully drained



Design: Data Handling Unit

• It is important to consider carefully the requirements of the software and to specifythe simplest system that can meet operational requirements (best to have simple

system)

• The following features can be useful in automated purification systems:

a. The data handling unit should be remote from the wet liquid handling unit, ideally at roomtemperature and in a dry environment

b. The software should record the continuous outputs from al the monitors and detectors

c. The software should be able to integrate the output from the UV monitor to give an estimate

of the quantity of UV absorbing material in each fraction

d. Key parameters (such as UV absorption) should be displayed n real time to inform the

operator the progress of the run

e. A real time mimic of the fluid path showing valves status should be available

f. The system should be able to distinguish between warnings (such as low fluid level in the

bubble trap) and alarms (such as incorrect valve position)

g. The system should always be fail-safe

h. The software should enable the monitor outputs from a run to be overlaid and compared

with a previous run recalled from the system’s memory



Design: Data Handling Unit (contd.)

i. Access to the program should be restricted (for example, by password) j. The facility for manual intervention should be available to supervisors, but all such

interventions should be recorded

k. After each run the systems should print out a record of all the events and monitor outputs

associated with that run for inclusion in the batch records. Estimated volumes and

quantities of UV absorbing material in each fraction, and any manual interventions, alarm

overrides, etc., should also be printed out

l. The automated system should be able to communicate with other computer basedsystems



Operation

The operation of automated systems requires more than just the connection of sample/buffer tanks and the initiation of purification program

A sequence operations similar to the operation of fermentors must be performed

The purification system and the gel must be cleaned before and after each purification run

Sodium hydroxide solutions are frequently used to clean and store both equipment and gels

Once the purification system has been cleaned and sanitized, new filters must be installed

and their integrity tested, the system primed and checked for leaks, and detectors tested

and calibrated The column needs to be packed and the quality of the packing testes by means of HETP

(height equivalent to a theoretical plate)

Column packing procedure depends on the physical properties of the gel/matrix, but in all

cases the overall aim is to achieve a uniform packing of the gel throughout the column

without the introduction of air into the system

This generally requires the gel to be slurried and poured into the column in one operation The slurry should be thoroughly mixed and ideally degassed to ensure homogeneity and to

remove any air pockets that may be trapped

The gel should then be packed under pressure either by pumping buffer through the

column or by applying gas pressure

An HETP test is used to examine the efficiency of the column packing procedure, and is

particularly important in gel filtration applications



Operation (contd.)



Installation, Commissioning, and Validation

Once purification equipment has been designed and constructed, it needs to be tested to

ensure that it operates consistently and reproducibly within the limits required by the

process

Process validation is a requirement of the Current Good Manufacturing Practices (CGMPs)

for pharmaceuticals

The validation of entire pharmaceutical manufacturing process requires validation of all the

individual equipments of that process, including analytical procedures, equipments,facilities, etc.

Equipment validation is generally divided into a number of phases through life cycle

approach: a prequalification phase involving the specification and design of the equipment,

installation qualification (IQ), operational qualification (OQ), process qualification, and

ongoing evaluation

Installation qualification (IQ) has been defined as “documented verification that all key

aspects of the installations adhere to manufacturer’s recommendations, appropriatecodes, and approved design intentions”

Models and serial numbers must be listed and checked to verify that all equipment in the

plant corresponds to the actual equipment specified

Materials of construction (for example, gaskets, seals) and pipe material certificates should

be checked to ensure that they conform to the appropriate grade of materials

Installation Commissioning and Validation



Installation, Commissioning, and Validation

(contd.)

Operational qualification (OQ) is “documented verification that the system or subsystemperforms as intended throughout all specified operating ranges”

OQ requires protocols to be written before studies are initiated, detailing the scope of the

investigation and the tests to be performed

The protocols should specify acceptable ranges for the measured parameters with clear

pass/fail limits

Particular attention must be paid to the safety and alarm systems, to verify that they trip

when the present levels are reached and that the system fails safe as intended All alarms must be shown to cause the expected effects on the system as specified in the

functional specification, and not cause any unintended actions to occur during shutdown or

while the system recovers to restart its operation

For example air should be deliberately introduced into the system to ensure that the bubble

trap prevents air from reaching the column

Standard Operating Procedures (SOPs) also need to be written to enable the operators touse the equipment correctly

The instructions must be clear and unambiguous

When alarm functions are incorporated into the system, the instructions must state what

action to be taken and by whom

#2C Chromatography Systems

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