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Vaccine Manufacture: Bringing Down Costs

Rodolfo Valdés and the team at the Center for Genetic Engineering and Biotechnology, Havana, Cuba, assess the stability of immunoaffinity chromatography of hepatitis B surface antigen using a mouse monoclonal antibody as a model ligand

Pichia pastoris has often been employed as a host for large-scale production of hepatitis B surface antigen (rec-HBsAg). Characterisation of rec-HBsAg indicates that the Pichia pastoris-synthesised HBsAg exists in a form similar to human serum-derived HBsAg 22nm particles. Chromatography is a group of techniques used to isolate substances (such as rec- HBsAg) from complex biological sources. One of the most commonly employed chromatographic procedures for rec- HBsAg purification is immunoaffinity chromatography (IAC) using anti-HBsAg monoclonal antibodies (mAb) as the ligand (1). However, due to the high cost of mAb production, an effective IAC method requires, among other factors, high stability of both the ligand and the immunosorbent under preservation conditions. Several papers have been published on CB.Hep-1-immunosorbents, but none of them have focused on ligand and immunosorbent stability. This article assesses CB.Hep-1 mAb and immunosorbent stability, used to purify the Pichia pastoris-HBsAg for vaccine purpose for 24 and 21 consecutive months respectively.

mAb & ANTIGEN SOURCES

mAb secreted by hybridoma CB.Hep-1 and purified from ascites with a purity greater than 95 per cent was used in these experiments (2). rec-HBsAg was produced by fermentations of a Pichia pastoris strain in a saline medium supplemented with glycerol. Its expression was induced with methanol and samples from the initial purification steps, and contained rec- HBsAg were used for IAC assessments (1).

mAb PURIFICATION METHOD

Ascitic fluid underwent two ammonium sulfate precipitations and was centrifuged at 4,800xg for 20 minutes at 4°C. Pellets were desalted by sizeexclusion liquid chromatography (LC) on Sephadex G-25 at 130cm h-1. Phosphate buffered saline (PBS) solution, 150mM, pH 8, was used as mobile phase. This desalted material was then purified by protein A-Sepharose affinity chromatography using 150mM PBS, pH 8; and 100mM citric acid, pH 3, as adsorption and elution buffers, respectively. Affinity columns were operated at 100cm h-1. Subsequently, incubation at acid pH was performed for one hour and then neutralised with 2M Tris-HCl. Next, the sample buffer was exchanged to 20mM Tris/150mM NaCl, pH 7.6, by size-exclusion-LC using Sephadex G-25 in a chromatographic column, operated at 130cm h-1, ultrafiltrated and filtered under sterile conditions (see Figure 1).

PROTEIN QUANTIFICATION

mAb concentration was determined according to Lowry’s method (3). The concentration of purified rec- HBsAg was determined by ultraviolet measurement using a molar extinction coefficient of five [A280 (1cm, 1mg mL-1) = 5]. mAb

CONCENTRATION ESTIMATION BY ENZYMELINKED IMMUNOSORBENT ASSAY (ELISA)

A polystyrene microplate was coated with 10μg well-1 of rec- HBsAg in 100mM NaHCO3 for 20 minutes at 50°C. After that, samples were added to the plate in 0.05 per cent Tween 20/150mM PBS and incubated for one hour at 37°C. Following several washings with 0.05 per cent Tween 20/150mM PBS, the plate was incubated for one hour at 37°C with an anti-mouse IgG-horseradish peroxidase conjugate. The reaction was revealed using 0.05 per cent OPD and 0.015 per cent H2O2 in citrate buffer, pH 5, and stopped with 1.25M H2SO4. Absorbance was measured in a Multiskan ELISA reader using a 492nm filter.

ESTIMATION OF RELEASED ANTIBODY CONCENTRATION BY ELISA

A polystyrene microplate was coated with 10μg well-1 of a sheep anti-mouse polyclonal immunoglobulin overnight at 4°C. The plate was blocked for 30 minutes at 37°C. Immunosorbent samples were added and incubated for three hours at 37°C with one per cent non-fat milk/150mM PBS. After washing the plate, it was incubated with 100μL well-1 of a goat anti-mouse polyclonal immunoglobulin-horseradish peroxidase conjugate. The reaction was revealed using 0.05 per cent OPD and 0.015 per cent H2O2 in citrate buffer, pH 5, and stopped with 1.25M H2SO4. Absorbance was measured in a Multiskan ELISA reader using a 492nm filter.

