The properties research of polymerized human placenta hemoglobin before and after lyophilization

Abstract

In this study, lyophilization was applied to polymerized human placenta hemoglobin (PolyPHb) solution. The lyophilization process was carried out with freezing at − 70°C for 5 h and two phases of drying: the first phase of drying was carried out at −35°C–35°C for 16 h and the second phase at 35°C for 8 h. Hemoglobin (Hb) concentration, methemoglobin (MetHb) content, oxygen-carrying capacity, Fe³⁺ content, pH, UV spectrum, average molecular weight and its distribution, circular dichroism, SDS–PAGE, P₅₀, crystal osmotic pressure, colloid osmotic pressure, zeta potential, average particle size, and other indicators were measured before and after lyophilization. Residual water content and rehydration time of the lyophilized products were also evaluated. All the indicators of lyophilized samples showed that the physical and biochemical properties of PolyPHb are not markedly changed before and after lyophilization. In this light, lyophilization may be a promising method for the preservation of PolyPHb solution.

Keywords::

• lyophilization
• methemoglobin
• polymerized human placenta hemoglobin solutions
• properties

Introduction

Attentions paid to the hemoglobin-based oxygen carriers (HBOCs) have been on a vivid rise during the past few decades in both the academia and the industry, which could be attributed to their potential application in the treatment of hemorrhagic shock and other cardiovascular diseases (Kim and Greenburg 2004, Greenburg and Kim 2004, Lok 2001, Goodnough et al. 2003, Goodnough 2003). Polymerized human placenta hemoglobin (PolyPHb) solution, first reported by ChengminYang group (Li et al. 2009), is a particularly promising HBOC, which allows transporting a greater amount of oxygen to hypoxic tissue owing to its higher oxygen affinity, lower viscosity, and smaller mean diameter comparing to human red blood cells (Li et al. 2006, Standl et al. 2003, McNeil et al. 2001). It is, therefore, likely to be helpful in providing sufficient microcirculation perfusion, alleviating hemorrhagic shock, and treating myocardial infarction, etc (Deem et al. 2002, Levy et al. 2002, Moore et al. 2005). However, PolyPHb is unstable in liquid formulations. Despite the progress in the preservation of PolyPHb solution, it remains a challenge to maintain protein activity and low toxicity. So as to overcome the barrier of its instability, PolyPHb could probably be made into solid forms.

Lyophilization is often a desired procedure for protein products insufficiently stable in liquid formulations (Heller et al. 1996). It has certain apparent advantages over the traditional cryopreservation methods: the probable maintenance of biological activity of proteins in cryogenic vacuum conditions; the formation of “skeleton” structure which remains in its shape and volume; the better rehydration; the complete dehydration of products permitting longer storage; the elimination of the need for low temperature storage (low cost); and the reduced weight (easy for storage and shipment) (Yu et al. 2010).

So far, studies on the lyophilization of PolyPHb have rarely been reported. Therefore, on the basis of this and our previous experiments, we have designed this study to examine the influence of lyophilization on the properties of PolyPHb and to evaluate the feasibility of lyophilization to be used in the preservation of PolyPHb products.

Materials and methods

Chemicals and reagents

All chemicals used in this study were of analytical reagent grade.

Main instruments

Labconco freezone¹² lyophilizer (US labconco); Thermo scientificforma ultra-low temperature refrigerator (Thermo); DU800 visible/UV spectrophotometer (Beckman); 2695 analytical high-performance liquid chromatograph (Waters); J-815 circular dichroism points apparatus (JASCO); HEMOX^TM-ANALYZER (TCS Scientific corp); and NanoC zeta potential (Beckman) were the main instruments used in this study.

PolyPHb preparation

PolyPHb was prepared as we previously described (Yang et al. 2002, 2001). Briefly, Hb from fresh human placenta blood (donated by Sichuan New Life Stem Cell Biotech Inc., Sichuan, China) was purified and virally inactivated by heat treatment, and then, cross-linking was performed using glutaraldehyde (GDA).

Lyophilization process

PolyPHb solution was filled into 10-ml serum vials (two-milliliter fill volume). Sucrose was added as a protective agent to the PolyPHb solution (Wsucrose/WHb = 1:1) as we previously described (Wang et al. 2008). Then, protein was diluted to a concentration of 5.0 g/dl with ultrapure water. PolyPHb solution was lyophilized following deoxygenation. The lyophilization process was carried out with the freezing at − 70°C for 5 h and two phases of drying: the first phase of drying was carried out at − 35°C–35°C for 16 h and the second phase at 35°C for 8 h. After the end of the lyophilization procedure, the vials with aluminum foil composite membrane packaging.

