Alejandro Beldarraín Iznaga*
Blas Tejera González
Financial support: This research was supported by CIGB grants.
Keywords: DNA-clearance factor, process characterization, rHBsAg purification process, spiking experiments.
We studied the capacity of an API-rHBsAg purification process to eliminate DNA contamination from yeast-host cell. Firstly, was demonstrated consistency of manufacturing purification process to remove DNA, from (3.9 ± 1.9)108 pg/dose in starting material to (3.4 ± 1.6) pg/dose, equivalent to 8.2 log in Active Pharmaceutical Ingredient (API), measuring DNA quantity in several unit operations along manufacturing process for twenty batches, five consecutive in 2000, 2001, 2003 and 2005. These values for API, lower than 10 pg/dose, accomplish current WHO requirements for Hepatitis B vaccines obtaining by recombinant DNA technology (WHO, 1989; European Pharmacopoeia, 2001a). The main removal factor for manufacturing process, equivalent to 6.4-log, was reached in negative anion-exchange chromatography. Then, the capacity of immunoaffinity chromatography and positive anion-exchange chromatography to remove chromosomal DNA purified from yeast-host cell was assessed using a scaled-down chromatographic process which was shown to yield product meeting purity criteria set for the manufacturing process. Log10 reductions for DNA through the immunoaffinity chromatography and positive anion-exchange chromatography were 7.3 ± 0.1, and 5.8 ± 0.1 respectively. Overall, these studies indicate that total DNA clearance factor for API-rHBsAg manufacturing process was 19.4 log, 2.4 times higher than the real DNA contamination, indicating that API-rHBsAg manufacturing as described here have sufficient DNA reducing capacity to achieved a high margin of DNA safety.
Regulatory agencies states that each unit operation of a cGMP manufacturing process must be controlled to maximize the probability that the finished product meets all quality and design specifications. Biopharmaceutical manufacturers must ensure that their products are free from impurities like nucleic acids, viral particles, in-process intermediate, endotoxins, and host cell proteins. It is generally recognized that downstream processing operations used in purifying and manufacturing a biopharmaceutical product must be validated. The ability of the processing operations to remove specific substances must be demonstrated and in some cases, the ability to effectively and consistently remove a contaminant may be demonstrated by direct measurements of material sampled from actual process, if analytical methods have sufficient precision and sensitivity (ICH, 1999).
DNA considered as cellular contaminant rather than risk factor which required removal to extremely low level and in biopharmaceutical productions, the principal source is host cell DNA. Fermentations feed streams can contain large quantities of nucleic acids that can be removed during purification process by passing a feed stream through chromatography resins. In this regard, regulatory authorities state that level of DNA from the host cell can be detected by direct analysis on the product considering that levels of 10-100 pg of residual DNA of host cell origin per dose of a parenterally administered biopharmaceutical is considered to be an acceptably safe level by most of the medicinal agencies (FDA, 1985; Griffiths, 1997; Trouvin, 1997). Nevertheless, in general guidelines for recombinant proteins, there is no precise specification of what an acceptable or tolerable level of contaminating DNA might be, but recommendations are given in some specific monographies as for recombinant hepatitis B vaccine (WHO, 1989; European Pharmacopoeia, 2001a; European Pharmacopoeia, 2001b). Moreover, it has been recently recommended that batch to batch testing should be performed to determine DNA content in biological products produced in cell lines, while the elimination of bacterial or yeast DNA may be validated and does not have to be checked routinely (Trouvin, 1997). Regulatory authorities challenge manufacturers to reduce the amount of residual DNA in particular in ‘‘well-characterized’’ biopharmaceutical products combining determination of DNA amount in the final product, calculation of clearance factor for each unit operation and demonstration of DNA removal in purification process by clearance experiments with spiked DNA (FDA; European Medicines Evaluation Agency (EMEA) or the World Health Organization (WHO)).
The Center for Genetic Engineering and Biotechnology produces since 1990s an API for a yeast-derived vaccine providing protection against Hepatitis B infection and commercialized under trade mark HEBERBIOVAC HB (Pentón et al. 1992). The downstream process for rHBsAg purification, which includes several chromatographic operations, is potentially effective to remove DNA contamination and it has proven to be safe and efficacious (Pérez et al. 1994; Hardy et al. 2000). However, the effectiveness of manufacturing process to remove DNA contamination has not been studied yet.
