EMERGENCIA EN EL EDIFICIO DE CIENCIAS AMBIENTALES
INCLUIDO) 1 Planta el´ ectrica Cummis Po-
5.3.1 Background
In this section, the study o f the fractionation diagram approach will be further extended from laboratory-scale chromatographic results to industrial scale data. The operation o f investigation is the removal o f an endotoxin contaminant from a plasmid DNA product by ion-exchange chromatography. The plasmid DNA products o f study are destined for vaccination purposes. The data was kindly provided by the
Biopharmaceutical Product Development Department o f GlaxoSmithKline
(Beckenham, Kent, United Kingdom).
The following material provides a overview to the biological system o f study. The principle o f gene therapy entails the stable introduction o f a gene into the genetic- compliment o f a cell, such that subsequent expression o f the gene achieves a therapeutic goal. The use o f purified plasmid DNA constitutes a new approach to vaccine development. Plasmid DNA vaccines may find applications as the preventive vaecines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation o f hyperimmune globulin products; therapeutic vaccines for
infectious diseases; or other indications such as cancer
Recombinant DNA (rDNA) is a deoxyribonucleic acid sequence produced artificially by pieces o f DNA from different organisms (recombinant DNA technology). Recombinant DNA must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporatirtj the rDNA in a vector. Plasmids in this case study are used as the vectors to transfer a recombinant DNA sequence into cell o f another organism. A plasmid is a circular, self-replicating form o f double stranded DNA molecule found in many species o f bacteria separate from the bacterial chromosome. They have a few thousand base pairs and usually carry
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only one or a few genes. After the insertion o f rDNA into the plasmid ring, it is then ready to be inserted into a host cell for a biotechnological application. E. coli is the most widely used host cell for plasmid DNA production.
Endotoxins are cell wall constituents o f gram-negative bacteria such as E. coli. The endotoxins can be liberated during bacterial growth and when bacterial die. The endotoxin is a lipopolysaccharide (EPS), consisting o f three regions: an innermost lipid moiety, called lipid A, an intermediate core polysaccharide and an outermost polysaccharide side-chain. The lipid A exerts most o f the biological activity (pyrogenicity). Endotoxin is the most common o f the pyrogens which they enter the bloodstream will influence hypothalamic regulation o f body temperature resulting in fever (Walsh, 1998). In humans, a pyrogenic response may occur if 5 endotoxin units (EU) per kilogram o f body weight are administered (Sofer and Hagel, 1997). It is a loiown fact that it is very difficult to control medically the pyrogen-induced fever, and in severe cases the consequences can be fatal.
One o f the major drawbacks in using E. coli as the host cell system is the presence o f endotoxins on its surface. Endotoxin removal is therefore a key challenge in the purification o f recombinant DNA proteins and peptides produced in E. coli. Endotoxin molecules exhibit a highly negative charge, and their effective removal from a process stream can be achieved either by allowing them to bind to anion exchange chromatography or alternatively, the product can be bound by a cation exchange chromatography. The common process sequence to produce a plasmid DNA product can be represented by Figure 5.7.
This case study demonstrates a common additional problem where the product and impurity content are measured by different assay techniques and are expressed in different units, and also where the quality o f process information is limited by the small number o f fractions collected. In reality it is not uncommon for the product and impurity o f a chromatographic separation to be expressed in different units and where there may be no mass equivalence. For example, in this case study: the DNA level is expressed in m gm f' (mg o f DNA per ml o f product solution) whereas that o f
Chapter 5: Application o f FD Approach to Single Experimental and Industrial Chromatographic Step
the major contaminant, endotoxin, is expressed in EU (endotoxin unit). Conversion to consistent units is often not straightforward. For such a system the fractionation diagram has been modified to represent the results accordingly.
— tt< -B #. -B H»'
ferm en tation cen trifu gation filtration
v ia lin g ch rom atograp h y cen trifu gation precip itation
Figure 5.7: Schem atic flowsheet for the production of plasmid DNA product.
5.3.1.1 Materials and Methods
The chromatographic elution data in this case study was kindly provided by Biopharmaceutical Product Development group o f GlaxoSmithKline (GSK) (Beckenham, Kent, United Kingdom). Due to issues o f intellectual property and confidentiality o f the process data specific details o f the products and actual chromatographic runs can not be disclosed but pertinent information will be given where appropriate.
The separations o f study was carried out on an anion exchange chromatography (endotoxin clearance column) where negatively-charged endotoxin was bound to the solid phase and plasmid DNA was collected in eluted pool. The size o f the endotoxin clearance column was approximately 3.5L. Four sets o f chromatographic data were provided corresponding to different fermentation batches where each had been harvested and then purified by the same purification process. Amounts o f plasmid
C h ap ter 5: Application o fF D Approach to Singie Exp erim en tal a n d Ind u s tria l C hrom atographic Step
DNA and endotoxins o f study have been quantified by GSK using HPLC and LAL
(Limulus Amebocyte Lysate) assays respectively. In these particular
chromatographic runs the target was to achieve a minimum specification in terms o f the clearance o f endotoxin from the DNA product. The precise value for endotoxin contamination is related to the dose regime envisaged for the final product. As an illustration a level o f less than lEU/m g DNA was selected as being typical o f a medical medication requiring a repeated dose [for example Heparin, an anti coagulant injected repetitively during pregnancy to avoid blood clots from entering the placenta and causing the baby^deprived Qr oxygen.].
