Bioanalytical Technique Practical

3404 words (14 pages) Essay

2nd Aug 2018 Sciences Reference this

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Introduction

Improvement in technology has widened the domain of bioanalytics, reliable and reproducible data can be obtained from several instruments and protocols. The drug arena has become really competitive and it is thus imperative that an understanding of the different techniques is crucial to the isolation and analysis of biomolecules.

This report is focused on the BCA assay for protein estimation and data analysis of SEC using a UPLC system. The BCA assay is a modified assay that is used for the detection and quantification of total protein in a given sample. The assay generates a purple colour which is as a result of the chelation reaction of bicinchoninic acid with cuprous ions. The complex formed as a result of the reaction is known to exhibit a very strong absorbance at a wavelength of 562nm and this shows an increasing linearity with the amount of protein in a given sample. Two main components make up the assay; the standard curve and the unknown protein sample. The BCA assay is widely used because of its sensitivity and compatibility with detergents and several other buffer types. The drawback however with the assay is that it is not as rapid as some other estimation method such as the Bradford due the incubation time required and moreover it is not an endpoint reaction as colour continues to develop even after incubation.

The second part of this report is concerned with running a system suitability test on the waters BEH200 SEC UPLC instrument. The American and European Pharmacopeia specifically mentioned that the requirements for a system suitability testing on the day of analysis showing that it is fit for its intended use. It is worth mentioning that this has no bearing with the qualification of the instrument. Failure of any of the parameters simply means that an assay cannot commence. This testing is concerned more about the method on the day of analysis rather than the instrument per se.

1.1 Materials:

  • Pipettes and appropriate tips
  • Microcentrifuge tubes
  • Microwell Plates
  • HPLC Vials
  • Bovine Serum Albumin Protein 2mg/ml
  • BCA Reagent
  • Deionised Water
  • Perkin Elmer Plate Reader
  • 100Mm sodium Phosphate Buffer
  • Waters Aquity H Class Bio UPLC instrument
  • Waters BEH200 SEC UPLC Column

1.2 Preparation of Standards:

Standards were prepared as per instruction manual

Table 1: Preparation of Protein Standards

[Protein Standard] (µg/ml)

Dilution factor

Volume Protein Standard (µl)

Volume dH2O (µl)

Final volume (µl)

0

0

0

150

200

25

1 in 80

2.5

197.5

200

125

1 in 16

12.5

187.5

200

250

1 in 8

25

175

200

500

1 in 4

50

150

200

750

1 in 3

75

125

200

1000

1 in 2

100

100

200

Calculations:

Dilution factor = concentration of stock solution / concentration of diluted solution

Volume of stock to add to water = Required volume of diluted solution/ Dilution factor

Volume of water to add = required final volume / Volume of stock required

1.3 Preparation of Sample:

The sample was prepared as per the instructions on the practical manual.

Table 2: Test Sample Dilution

Sample

Dilution Factor

Volume (µl)

Sample

Volume (µl)

Added Water

Final Volume (µl)

Biopharmaceutical

1 in 5

20

80

100

1.4 Preparation of BCA Reagent and Well:

The BCA reagent was prepared and the 96 –well microplate was prepared and read in the [email protected] 562nm as per the instruction manual.

1.5 Data Analysis:

Concentration mg/ml

Absorbance 1

Absorbance 2

Absorbance 3

Average

0

0

0

0

0

25

0.085141386

0.050185522

0.149322437

0.094883

125

0.014410967

0.117177903

0.100239697

0.077276

250

0.051610414

0.087607308

0.174962059

0.104727

500

0.53853473

0.338323087

0.351367406

0.409408

750

0.622078392

0.563260249

0.452668921

0.546003

1000

0.773383051

0.85954018

0.870560367

0.834495

Sample (x)

0.155851667

0.056575722

-0.105354519

0.035691

Calculation:

Equation of the linear least square fit can be represented as outlined below.

