Use of the Labscale Tangential Flow Diafiltrator to Remove Tetrameric Hemoglobin from Polyhemoglobin, Purify Hemolysate, and Concentrate Polyhemoglobin

Figures & data

Figure 1.  Schematic representation of a Tangential Flow Filtration filter. The direction of the flow through the filter is parallel (or tangential) to the membrane, which reduces the clogging of the filter and improves its efficiency. The pressure difference between the retentate out and feed in ports provides driving pressure for passage through the filter.

Figure 2.  Schema of experimental setup. A: Outflow from reservoir. B: Feed In port of filter. C: Permeate Out port of filter. D: Retentate Out port of filter. E: Retentate In port of reservoir. F: Diafiltration medium In port of reservoir. Upper left: additional elements for diafiltration setup (vessel with distilled water, tubing attached to airtight opening in reservoir).

Figure 3.  Removal of tetrameric Hb from polyHb with 100kDa filter. Chromatographic results. Left column: Initial Hb mass distribution. Right column: mass distribution post-diafiltration. The best result is obtained with a filter integrity of 0.075 using a flow rate of 0.020ml/min.

Figure 4.  Relationship between filter integrity and percent change in tetrameric Hb in the solution (change calculated as [(Final-Initial)/Initial]%Hb < 100).

Table 1. Removal of tetrameric Hb from polyHb (100kDa filter)

								Chromatography
Filter Age (weeks) Integrity (ml/min)	Ret initial [Hb] (g/dL)	Ret Final [Hb] (g/dL)	PRESSURE (bar)	Flow (ml/min)	Time diafil (hr)	Time conc (hr)	DV	Sample Date	% > 450 kDa	100-450	% < 100 kDa
0	1.0	0.94	Pf = 1.4	0.2	10	0	1.3	Initial
								06/11/07	69.4	20.4	11.2
0.075			Pret=1.1					Ret final
								19/11/07	85.1	13.6	1.3*
0	0.23	0.64	Pf = 0.8	2.2	5	0.25	13.5	Initial
								26/11/06	74.5	17.9	7.6
0.075			Pret=0.55					Ret final
								26/11/06	80.1	14.7	5.2
6	2.9	0.29	Pf = 0.4	0.75	1.75	0.75	2.4	Initial
								4/5/06	62.4	19.7	17.8
0.06								Ret final
								26/11/06	67.9	18.8	13.3
4	0.45	0.70	Pf = 1.4	1.75	8.5	0.5	14.5	Initial
								28/11/07	63.5	12.6	23.9
0.05			Pret=1.1					Ret final
								27/11/07	64.5	13.1	22.4
19	0.26	1.36	Pf = 1.5	0.5	8	1.5	2	Initial
								18/03/08	33.5	17.7	48.8
0.05			Pr = 1.3					Ret final
								19/03/08	37.7	17.4	44.9
15	1.0	0.9	Pf = 1.4	0.75	8	0.5	3	Initial
								14/02/08	54.2	14.2	31.6
0.045			Pret=1.1					Ret final
								21/02/08	58.3	12.4	29.3
28	4.65	0.49	Pf = 0.85	0.5	5	0	5.3	Initial
								14/09/06	66.6	23.1	10.2
								Ret final
0.03			Pret=0.55					20/10/06	57.7	27.3	15

Table 2. Diafiltration SFHb with 500kDa filter

	Ret initial [Hb], mass						Ret final [Hb], mass		Perm final [Hb], mass
Sample	g/dL	g	Time hr	Press Feed Ret Bar	Flow mL/ min	DV	g/dL	g	g/dL	g	Mass PermF/ TotF* %	Mass PermF/RetI %	Hb yield** %
A1	1.31	1.31	0.5	0.4	3.1	0	1.48	0.14	1.23	1.14	89	90	98
				0.4
A2	1.20	1.20	0.5	0.4	3.2	0	1.21	0.09	1.09	1.03	92	90	94
				0.4
A3	1.50	1.50	1	0.2	1.6	0	1.48	0.07	1.16	1.13	94	80	80
				0
A4	0.45	0.13	0.5	0.3	0.8	0.1	0.46	0.03	0.31	0.08	75	60	81
				0
A5	1.3	1.3	1.5	0.3	1.35	0.8	0.46	0.13	0.61	0.9	90	70	80

* PermF = final permeate Hb mass.