5.3.1.2 Experimental Data Treatment
As discussed in Section 5.2.1.2, it was necessary to curve fit experimental data to generate a smooth fractionation curve. For the endotoxin and the DNA data the small number o f data points available were fitted with a smoothed line obtained by applying a polynomial curve fitting function (to the degree o f one) in MATLAB (The M athworks Inc., Natick, MA, USA, 2000). The purpose o f this fitting was to form a continuous function for subsequent estimation o f the tie-line gradients which form the basis o f the contamination index versus yield plots (Section 3.3.3). After curve-fitting, the minimum operating tie-line gradient for a particular yield was derived using the polynomial relationships to describe the fractionation diagram.
5.3.2 Results and Discussions
Data for the concentration o f endotoxin and plasmid DNA in each o f the chromatograms generated from the ion exchange endotoxin clearance column are presented in Figure 5.8 for the four different fermentation batches. The mass of endotoxin (in terms o f endotoxin unit EU) and plasmid DNA (in terms o f mg) corresponding to each fraction collected were determined respectively. These data were processed to produce a modified fractionation diagram (Figure 5.9) that shows
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the relative change in the mass o f endotoxin and plasmid DNA. In Figure 5.9 are plotted the cumulative fractional mass o f plasmid DNA and the corresponding fractional amount o f endotoxin eluted respectively. For each set o f data, a fractionation line was obtained by employing linear-line-fitting to generate a continuous function. The fractionation lines do not pass through the origin, since only endotoxin was eluted in a significant amount in the initial portion o f the cliromatogram. So for Batch 2 about 63% o f the total endotoxin collected appears in the first fraction whilst for the other batches about 85% (±3%) elutes in the first fraction. For all but Batch 1 the fractionation plots are fairly flat between 0 and 0.6 cumulative DNA eluted. This indicates that following the first fraction there is a period where no significant amount o f endotoxin was eluted while the amount o f DNA collected increased from 0 to 60%. For Batch 1, a gradually increasing tangent shows the constant co-elution o f endotoxin and DNA. The large amount o f endotoxin found in the last fractions for all batches gives rise to the abrupt increase in slope o f the fractionation curves at about 0.9 on the cumulative DNA axis.
From a regulatory viewpoint it is crucial to be able to determine a suitable operational trade-off between achieving a desired minimum level o f contaminant removal whilst realising an acceptable product yield. This feature is explored in Figure 5.10 which shows the minimum contamination index against product yield. A typical specification for a DNA product might be related to the endotoxin level. W hilst the precise amount that may be tolerated is dose-specific a limit o f 1 EU m g'’ DNA was taken as the target for the study. Batch 1 gives an endotoxin level higher than the specification and much greater than the other three batches. Batch 2, 3 and 4 display constant impurity levels for product yields less than 60% with Batch 3 achieving nearly an order o f magnitude greater contaminant removal in this interval. For the principal region o f interest i.e. product yields which are greater than 90%, the changes o f contamination index are abrupt. O f the three batches which satisfy the <lEUm g"' criteria in this region, batches 3 and 4 out-perform Batch 2 by realising an in-specification product at 95% yield. Data for Batch 2 shows that this will be out of specification at a yield o f >92% and that further steps to reduce the endotoxin level will be needed if higher yields are sought. The figure therefore provides an analysis
Chapter 5: Application o f FD Approach to Single Experimental and Industrial Chromatographic Step
of the process trade-offs and an indication o f the impact on further processing o f the cut-point decisions that are made.
Finally the different sample collection retention volumes corresponding to the required product yield or contamination index for batch 3 product were tabulated in Table 5.2. The results show how the volume o f material to be collected increases with the desired yield but at the expense o f the degree o f contamination. For a known value o f fraction volume and flow rate of the chromatographic run, the sampling points can be re-defmed on the chromatogram. For example, if product yield o f 90% and minimum contamination index of lEum g are posed as the constraints o f the batch 3 product, then sample collected for the product yield o f 95% fulfils these requirement. The sample can be collected between elution volumes o f 138ml and 272ml in order to achieve a specification o f product that meet all the criteria posed.
2 5 0 2 0 0 1 50 1 o o 50 O 140 2 1 O V^oliutie (iiil) Batcli 1 CEU/iiil) Batch 3 CEU/iiiI) — Batch 2 CEU/iiil) Batch 4 CEU/ml) 1 2 lO 8 & 2 1 O (nil)
Figure 5.8; Fraction data for the amount of (a) endotoxin and (b) plasmid DNA and collected from an ion exchange chromatographic separation of endotoxin from DNA product. The batch numbers refer to separate ferm entation batches where the product has been processed by a common purification sequence. [Data from Biopharmaceutical Product Development, GlaxoSmithKline, Kent, UK].
Chapter 5: Application o f FD Approach to Single Experimental and Industrial Chromatographic Step ■o 0) 3 Œ '= 3 Q.