ANALYSIS OF PROTEIN PURITY BY SDS-PAGE & SIZE EXCLUSION-LC

mAb and antigen purities were analysed by SDS-PAGE (12.5 per cent) with Coomassie blue staining, followed by densitometric scanning using a laser densitometer (4). A size-exclusion-LC column TSK G3000 PW (600mm/7.5mm (inner diameter) was also used to determine mAb purity (see Figure 1). Chromatographic mobile phase was 150mM PBS, pH 7, and 100μL of samples were directly applied onto the column. The linear flow rate employed was 27.2 cm h-1. Absorbance was measured at 226nm.

MATRIX ACTIVATION, IMMUNOSORBENT PREPARATION & IAC

Sepharose CL-4B was activated with CNBr, as described (5). The matrix was washed with three-fold matrix amount of purified water and dried. It was resuspended in one-fold of matrix amount with purified water and mixed with CNBr in acetonitrile, controlling the pH and temperature. Next, it was washed with three-fold matrix amount of purified water, two-fold 100mM acetic acid and five-fold of purified water. The activated matrix was dried using 40 per cent (v/v) acetone, wetted in 1mM HCl and washed with 100mM Na2CO3/100mM NaHCO3/500mM NaCl pH 8.3 for mAb immobilisation. mAbs were coupled on CNBr-matrix at pH 8.3, 25°C for two hours and free active groups were blocked by adding 100mM glycine pH 8.0. Subsequently, five alternative washings with 100mM CH3CO2Na/500mM NaCl, pH 4, and 100mM Na2CO3/100mM NaHCO3/500mM NaCl, pH 8.3 were performed. Finally, the immunosorbents were stored in 150mM PBS, pH 7.2, at 4°C (see Figure 1). IAC was performed in PD-10 columns packed with 6mL of immunosorbents using 20mM Tris/3mM EDTA/1M NaCl, pH 7. As elution buffer 20mM Tris/3mM EDTA/1M NaCl/3 M KSCN, pH 7 was employed. Linear flow rate employed was 10.5cm h-1 and semipurified rec-HBsAg was used as column loading material.

mAb STABILITY STUDY

Three mAb-independent purified samples formulated in 20mM Tris-HCl/150 mM NaCl, pH 7.6 ± 0.2 were dispensed in 1mL glass vials and placed at 4°C and -20°C, respectively. The total amount of vials distributed for each temperature allowed performing assays to duplicate in each sampling time. Time periods evaluated were 0, 1, 5, 7, 9, 11, 13, 18, 21 and 24 months (see Stability study results).

Stability study results

mAb stability study
mAb concentration (a); protein concentration (b); and purity values (c) of initial samples were:

  • I(a) = 6.86mg mL-1, I(b) = 6.76mg mL-1, I(c) = 95.2 per cent
  • II(a) = 6.60mg mL-1, II(b) = 7.18mg mL-1, II(c) = 97.0 per cent
  • III(a) = 7.87mg mL-1, III(b) = 5.98mg mL-1, III(c) = 95.0 per cent

We have also noted in preliminary experiments that this mAb formed aggregated at concentrations ≥ 9 mg mL-1 in this buffer. Pre-established limits were: (a) ≥ 70 per cent, (b) ≥ 90 per cent, (c) ≥ 95 per cent, size-exclusion-LC retention time = 27 ± 3 min pH 7.6 ± 0.2, all samples were sterile, without presence of aggregates and not opalescent. mAb specific content (SC) was determined as follows:

mAbSC = (Elisa/Lowry) x 100 per cent 

Immunosorbent stability study
Immunosorbent elution capacity (a); rec-HBsAg purity (b); and released IgG values (c) of initial samples were:

  • I(a) = 83μg mg-1 mAb, I(b) = 90 per cent, I(c) = 0.28ng IgG μg-1 rec-HBsAg;
  • II(a) = 89μg mg-1 mAb, II(b) = 100 per cent, II(c) = 0.50ng IgG μg-1 rec-HBsAg);
  • III(a) = 102μg mg-1 mAb, III(b) = 91 per cent, III(c) = 0.96ng IgG μg-1 rec-HBsAg.