TotF = total final Hb.

RetI = initial retentate Hb.

**Hb yield: calculated as [Final (perm + ret)Hb mass]/ [Initial ret Hb mass].

Table 3. Diafiltration SFHb 100kDa filter

	Ret initial [Hb], mass				Ret final [Hb], mass		Perm final [Hb], mass
Sample (SF Hb)	g/dL	g	Pressure Feed Retentate (bar)	DV	g/dL	g	g/dL	g	Hbmass perm F/ retI %	Hbmass Perm F/ TotF %	Flow mL/min	Hb yield*** %
A5	0.84	0.84	Pf = 0.28	1.1	0.41	0.41	0.24	0.25	30	40	2.2	80
A6	0.86	0.86	Pf = 0.28	3.1	0.19	0.18	0.20	0.63	73	80	0.5	95
A7	0.84	0.84	Pf = 0.3-0.5	4.1	−.087	∼ 0	0.15	0.65	77	100	4	77
			Pret = 0.1-0.5
A8	1.89	1.89	Pf = 0.3-0.5	1.5	0.92	0.92	0.62	0.93	50	50	1.25	100
			Pret = 0.1-0.3
A9	0.96	0.96	Pf = 0.3-0.5	2.0	0.25	0.25	0.39	0.77	80	80	0.75	100
			Pret = 0.3-0.5
A10	1.7	8.5	Pf = 1.0-1.4	4.5	0.6	0.5	0.3	8.2	97	94	3.5	100
			Pret = 0.7-1.0
A11**	2.1	5.2	Pf = 1.2-1.4	0.9	3.2	3.5	0.4	1.4	27	28	0.5	95
			Pret = 1.1-1.2
A12**	2.2	5.5	Pf = 1.2-1.4	0.9	3.2	4	0.3	1.1	20	22	0.5	90
			Pret = 0.7-1.1
A13	0.9	2.6	Pf = 1-1.2	0.1	2.6	0.8	0.4	1.1	42	59	1	70
			Pret = 0.6-1.0
A14	0.36	1.1	Pf = 0.5-1.0	0.04	0.2	0.7	0.1	0.3	28	29	0.6	90
			Pret = 0.3-.5
A15	1.75	1.68	Pf = 0.7-1.1	4	2.69	0.54	0.25	1.14	68	68	0.5	100
			Pret = 0.4-0.5
A16	0.6	2.4	Pf = 1.2-1.4	0.2	0.8	1.9	0.1	0.3	12	14	0.5	90
			Pret = 1.1-1.2
A17	1.3	6.6	Pf = 1.1-1.4	1.1	0.6*	0.4*	0.6	5.9	89	94*	1.3	95*
			Pret = 0.5-0.7

* Values calculated, assuming 95% yield.

** Analysis of catalase concentration in retentate pre and post-filtration available (Table 3).

***Hb yield: calculated as [Final (perm + ret) Hb mass]/ [Initial ret Hb mass].

Table 4. Diafiltration with 100kDa filter, catalase assay

	Initial Catalse (ret)			Final Catalase (ret)			Final Catalase (permeate)				Cat Pf/Ri
Sample (SFHb)	U/mL	U*10^3	U /mg Hb	U/mL	U*10^3	U /mg Hb	U/mL	U*10^3	U /mg Hb	Cat Units recovery %	%U	%U/ mgHb
A11	1700	440	85	1100	120	34	10	4	3	30	0.9	3.5
A12	1500	380	64	2000	250	63	13	4.4	4	70	1.2	6

Table 5. To concentrate PolyHb solution

Filter NWML (kDa)	Retentate Initial [Hb] (g/dL), mass (g)	Retenate Final [Hb] (g/dL), mass (g)	Time (hr)	Flow (mL/min)	Pressure (bar)	Perm final (dL)	[Hb] Final/Initial (retentate)
100	0.29	1.3	0.75	0.6	Pf = 0.4	0.35	4.5
	0.12	0.094
100	0.47	2.8	1	0.7	Pf = 0.3-0.7	0.77	6
	0.44	0.43			Pret = 0-0.7
100	0.41	3.9	1	0.9	Pf = 0.4	0.84	9.5
	0.66	0.35
5	0.46	4.8	1	1.3	Pf = 0.4	0.93	10.4
	0.46	0.34			Pret = 0.7