Determination of Hb and methemoglobin (MetHb) concentration

The concentration of Hb was determined using an automatic blood count machine (CA620, Medonic) and that of the MetHb using standard spectral analysis methods described in the reference (Li et al. 2006).

Determination of oxygen-carrying capacity and P₅₀

Oxygen-carrying capacity assay and P₅₀ determination were performed according to the reference (Zhu and Chen 1978). UV spectral assay was conducted at a wavelength of 200–700 nm with Hb concentration at 0.01 g/100 ml (Li et al. 2006). Circular dichroism scan was conducted at a wavelength of 190–250 nm with Hb concentration at 0.3 g/100 ml.

Determination of freeze-dried solid

The solution color of the lyophilized product was observed with naked eyes. Rehydration time was counted using stopwatch. According to Loss on drying assay of Chinese Pharmacopoeia (2010) Appendix VIII L, the residual water content was tested. According to Appendix IX B, the clarity was tested.

Determination of average molecular weight and molecular weight distribution

The average molecular weight and molecular weight distribution was detected using high-performance liquid chromatography (HPLC) using Superdex-200 Column (10 mm × 300 mm) with 0.1 M PBS (pH, 7.2–7.3) at a flow rate of 0.5 ml/min. The HPLC assay was conducted at 25°C and monitored at a wavelength of 280 nm. The average particle size was determined using zeta potential analyzer (NanoC, Beckman) with Hb concentration at 5 g/100 ml.

Determination of other indicators

SDS–PAGE was detected using 10% gel. Crystal osmotic pressure was determined using Crystal Osmometer (5520, Wescor) and colloid osmotic pressure using Colloid osmotic pressure analyzer (4420, Wescor). Free ferric ion (Fe³⁺) content determination methods were described in the reference (Jan et al. 2009).

Statistical analysis

All values in this study were presented as mean ± SD and analyzed using one-way ANOVA followed by LSD correction for post hoc t-test (SPSS 13.0 software). P values less than 0.05 were considered statistically significant.

Results

The quality of product before and after lyophilization

As shown in Table I, the MetHb concentration of products was within 3.12% after lyophilization. The Hb concentration, Fe³⁺ concentration, pH, crystal osmotic pressure, colloid osmotic pressure, Zeta potential, and average particle size had no significant changes before and after lyophilization (P > 0.05). The oxygen-carrying capacity and P₅₀ had no significant changes either (P > 0.05), which indicated that the oxygen-carrying capacity of PolyPHb was preserved. Other indicators such as reconstituted time, residual water content, solution color, and clarity of lyophilized products all met the general standards. In short, the physical and chemical indicators of the sample before and after lyophilization had no significant changes.

Table I. The quality of product before and after lyophilization (n = 5).

S. No.	Indicators	Before	After
1	Hb Concentration (g/dl)	5.0 ± 0.1	5.04 ± 0.09
2	MetHb (%)	2.87 ± 0.01	3.02 ± 0.10
3	Oxygen-carrying capacity (ml/g)	1.1 ± 0.05	1.06 ± 0.01
4	Fe^3+ (ug/ml)	0.96 ± 0.016	0.84 ± 0.54
5	pH	7.40 ± 0.01	7.61 ± 0.09
6	Crystal osmotic pressure (Osmol/kg)	312.7 ± 0.58	310.0 ± 6.0
7	Colloid osmotic pressure (mmHg)	5.95 ± 0.21	5.92 ± 0.18
8	Zeta potential (mv)	− 16.44 ± 0.51	− 14.48 ± 1.11
9	P50 (mmHg)	11.16 ± 0.12	11.82 ± 1.14
10	Average particle size (nm)	30.53 ± 0.67	31.65 ± 0.45
11	Rehydration time (min)	/	1.5 ± 0.07
12	Residual water content (%)	/	1.19 ± 0.34
13	Solution color	Purple red	Purple red
14	Clarity	Clarify	Clarify

UV spectrum and circular dichroism

The changes in the biological activities of a protein are highly related to the changes in its structural conformation. Spectral analysis showed that PolyPHb after lyophilization had almost identical absorption spectra when compared with PolyPHb before lyophilization on a maximum absorption of 415 nm (Figure 1). Similarly, the results of circular dichroism scan showed that there were no significant changes at characteristic absorption wavelengths (208, 222, and 192 nm) before and after lyophilization, indicating the absence of significant secondary structural changes (Figure 2). It could therefore be concluded that the biological activities of PolyPHb could be reserved during the lyophilization process.

Figure 1. The UV spectrum before and after lyophilization (n = 5). A: UV spectrum before lyophilization; B: UV spectrum after lyophilization.