Y = 0.0008 (X) – 0.0051

0.035691= 0.0008 (X) – 0.0051

X = 0.035691 + 0.0051/0.0008

X = 0.040791/0.0008

X = 50.98875

Taking the dilution factor into account we multiply by 5

The protein concentration is thus 50.98875 x 5 = 254.94mg/ml

Discussion:

The sample data had an anomaly, showing a negative reading on the third well. This is suggestive of contamination. The possibility of interference from the reagent can be ruled out because the standard was treated the same way and also taking into cognisance the fact that the experiment was not carried out under a non-denaturing condition. The likely cause could be due to dirt on the Microwell thus blocking out the necessary wavelength for the absorbance reading or the sampling pipette not delivering the right amount of reagent. The intensity of the colour change for the third well was observed to be less than the other two wells. There is also the possibility of the sample not being vortexed properly or sample settling to the bottom of tube.

As mentioned earlier there seem to be an anomaly with our absorbance reading and this can be validated from our standard curve as it is not quite linear and on this basis we cannot absolutely rely on the result of the experiment.

2.1 Size Exclusion Chromatography Experiment

The priming and purging of the UPLC instrument was carried out by the trainer as per the instruction manual. System suitability testing was then carried out to ensure that it is fit for purpose. The test serves to assure the reproducibility of the instrument and the method. It is a regulatory requirement which was mentioned in both the EU and US pharmacopoeias. The testing is important as it can allow for critical factors that could affect the performance of the instrument to be adjusted to meet the test criteria. Parameters such as the resolution, efficiency of the column, tailing factors, relative standard deviation etc. are used as criteria for comparison with regards to standards and test samples.

The table below details the results obtained from the system suitability testing, reference standard and our test sample. The UPLC system used in our experiment can be said to be fit for purpose taking into consideration, the system suitability test. The results obtained were within our test criteria. The resolution of the peak and standard deviation of the different retention time was less than 1 which as a rule of the thumb is quite acceptable.

Comparing the test sample to the reference standard one would not fail to notice that the first peak in the reference standard was a dimer while the second peak was a monomer but in our test sample the retention time of the first peak was really short showing evidence of a high molecular weight aggregation .Also from our result the second peak was our product dimer while the third peak was our product monomer. This result serves to highlight the mechanism of protein aggregation and the reason why it should be minimised as it impacts on the yield of the product and moreover it can affect the potency and therapeutic potential of the parenteral. It is also worth mentioning that sometimes early elution may not necessarily mean that there is aggregation, it could be for the simple reason that sometimes intrinsically unstructured proteins can elute so fast that they tend to behave like aggregates. A molecular weight comparison testing can be used to differentiate them.

From our experiment the test sample showed increase aggregation and this can be explained given the fact that the experiment was not carried out under a non-denaturing environment. The possibility of column contamination or buffer contamination can help to encourage aggregation. It is also important that samples should be free of extraneous particles during injection as this can also be a determining factor. The changing environment of the mobile phase can also be construed as a possible cause of the aggregation observed. The temperature of the instrument is another factor that can play a role and as we all know that the Arrhenius theory of a 10°C increase in temperature speeding up a reaction does not relate to proteins as it rather opens up the pathway of denaturation and aggregation.

 

RT

% Area

Pass?

Peak

MEAN

RSD

MEAN

RSD

(Y/N)

 

1

3.035

0.5

18.00

0.8

Y

 

2

3.475

0.5

1.88

0.8

Y

 

3

3.817

0.5

27.83

0.8

Y

 

4

4.295

0.5

19.14

0.8

Y

 

5

4.885

0.5

3.54

0.8

Y

 

6

5.279

0.5

16.36

0.8

Y

 

7

5.957

0.5

0.59

0.8

Y

 

8

6.694

0.5

11.73

0.8

Y

 

9

8.035

0.5

0.04

0.8

Y

 

Reference Standard

Pass?

 

RT

% Area

 

Peak

MEAN

RSD

MEAN

RSD

(Y/N)

1

3.754

0.5

6.92

0.53

Y

2

4.251

0.5

93.08

0.53

Y

Test sample

Pass?