This study describes the assessment of the DNA removal capacity of an API-rHBsAg manufacturing process combining direct DNA stepwise measurements along downstream purification stream with spiking experiments of two chromatographic separations. Finally, total clearance factor was calculated, demonstrating the high degree of security for an API-rHBsAg purification process to remove DNA burden.
Hybond-N filter, ATP 32 and X-ray the films were supplied by Amersham
All biological samples were taken after cellular disruption from yeast derived API-rHBsAg manufacturing process. Buffers were prepared according to manufacturing SOPs.
determination by staining with ethidium bromide in agarose gel.
DNA determinations by ethidium bromide (
determination by dot-blot hybridization. Hybridization, which
use probe too specific to measure total DNA, can detect DNA at the
picogram level (Eaton, 1989; Per et
al. 1993, DiPaolo et al. 1999). Specifically,
we use a radioactivity probe with αdATP32 to quantify
residual DNA from P. pastoris host, developed and validated
to API-rHBsAg manufacturing process in correspondence to current regulations
(Candelario and Calvo, 1999). A standard curve was
prepared with DNA from host cell, P. pastoris in 1/2 serial
dilutions from 250 pg to 1.9 pg. Usually 100 µg of problem samples,
reference material for API-rHBsAg and negative control, BSA fraction
V, were always assayed. DNA curve, samples and controls were heated
biochemical assays. The HBsAg concentration was measured by an
enzyme-linked immunosorbent assay (ELISA) system (González et al.
1993) or by UV absorbance at 280 nm using an adsorption coefficient
(1 mg/ml) of 5. Carbohydrates were determined by anthrone method according
to Carney (1986), lipids according to Woodman
and Price (1972). The concentration of mouse IgG was determined
by a sandwich ELISA using a commercial (Sigma,
DNA was purified according to procedure described elsewhere (Sambrook
et al. 1989). 50 ml of YPG culture (yeast extract 5 mg/ml, bacteriological
peptone 10 mg/ml and glucose 2%) was inoculated with a yeast colony
of P. pastoris and grown at
Concentration of purified yeast- DNA, diluted in purified water, was calculated according to equation:
[DNA] = Abs260nm x Factor x Dilution 
where: [DNA], DNA concentration in μg/ml; Factor = 50 µg/ml.
Pure and nodegradated fractions with 7 mg/ml approximately were obtained free of proteins, solvent and RNA contaminant, measuring absorbance radio at 260 nm to 280, 270, 230 and 320 nm respectively (Laws and Adams, 1996). Molecular integrity and absence of tRNA was verified by agarose gel electrophoresis at 0.8%. Only one band was observed (results not shown).
The laboratory scale systems for DNA-clearance studies were performed at 0.1% of manufacturing scale, using well-known principles of chromatography scale-down and varying columns diameters, process volumes, and volumetric flow rates (Sofer, 1996; Sofer and Hagel, 1997; Rathore et al. 2005). For immunoaffinity chromatography, experimental conditions were adjusted to maintain constant initial protein load per millilitre of gel and optimal diffusion time in correspondence to manufacturing scale; approximately 2 hrs (Pérez et al. 1994, Hernández et al. 2005). For Ion-exchange chromatography were maintained constant initial protein load, 1 mg per ml packed matrix, and lineal flow at 35.4 cm/h (Pérez et al. 1994). Five runs were performed on the scale down systems with and without DNA. Process outputs of chromatographic profile, product yield, contaminants and product purity were compared to manufacturing-scale systems values.
runs were performed according to manufacturing procedures (Pérez
et al. 1994). DNA removal by immunoaffinity chromatography was
determined by spiking approx. 5.5 mg rHBsAg with 6 mg purified DNA,
equivalent to 2.2 x 107 pg/dose. Samples were applied to
a 20 ml of immunoaffinity matrix (CIGB,
ion-exchange chromatography, 18 mg rHBsAg was spiked with 1 mg purified
DNA, equivalent to 1.1 x 106 pg/dose, and applied to a
25 ml-DE-52 matrix (Whatman, England) packed into a XK26/40 (Amersham
Bioscience, USA). Binding buffer was
For further comparison, DNA quantities were expressed in picogram per adult dose of 20 µg rHBsAg according to:
where DNA and rHBsAg represents it respective quantity.