Pre-established limits were: (a) 80-100μg rec-HBsAg mg-1 mAb, (b) ≥ 90 per cent and (c) 3ng IgG μg-1 rec-HBsAg. 

 

IMMUNOSORBENT STABILITY STUDY

Three immunosorbent independent samples formulated in 150mM PBS pH 7.2 ± 0.2 /0.01 per cent Tiomersal were dispensed in 10mL glass vials and placed at 4°C. The total amount of vials allowed the performance of respective assays in each sampling time. Experimental times evaluated were 0, 3, 6, 9, 12, 15, 18 and 21 months (see Stability study results).

RESULTS & DISCUSSION

A major challenge of bioseparation is identifying scalable and economic procedures for protein recovery, which is complicated greatly by the uncharacterised nature of the contaminants. In general, chromatographic methods can not accomplish satisfactory selectivity in a single step. However, affinity chromatography might overcome this by exploiting the specific interaction of one molecule with its respective ligand. Since mAb discovery, several proteins have been isolated by using this procedure (6). Nevertheless, this highly biospecific procedure is very expensive and only applicable in particular cases. In this sense, ligand and immunosorbent lifetime is one of the most important contributing criteria to operation cost of the target protein being purified. A short ligand lifetime due to lability of ligand and a short immunosorbent lifetime under preservation conditions would make this procedure unsuitable for isolating pharmaceutical proteins with low aggregated values.

In this study, the stability of CB.Hep-1 mAb and CB.Hep-1- immunosorbent was assessed during several consecutive months. During freezing in saline buffer solutions, a change in pH might have an effect on protein stability because protein denaturalisation is governed by conformational changes (7). To avoid physico-chemical factors that could affect mAb stability during preservation time, a comparative study for measuring the stability of the highly pure mAb CB.Hep-1 at 4°C and -20°C, respectively was performed. Several parameters (mAb concentration, mouse DNA content, isoelectric pH, sterility and purity measured by SDS-PAGE and sizeexclusion- LC) were studied in these assessments. From this we can consider mAb specific content to be a convincing manner in which to express mAb stability during preservation time. Figure 2A illustrates average mAb specific content and confidence intervals of three independent experiments. Since the P-value of F-test was P = 0.8639, α = 0.05, there was not a statistically significant difference among the results of six independent experiments (three at each temperature). It demonstrated that this mAb is quite resistant to the pH changes produced during the freezing process, which aligns with reports demonstrating that mAbs are resistant to acid conditions, but is in contradiction with other reports which state that inactivation by pH changes had been observed (7). mAb inactivation has also been observed after a long period of incubation at low pH. Stability of this molecule was also corroborated measuring mAb purity by SDS-PAGE and size-exclusion-LC. To summarise, all studied samples (at 4°C and -20°C) evidenced a high level of purity measured by both assays (>95 per cent) (see Figure 1B and 1C), allowing the application of this mAb for rec-HBsAg purification for 24 months under these preservation conditions.

Covalent antibody immobilisation on sorbents by CNBr method results in binding of antibodies in a random way (8). Thus, only a small part of immobilised antibodies retains the ability to interact with antigens. Perhaps this will be one of the most critical factors contributing to immunosorbent efficiency and also to the cost. Nevertheless, another important factor contributing to the cost and quality is the immunosorbent stability in terms of purification cycles and preservation time. The number of purification cycles of CB.Hep-1- immunosorbents has been addressed previously, but stability of this immunosorbent under preservation conditions has not yet been reported. In this study, three parameters were evaluated to assess immunosorbent stability: antigen elution capacity; antibody leakage; and antigen purity. Results demonstrated that elution capacity was statistically similar over 21 months. Values ranged throughout the study limit of 80 to 100μg rec-HBsAg mg-1 mAb (see Figure 2B). Values corresponding with months 15 and 21 were slightly lower, but without significant differences to the values of the initial samples (P = 0.7587, α = 0.05 (ANOVA).