 

RT

% Area

 

Peak

MEAN

RSD

MEAN

RSD

(Y/N)

1

3.095

0.5

47.93

0.8

Y

2

3.754

0.5

3.14

0.8

Y

3

4.251

0.5

48.93

0.8

Y

Questions:

Estimation of protein concentration is important as we have to know the amount of protein in our final product after fermentation to know if the bioprocess has to be optimised with regards to the expected titre value. The concentration of the protein can also allow for the portioning of the product into the right dosage formulation, certain therapeutic proteins are required in a very high dosage form and their production can be sometimes targeted at a particular section of the population e.g. during an epidemic outbreak to ensure potency and biological activity. The knowledge of the concentration of proteins can also allow us to work out the economy of scale with regards to the profit margin taking into account, the expense incurred in research and development and other aspect of the production process. It is also important to estimate the amount of protein in our biomass so as to be able to optimise our subsequent purification steps. The estimation of the protein concentration can also give us an idea of product related impurities and those associated with the process.

Proteins are very complex molecules and are prone to several types of condition than cause instability from the starting stage of production to the end of their shelf life. Aggregation can be described in a layman’s term as the propensity for proteins to stick together under conditions such as a slight increase in temperature, pH, shear force, ionic strength of the solution they are contained in etc. Aggregation have been seen to cause delay in several novel biologics due to the debilitating effect on the health of the population that the drug is directed at and also in the context of compliance to regulatory authority as there is a specification to the amount of aggregates that can be allowed. It is extremely difficult if not impossible to totally eradicate aggregation from the process. It is worth mentioning however that the mechanism of aggregation is still subject to debate as it has not been fully understood. Aggregation can be reversible or irreversible depending on the stage it has attained as can be loss of primary structure.

The potency of biologics as we all know are normally related to them being in their native structure, in most instances aggregation leads to the loss of activity and moreover the overall yield of the biotherapeutic is greatly affected. Aggregation has also been known to spur immune response in patients that have been administered with protein therapeutics affected by aggregation this could be by way of the neutralisation of antibodies that helps to ensure the effectiveness of the drug. In a worst case scenario the immunogenic reaction can lead to incurable conditions such as seen in patients with pure red cell aplasia where the red blood cells are attacked and blood transfusion is needed for life. The route of administration of biologics is intravenous and the presence of aggregation especially those of very high molecular size can result in the blockage of blood vessels. It is thus very important that at each stage of our production testing should be carried out to check for aggregation.

Size exclusion chromatography is a purification system that exploits the molecular size of the compound of interest. Simply put it works just like a molecular sieve, smaller particles passes through the sieve which is the stationary phase and could be a bead coupled to a resin. The pore size of the beads are defined and on this basis it will only allow certain particle sizes to pass through while excluding those that are too large for the pore. The larger particles because they are not passing through the beads are thus excluded quickly, their retention time is thus said to be short. The smaller particles are retained longer while the larger particles earlier mentioned are eluted through the void volume. Different gels in use would typically have different pore sizes and can be used to determine the size of the molecules to be separated.

Despite all the numerous advantages of size exclusion chromatography which has made it the gold standard over the years for analysing protein aggregation there are still some limitation associated this method. The possibility of the stationary phase and the analyte reacting together can be sometimes rife thus leading to a longer retention time which serves to mimic the compound as being of low molecular size. The cost associated with running this type of separation technique can also be enormous due to the fact that large columns and eluents are required and this serves to add to the overall cost of the unit operation. In comparison to other modes of separation, size exclusion chromatography can be said to have an inherent low resolution as there is a limited range of molecular weight that can be separated as a result of dependence on the pore size of the beads in use. There is also the possibility of proteolytic degradation as the protein of interest can become targets for proteolytic enzymes still present in solution. The accuracy of this technique can sometimes come into question due to the fact some aggregates will remain in solution and as such would not be detected. Also taking into consideration the fact that larger molecular aggregate leaves the column through the void volume, there is also the possibility. The possibility of the polymer in use to degrade is also a drawback as this can occur at a very high flow rate. The high flow rate as mentioned earlier can degrade the polymer and it also has the ability of altering the geometry of the beads in use making the separation technique inefficient

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