The DNA log10 reduction factor (RF) was calculated individually for each operation according to:
where DNAinitial is the starting DNA quantity in pg/dose and DNAfinal is output DNA quantity after performing purification operation. The total DNA reduction factor was calculated as a sum of the individual log reduction factor.
Initially, we investigate DNA removal in manufacturing purification process of API-rHBsAg taking into account the potential ability of some operations to eliminate DNA contamination (Pérez et al. 1994). Figure 1 and Table 1, shown the results obtained during DNA-testing for twenty manufacturing batches, 5 consecutive released in 2000, 2001, 2003 and 2005.
After fermentation process and cellular disruption of yeast broth, the first downstream purification operation is an acid precipitation by adding HCl (Pentón et al. 1992). After acid precipitation (AP), DNA quantity is very high, (1.5 ± 0.81) x 109 µg, equivalent to (5.1 ± 2.7) x 108 pg/dose. These values can be reasonable in correspondence to a hundred kilograms of biomass processed during manufacturing fermentation process (Hardy et al. 2000). Significant DNA quantities should be removed after acid precipitation, however, the high viscosity in starting material from cellular disruption impossibilities precise DNA quantification. Nevertheless, values obtained from DNA determination in acid precipitation, equivalent to 8.7 log, could be considered as target DNA contamination to remove in downstream purification process (Figure 1 and Table 1).
The next step evaluated was a sequence of manufacturing operations called primary purification (PP), that include an ion-interaction with a diatomaceous earth matrix, tangential flow filtration to reduce volume by concentration and centrifugation (Penton et al. 1992). In these operations DNA quantity decrease to (3.1 ± 2.1) x 108 µg, equivalent to (1.7 ± 1.1) x 108 pg/dose, which are not significant comparing these values to previous operation (Figure 2 and Table 2). In principle, these results were expected due primary purification is designed to eliminate carbohydrate, host proteins and lipids contamination basically from process stream and increased HBsAg purity (Hardy et al. 2000). After primary purification we start to operate with chromatographic resins and detect DNA level by dot-blot hybridization expecting significant DNA removal to microgram levels.
Multiple assays have been developed to quantify low DNA impurities in biopharmaceuticals (Workman, 1995) such as DNA probe hybridization techniques involving radioactive (Eaton, 1989; Kuroda et al. 1988; Yoneyama et al. 1989), biotinylated or otherwise modified probes, enhanced chemiluminescence, ECL (DiPaolo et al. 1999), PCR (polymerase chain reaction) and the total DNA Threshold assay (Per et al. 1993). Because DNA levels and matrix conditions vary during purification, residual DNA analysis in biopharmaceuticals can be very difficult and the methods have to be carefully validated before use. For manufacturing proposes, DNA is usually determined by DNA dot-blot hybridization methods or by a commercially available Threshold system. The hybridization methods are sensitive and specific but time consuming and difficult to validate. The Threshold Total DNA Assay is very sensitive and much easier to use. In contrast to the hybridization methods, it is not sequence specific. The hybridization method developed at CIGB for determination of DNA traces in manufacturing process of API-HBsAg is very specific for ours proposes, with very low detection limit, validated according to current regulations (Candelario and Calvo, 1999) and approved for WHO authorities (http://www.who.int/immunization_standards/en/). A typical radiogram obtained using the dot blot methods is shown in Figure 2. It clearly showed the elimination of residual DNA during chromatographic purification process of API-HBsAg.
