Hydroxylapatite (HA) Powder for Autovaccination Against Canine Non Hodgkin’s Lymphoma - Advances in Biomaterials Science and Biomedical Applications - page 504

Powder characteristics

Nature of the charged groups PO 4 3- , OH - , Ca 2+

Electrocinetical potential (mV) -35

Hydrophobicity +

Surface pH 7,8

Granulometry (µm) 0-25

Surface area (m 2 /gr) 4

Shape spherical

Table 1. Characteristics of the powder

The sintering considerably reduced the surface area making the amount of proteins adsorbed
on the powder lower. It also stabilized the powder structure. The powder was submitted to
dissolution/ precipitation processes when soaked inside a saline solution [13]. The modifica‐
tion occurring at the powder surface affected the adsorption properties of the powder. The
reduced surface area decreased the interactions with the saline solution containing the
proteins. There was a microporosity between the ceramic grains inside the same particle
allowing the protein solution to diffuse inside the particles. The characteristics of the powder
used in this experiment was given in table 1

As the chromatography was carried out under atmosphere, the granulometry of the powder
was an important factor in order to avoid any plugging of the column. The HSPs could be
eluted from the powder by 200-300 mM NaCl solutions. The powder solution in NaCl was not
stable enough to be injected as it decanted too fast in the syringe. Thus to improve the
injectability the powder was put in suspension in a 2% solution of carboxymethylcellulose in
20 mM NaCl.

2.4. Vaccine manufacturing protocol

The tumor tissue and all the materials used to prepare the vaccine were handled in sterile
conditions under a laminar flow. The frozen tumor (200 mgr) tissue was homogenized using
a bead tissue homogenizer. 1 ml of NaHCO 3 (30 mM, pH 7) was added for 1 ml of homogenate.
The resulting homogenate was then centrifuged at 1000 g for 15 mn at 4°C to remove all cell
fragments.

The supernatant containing the cytoplasmic proteins was used for protein purification by HA
column chromatography as follows: a) two precipitations with ammonium sulphate (first at
50% and then at 70%) recovering the pellets. The last pellet was resuspended in 1 ml phosphate
buffer (20 mM, pH 7). The column was filled (chromatography columns, Poly-prep, Cat.
731-1550, Bio Rad) with 0.2 gr of HA (0-25 μm), equilibrated with 10 volumes of phosphate
buffer (20 mM pH7). The resuspended pellet was then added. The column was then washed
with 3 ml of a 100 mM NaCl solution (fig.2).