Temperature stability of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] in the form of a solution or in the lyophilized form during storage at −80 °C, 4 °C, 25 °C and 37 °C or pasteurization at 70 °C

Figures & data

Table I. The change in enzyme activities after freeze drying.

Enzyme type	SOD	CAT	CA
Percentage of original after freeze-dry	100 ± 2	93 ± 3.5	100 ± 2

Table II. Stability at different temperatures.

STABLILITY AT DIFFERENT TEMPERATURES
Donor Red Blood Cells
Maximal time of storage allowed
	20–25C	4C
	1 day?	42 days
Poly-[Hb-SOD-CAT-CA] solution
T1/2 of enzyme activities:
	20–25C	4C
CAT	172 days	380 days
SOD	92 days	198 days
CA	51 days	231 days
Poly-[Hb-SOD-CAT-CA] freeze dried:
Day 320 % enzyme activity
	20–25C	4C
CAT	73% (day 320)	85% (day 320)
SOD	67% (day 320)	76% (day 320)
CA	73% (day 320)	85% (day 320)

Figure 1. Storage stability of carbonic anhydrase (CA) at −80 °C, 4 °C, 25 °C and 37 °C for: (1) un-cross-linked free solution of Hb, SOD, CAT and CA; (2) polymerized solution of Poly-[SFHb-SOD-CAT-CA] and (3) freeze-dried polymerized Poly-[SFHb-SOD-CAT-CA].

Figure 2. Storage stability of superoxide dismutase (SOD) at −80 °C, 4 °C, 25 °C and 37 °C for: (1) uncross-linked free solution of Hb, SOD, CAT and CA; (2) polymerized solution of Poly-[SFHb-SOD-CAT-CA] and (3) freeze-dried polymerized Poly-[SFHb-SOD-CAT-CA].

Figure 3. Storage stability of catalase (CAT) at −80 °C, 4 °C, 25 °C and 37 °C for: (1) uncross-linked free solution of Hb, SOD, CAT and CA; (2) polymerized solution of Poly-[SFHb-SOD-CAT-CA] and (3) freeze-dried polymerized Poly-[SFHb-SOD-CAT-CA].

Table III. Molecular distribution of freeze-dried samples stored for 12 months.

Freeze-dried Poly-[SFHb-SOD-CAT-CA]	Molecular weight distribution (%)
		>450	100–450	<100
−80 °C	82	18	–
4 °C	78	22	–
20 °C	74	26	–
37 °C	63	37	–

Table IV. Molecular distribution of Poly-[SFHb-SOD-CAT-CA] solution stored for 4 months.

Solution of Poly-[SFHb-SOD-CAT-CA]	Molecular weight distribution (%)
		>450	100–450	<100
Before storage	83	17%
4 months stored in −80 °C	83	17	–
4 months stored in 4 °C	82	18	–
4 months stored in 20 °C	60	21	13
4 months stored in 37 °C	48%	23	31

Table V. Stability with pasteurization of the soluble and freeze-dried samples.

Enzyme activity samples		SOD	CAT	CA
Poly-[SFHb-SOD-CAT-CA]  solution	0.5h (65°C)	83.4 ± 4%	28 ± 1%	87.6 ± 4%
	1 h (70°C)	81.5 ± 3%	0%	77 ± 6%
	2 h (70°C)	64 ± 7%	0%	62 ± 6%
	3 h (70°C)	57.8 ± 10%	0%	50 ± 6%
Freeze-dried  Poly-[SFHb-SOD-CAT-CA]	0.5 h (65°C)	101 ± 4%	78 ± 5%	102 ± 4%
	1 h (70°C)	100 ± 3%	67.4 ± 1%	100 ± 5%
	2 h (70°C)	100 ± 2.5%	63.8 ± 4%	97 ± 4%
	3 h (70°C)	100 ± 4%	46.7 ± 5%	94 ± 5%

Table VI. The P50 value of the solution and freeze-dried sample of Poly-[SFHb-SOD-CAT-CA] stored for 4 months at the temperature shown.

Solution of Poly-[Hb-SOD-CAT-CA]	P50 value (mmHg)	Freeze-dried Poly-[Hb-SOD-CAT-CA]	P50 value (mmHg)
−80°C	19.15 ± 0.95	−80°C	14.94 ± 0.38
4°C	19.92 ± 0.74	4°C	14.22 ± 1.4
20°C	18.2 ± 1.0	20°C	14.57 ± 1.1
37°C	20.5 ± 0.28	37°C	13.32 ± 0.93

Table VII. The C3a complement activity of Poly-[SFHb-SOD-CAT-CA].

Sample	Average O.D.	Net O.D. (Av. O.D. – NSB)	Percent bound (%)	Human C3a des Arg (ng/ml)
Blank	0	–	–	–
NSB	0.102	0	–	–
Bo	0.676	0.574	100	0
Saline + plasma  (negative control)	0.639	0.537	93.55	0.035 ± 0.014
Zymosan + plasma  (positive control)	0.587	0.485	84.49	0.24 ± 0.04
PolyHb-SOD-CAT-CA + plasma	0.640	0.538	93.97	0.028 ± 0.07