The first chromatographic purification operation is a negative ion-exchange chromatography by using an anion matrix, DE-52 (Whatman, UK) where DNA removal is very significant, decreasing from (3.1 ± 2.1) x 108 µg in PP to 88.4 ± 56.5 µg, equivalent to 70.1 ± 39.3 pg/dose. Resulting values equivalent to 1.9-log demonstrate the efficiency of DE-52 resin to eliminate DNA, reaching a removal factor quite high, 6.4 log (see NAE in Figure 1 and Table 1). Anionic exchangers are very effective in binding DNA, because the highly charged DNA can bind to an anion exchange at ionic strengths at which proteins don't usually bind (Ng and Mitra, 1994; Sofer and Hagel, 1997). For this reason, the anionic-exchanges resins are considered as a good first purification step where the high content of DNA can increase the viscosity and decrease the efficiency of chromatographic column (Janson and Rydén, 1998). For these resins, cleaning factors equivalent to 3-6 log have been reported (Ng and Mitra, 1994; Sofer and Hagel, 1997; Sadana, 1998, Lightfoot and Moscariello, 2004).
The next chromatographic operation is an immunoaffinity chromatography (IAF) where DNA contamination still decreases to 11.3 ± 8.8 µg, equivalent to 19.38 ± 10.52 pg/dose. These values equivalent to 1.3 log, represent 30.2% of incoming DNA. Immunoaffinity chromatography like anion-exchange chromatography has been reported as a very effective to remove DNA (Ng and Mitra, 1994). In principle, we expect more DNA removal percentage; even reach consistently specification value for API, 10 pg/dose, although variation of rHBsAg quantity in production batches, relative low quantity of DNA in purified material or detection limit for DNA assay, can be have significant influence in these results. After immunoaffinity chromatography, the material is desalted by gel filtration chromatography (DS) and practically no DNA removal effect was detected due basically to low resolution of chromatographic matrix used, Sephadex G-25M (Table 1 and Figure 1).
No significant removal was apparent in positive anion-exchange chromatography (PAE) where DNA quantity decreases to 3.84 ± 2.04 µg, representing only 34.1% of incoming DNA. However, eluted material reaches consistently specification for API, 5.5 ± 2.2 pg/dose (Table 1 and Figure 1).
The last three operations, concentration-diafiltration (C-D), gel filtration by High Performance Liquid Chromatography (GF) and final desalting gel filtration chromatography to conform the Active Pharmaceutical Ingredient (API), conserve DNA quantity lower than the current specification (Table 1 and Figure 1). DNA mass remain close to 2 µg with no significant difference. However, because concentration of rHBsAg in C-D is 3-10 times higher that the final two purification operations, GF and API, and DNA quantification is performed in the lower detection range of curve, we observe a lightly decreasing when DNA content is expressed as a radio per dose.
DNA quantity for final material, API, is 3.13 ± 1.46 pg/dose, lower than maximum allowable value according to WHO regulations (WHO, 1989; European Pharmacopoeia, 2001a). These results, beside fulfils a regulatory requirement, demonstrate the high effectiveness and consistency for API-purification process to eliminate DNA contamination from (5.01 ± 2.72)108 pg/dose to 3.13 ± 1.46 pg/dose in API.
Despite previous results, the question related to real potentiality of API-rHBsAg purification process to remove DNA contamination has not been fully addressed so far. Considering that 106-107 pg/dose of DNA in starting material as a worst case, that correspond to those quantities before chromatographic purification, we performed spiking experiments at laboratory scale scaling down manufacturing operation for immunoaffinity and positive anion-exchange chromatography.
Qualification of scaled-down model for immunoaffinity and positive anion-exchange chromatography. The general approach towards qualification of a scale-down model is to run all operational parameters (inputs) at the centre of the operating range used at large scale (manufacturing production). The performance parameters (outputs) should be similar across scales (Sofer, 1996; Rathore et al. 2005). Taken into account previous consideration, we evaluate the comparability of the model by examining product elution profiles, at small and manufacturing scale, by analysis of chromatographic profiles and quality of purified material.