Figure 2. Circular dichroism before and after lyophilization (n = 5). A: Circular dichroism before lyophilization; B: Circular dichroism after lyophilization.

HPLC and SDS–PAGE

Average molecular weight and molecular weight distribution of PolyPHb were detected using HPLC (Figure 3). The distribution of the molecular weight was detected using a standard method with standard marker (molecular weight: 669, 443, 200, 150, 66, 29 kD).

Figure 3. HPLC before and after lyophilization (n = 5). A: HPLC before lyophilization;B: HPLC after lyophilization.

The average molecular weight = Σ Molecular weight * the percentage of this molecular weight in the HPLC picture.

At HPLC spectra, the first peak (16.1 min) is super-weight molecular (Mw > 600 kD). The second and third peaks (24.6 and 27.3 min) are cross-linked Hb (600 kD > Mw > 64 kD). The fourth peak (32.1 min) is the mixture of Hb tetramer and polyHb cross-linked with two Hb. The average molecular weight of products before and after lyophilization was both 210.7 kD. It can be seen from Figure 3 that HPLC peak time and peak height of the sample did not change significantly before and after lyophilization, which indicated that the average molecular weight and molecular weight distribution of the products have not been changed. Also, it can be seen from Figure 4 that Electropherogram of the samples has no significant changes before and after lyophilization.

Figure 4. SDS–PAGE before and after lyophilization (n = 5). 1: SDS–PAGE before lyophilization; 2: SDS–PAGE after lyophilization.

Discussion

Activity of PolyPHb

The most important biological function of PolyPHb is transporting oxygen, the function of which could be attributed to its Hb structure. Hb is an iron-containing oxygen-transporting metalloprotein with four subunits. The four subunits will be separated when Hb is separated from red blood cells. Each subunit consists of a globular protein with an embedded heme group. Each heme group contains one iron ion, which can bind one oxygen molecule through ion-induced dipole forces. The iron ion may be either in the Fe²⁺ or in the Fe³⁺ state. However, it is notable that MetHb (Fe³⁺) cannot bind oxygen. Therefore, when the heme iron is oxidized to the ferric form, oxygen can no longer bind reversibly and the PolyPHb is nonfunctional as an oxygen carrier.

The oxidation of ferrous Hb to a nonfunctional ferric Hb is hence a serious concern, to which contacts of oxygen and increase in temperature are the key factors. In our research, MetHb had no significant increase after lyophilization. This result may be attributed to four reasons: (1) The self-processing of lyophilization. An appropriate lyophilization program can reduce the damage of products. (2) Lyoprotectants–surcose. The interaction of the protein with the ice crystals could cause exposure of hydrophobic residues normally buried within the protein core. With the loss of moisture, protein hydrogen bonds were exposed. In the freezing process, sucrose can reduce the protein damage in contact with ice, stabilizing the protein structure; in the drying process, sucrose instead of sublimating water molecules forms hydrogen bonds with Hb to stabilize the protein structure. (3) The prevention of air contact throughout the lyophilization procedure, and (4) the temperature Control.

Stability of PolyPHb

The value of zeta potential indicates the stability of the colloidal dispersions. The zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in dispersion. A high zeta potential will confer stability, that is, the solution or dispersion will resist aggregation, whereas low zeta potential leads to increase in attraction, which leads to the break of flocculation of dispersion. So, the higher the zeta potential, the more stable the dispersion. In our experiments, the zeta potential (− 16.44 ± 0.51) before lyophilization has no significant changes compared with zeta potential (− 14.48 ± 1.11) after lyophilization (P > 0.05), which indicated that the stability of products was reserved.

Molecular weight of PolyPHb

Researchers in HBOC areas have been constantly focusing on the molecular weight of HBOCs. As shown in this research, low molecular weight may cause renal toxicity and a short intravascular residence time in vivo, and high molecular weight may have an effect on blood viscosity and immunogenicity of the body. Therefore, a suitable molecular weight is of great importance.

Ice crystal could be produced in freezing process. And freezing protein solutions at a faster cooling rate increases the ice crystal surface area, which leads to the higher degree of protein aggregation. In our study, average molecular weight and molecular weight distribution of PolyPHb has no significant changes. Therefore, it could be concluded that lyophilization has no adverse effect on PolyPHb molecular weight.

Conclusion

In summary, data from this report demonstrated that the physical and biochemical properties of PolyPHb are not markedly changed before and after lyophilization. It could be concluded that lyophilization has great potential in the preservation of PolyPHb products.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

Supported by National High-tech R&D Program of China (863 Program) (Grant No. 2012AA021903).