A crucial disadvantage of all immobilisation procedures is the leakage of immobilised antibodies contaminating the target protein. This situation is aggravated if antibody matrix interaction is produced by an active group different to cyanate steres and if harsh elution conditions are used. Figure 2C illustrates that IgG values never exceed 3ng IgG μg-1 rec-HBsAg, demonstrating the high stability of this parameter during the preservation time and the extremely low level of the coeluted ligand.

The most important problem that can be solved by IAC perhaps is the removal of contaminants to get a high purity level of the protein being purified. As a rule, high purity of the target protein cannot be achieved in a single step by using any other bioseparation procedure. This fact takes a further significance if it is important to separate nonnative and native forms of the target protein. The SDS-PAGE of the rec- HBsAg eluted from one of the immunosorbent assessed in this study is illustrated in Figure 3. As can be observed, antigen aggregation status identified in SDS-PAGE is the typical HBsAg profile under moderate reducing conditions and thus all samples showed more than 90 per cent purity of eluted antigen during 21 months of preservation.

CONCLUSION

CB.Hep-1 mAb maintained its properties in 20mM Tris-HCl/150mM NaCl, pH 7.6 ± 0.2 at both temperatures during 24 consecutive months. CB.Hep-1- immunosorbent is also stable for 21 consecutive months in 150mM PBS, pH 4. Thus, both the ligand and immunosorbent preserved under these conditions can be employed in vaccine manufacturing without affecting antigen quality. The IAC matrix is as stable as other chromatographic matrices, which drastically reduces vaccine manufacture operation costs.

Acknowledgement

The author would like to credit his colleagues Andrés Tamayo, José Montero, Sigifredo Padilla, Lamay Dorta, Tatiana Alvarez, Cristina García, Maribel Vega, Gerardo García, Cristina Rodríguez, Adisley Dorta, Jorge López, Susset Valderrama, Lena Hernández, Yurisleidy Aldama and Makis Torres for their equal contribution to this article.

References

  1. Hardy E, Martínez E, Diago D, Díaz R, González D and Herrera L, Large scale production of recombinant hepatitis B surface antigen from Pichia pastoris, Journal of Biotechnology 77: pp157-167, 2000
  2. Fontirrochi G, Dueñas M, Fernández de Cossio ME, Fuentes P, Pérez M, Mainet D et al, A mouse hybridoma cell line secreting IgG and IgM antibodies with specificity for the hepatitis B virus surface antigen, Biotecnología Aplicada 10: pp24-30, 1993
  3. Lowry DH, Rosembrough NJ, Farr AL and Randal RJ, Protein measurement with Folin phenol reagent, Journal of Biological Chemistry 193: pp265-269, 1951
  4. Laemmli U, Cleavage of structural proteins during the assembly of head bacteriophage T4, Nature 227: pp680-685, 1970
  5. Fernández E, Montero J, Valdés R, Arce M, Tamayo A, Geada D et al, Characterization and scale-up of Cyanogen bromide chemical activation of sepharose CL-4B in a stirred tank reactor to purify the rHBsAg, International Journal of Chemical Reactor Engineering 6: pA33, 2008
  6. Firer MA, Efficient elution of functional proteins in affinity chromatography, J Biochem Biophys Methods 49: pp433-442, 2001
  7. Muronetz I and Korpela T, Isolation of antigens and antibodies by affinity chromatography, Journal of Chromatography B 790: pp53-66, 2003
  8. Porath J, Axén R and Ernback S, Chemical Coupling of Proteins to Agarose, Nature 215: pp1,491- 1,492, 1967

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Rodolfo Valdés has a degree and a PhD in Biology from Havana University. He has worked on the production of monoclonal antibodies to be used as reagents in vaccine manufacturing for 20 years at the Center for Genetic Engineering and Biotechnology in Havana. He is currently the Head of Monoclonal Antibody Production Department, and his team, composed of several researchers and technicians, has published a range of research and technical papers regarding monoclonal antibody production, plant-derived antibody production, antibody immobilisation on chromatographic matrices, quality assurance of monoclonal antibody production and hepatitis B surface antigen purification and expression characterisation.
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