A typical chromatogram for immunoaffinity chromatography for both scales is shown in Figure 3, left. The feed load solution containing buffer and biological materials is compatible for loading the column, which is pre-equilibrated in buffer of similar composition (Material and Methods). Chromatographic resin is loaded at low flow rate (8 cm/h) with approximately 9 columns volumes of load. In correspondence to an immunoaffinity chromatography process, a high signal is observed corresponding to unbound material. The load is immediately followed with a wash buffer with approximately 3 column volumes and then the HBsAg elutes as a single peak collected from the stat of UV rise for 1.5 column volumes. The column is then regenerated by changing pH from basic to acidic solution before to re equilibration for next load. As is shown in Figure 2, left in both scales chromatographic profile are very similar and as would be expected unit operation provides excellent removal of host-cell impurities, including DNA with high HBsAg purity with no significant differences (Table 2).
typical chromatographic separation for positive anion-exchange chromatography
in both scales is shown in Figure 3, right.
All biological material is bounded to chromatographic resin at moderate
flow rate (approx. 36 cm/h) with approximately 4 columns volumes.
The load is immediately followed with a wash buffer with approximately
3 column volumes and then the HBsAg elutes as a single peak collected
from the stat of UV rise for 1 column volume. The column is then cleaned
and regenerated using a concentrated solution of KSCN and
These results suggest that current manufacturing scale immunoaffinity and positive anion-exchange process for purification of API-HBsAg, can be successfully scaled down with eluted product purity and yield comparable to samples manufactured at production scale. Therefore, the scales down models described were carried out as the basic for DNA-spiking experiment.
DNA removal in small scale spiking experiments for immunoaffinity and positive anion-exchange chromatography. To determine the DNA removing capacity of the immunoaffinity and positive anion-exchange chromatography, spiking experiments were carried out at reduced scale under conditions equivalent to those used in the full-scale manufacturing process. In both cases, the resulting chromatogram and product recovery were consistent to those seen in the full-scale manufacturing process and small scale process without contaminant (Figure 2).
For immunoaffinity chromatography, the starting material with 2.2 ± 0.5 x 107 pg/dose of DNA (7.3 ± 0.1 log) was applied onto the column (Table 3). DNA is largely removed during load and washing steps, consistent with an affinity chromatographic process, removing more than 99% of DNA contamination, equivalent to 8.1 ± 0.2 log. Eluted material with a highly pure rHBsAg, contain 1.1 ± 0 pg/dose DNA, less than 0.1 log detected by dot-blot hybridization assay, indicating that clearance factor for immunoaffinity chromatography is 7.3 ± 0.1 log (Table 3). Similar results for different contamination have been reported previously, where affinity matrices demonstrate high purification capacity, eliminating large quantities of impurities or contaminant, including DNA (Ng and Mitra, 1994; Rhemrev-Boom et al. 2001; Weller, 2000; Subramaian, 2002; Kelly et al. 2004; Lightfoot and Moscariello, 2004). These results beside fulfill DNA specification for API-HBsAg in correspondence to current regulations (WHO, 1989; European Pharmacopoeia, 2001a).
positive anion-exchange chromatography, the starting material with
1.1 x 106 pg/dose, equivalent to 6 log DNA, was applied
onto the column and all biological material is bounded to chromatographic
resin (Table 3). During washing, increasing
NaCl concentration to
In summary, total DNA-reduction factor for API-rHBsAg purification process is 19.4 log, corresponding to 6.4 log for negative-anion exchange chromatography, 6.2 log for immunoaffinity chromatography and 5.7 for positive-anion exchange chromatography. This value is 2.4 times higher than the real DNA quantity to be removed form API-HBsAg process stream, 8.2 log (see above), demonstrating a high degree of security for manufacturing process. Although, our study has been limited to three chromatographic operations, there are, at least, two other that may remove DNA contamination, concentration-diafiltration by tangential flow filtration (C-D) and gel filtration by HPLC. However, these were not evaluated because satisfactory results were achieved.
The efficiency and consistency of a biopharmaceuticals purification process determines drug quality, including which specific types and concentrations of process contaminants may remain. From regulatory point of view, we achieved an essential requirement for API-rHBsAg to conform the commercial vaccine HEBERBIOVAC HB, demonstrating the robustness of manufacturing process to remove DNA in correspondence to current regulations (WHO, 1989; European Pharmacopoeia, 2001a). Results reported in this study are supported for more than 150 million dose released from 1992 up to date without any adverse event reported.
We thank colleagues from the Hepatitis B, In-Process Control and Quality Control Departments at CIGB for their technical assistance.
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