CN105738322A - Affinity chromatography filler dynamic loading capacity determination method - Google Patents

Abstract

The invention discloses an affinity chromatography filler dynamic loading capacity determination method.The method comprises the steps that firstly, purified protein samples with different concentrations are prepared with flowthrough liquid; secondly, a filtering plate for filling of filler is prepared; thirdly, the samples are added onto the filtering plate and hatched; fourthly, suction filtration is carried out, the flowthrough liquid is collected, the protein concentrations are detected, and an adsorption isotherm is fitted; fifthly, chromatographic columns are filled and loaded with the samples, elution is carried out, the retention volume of ultraviolet outlet peaks and the retention volume of electric conductance outlet peaks are recorded, the chromatographic columns are short circuited, the retention volume of electric conductance outlet peaks is recorded, and the water drainage porosity outside the filler, the full-permeation porosity and the filler porosity are calculated; sixthly, the average radius of protein hydration molecules is determined; seventhly, the dynamic loading capacity is calculated.According to the affinity chromatography filler dynamic loading capacity determination method, according to the characteristics and natures of the affinity filler and monoclonal antibodies, an adsorption isotherm and adsorption dynamical model is studied through the high throughput screening technology, the dynamic loading capacity of the chromatography filler is predicted through the static loading capacity of the monoclonal antibodies, less time is consumed, cost is low, and the accuracy of a determination result is high.

Description

The dynamic carrying capacity assay method of affinity chromatograph filler

Technical field

The present invention relates to biological field, particularly relate to the assay method of the dynamic carrying capacity of affinity chromatograph filler in purifying process.

Background technology

Dynamic carrying capacity mensuration is process exploitation step important in affinity chromatograph.Traditional dynamic carrying capacity assay method needs to consider the factor such as different retention times and sample concentration, completes related assays work on laboratory scale affinity column.The shortcoming of this assay method is that the process time is long, and consumption of materials is big.

Adopt high-throughput techniques can be greatly saved process exploitation time and experiment material, save process exploitation cost.But, utilizing the affine filler carrying capacity that high-throughput techniques measures is all static carrying capacity, incomplete same with the result of dynamic carrying capacity, it is necessary to consider the dynamic behavior between albumen and affine filler, dynamic carrying capacity is predicted.

Adopt adsorption isotherm and kinetics model of biosorption can estimate the dynamic carrying capacity of affine filler.The research of current affinity chromatograph theoretical model is concentrated mainly on the models such as Langmuir adsorption isotherm, hole diffusion, diffusion into the surface and parallel diffusion.When determining tradition adsorption isotherm and adsorption parameters, it is generally adopted the research method of batch absorption or surface bed fluidised bed adsorption.It is known that this traditional power science study method sample requirements is big, tests length consuming time, be unfavorable for carrying out of macromolecules adsorption dynamic experiment.Additionally, in traditional power research, it is necessary to study different kinetics model of biosorptions and obtain effective diffusion cofficient accurately, thus convection current is worn curve and is fitted, it was predicted that the dynamically value of carrying capacity.But owing to monoclonal antibody structure is complicated, affine filling-material structure is also not quite similar, and causes that model fitting process is sufficiently complex, increases the difficulty of the process exploitation process applying to the bio-pharmaceuticals dynamic carrying capacity of downstream purification affinity chromatograph.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of dynamic carrying capacity assay method of affinity chromatograph filler, and the method is not only simple, and accurately.

For solving above-mentioned technical problem, the dynamic carrying capacity assay method of affinity chromatograph filler of the present invention, step includes:

1) albumen after affinitive layer purification is proportionally added into in affinity chromatograph process collect fermentation broth stream wear in liquid, be configured to the sample of variable concentrations gradient；

2) prepare affinity chromatograph filler and be loaded on screen plate, with affinity chromatograph equilibration buffer affinity chromatograph filler, the preservation liquid in displacement filler；

3) by step 1) sample prepared is added to step 2) loaded on the screen plate of affinity chromatograph filler, and hatch；

4) vacuum filtration, collects stream and wears liquid, and detect the protein concentration that stream is worn in liquid, and then with protein concentration for X-axis, the corresponding protein load being adsorbed onto on affinity chromatograph filler is Y-axis, matching adsorption isotherm；

5) chromatographic column of the Lab Scale of the corresponding affinity chromatograph filler of filling, respectively by PVP-K90 and NaCl loading, eluting, record ultraviolet goes out the retention volume at peak and conductance goes out peak retention volume, again by chromatographic column short circuit, record conductance goes out peak retention volume, and the calculating outer drain aperture rate of affinity chromatograph filler, full impregnated cross porosity and the porosity of this affine filler；

6) albumen after affinitive layer purification is changed liquid in affinity chromatograph level pad, carry out dynamic light scattering experiment, measure albumen hydrate molecule mean radius；

7) the dynamic carrying capacity of affinity chromatograph filler is calculated.

Above-mentioned steps 4), it is possible to use Langmuir adsorption isotherm is fitted, and fitting formula is:

$q=\frac{q_{m}cK}{K+c}$

Wherein, c is that stream wears protein concentration in liquid, and q is the protein load being adsorbed onto on affinity chromatograph filler, q_mFor maximum static carrying capacity, K is dissociation constant.

Above-mentioned steps 5), the computing formula of the porosity that the outer drain aperture rate of affinity chromatograph filler, full impregnated cross porosity and this affine filler is:

ε=(V-V_v)/(V_c-V_v)

ε_b=(V_b-V_v)/(V_c-V_v)

ε_p=(ε_b-ε)/(1-ε)

Wherein, V_cFor chromatographic column volume, V is the retention volume that ultraviolet goes out peak, V_bPeak retention volume, V is gone out for conductance_vDuring for short circuit chromatographic column, conductance goes out peak retention volume, and ε is the outer drain aperture rate of affine filler, ε_bFull impregnated for affine filler crosses porosity, ε_πPorosity for affine filler.

Above-mentioned steps 5), the sample concentration of PVP-K90 and NaCl respectively 0.05% and 1M, applied sample amount is 2% chromatographic column volume, and ultraviolet detection wavelength is 220nm.

Above-mentioned steps 7), the dynamic carrying capacity of affinity chromatograph filler is calculated according to below equation:

${\mathrm{\λ}}_m=\frac{r_m}{r_{pore}}$

K_p=0.865 (1-2.1 λ_m+2.09λ_m ³-0.984λ_m ⁵)

τ=(2-ε_p)²/ε_p

$D_e={\mathrm{\ϵ}}_p{D}_p=K_p\frac{s_{p}D}{\mathrm{\τ}}$

$N=\frac{15(1-s)D_{e}L}{{\mathrm{ur}}_p^2}$

As N τ₁During > 2.5, τ₁=1-1.03/N

As N τ₁During < 2.5, τ₁=0.364N-0.0612N²+0.00423N³

${\mathrm{DBC}}_{10\%}=\frac{q_m{c}_0}{K_d+c_0}{\mathrm{\&tau;}}_1(1-\mathrm{\&epsiv;})$

K_d=1/K (K_dAlso it is commonly used to evaluating protein and is combined, with filler, the situation of dissociating)

Wherein, r_poreFor affine filler aperture radius, Kp is space resistance coefficient, and De is effective diffusion cofficient, and D is m protein free diffusing coefficient, and L is that chromatographic column post is high, and u is linear velocity, r_pFor affine filler particles radius, the value of L/u is retention time, c₀For the initial protein concentration of fermentation liquid, r_mFor albumen hydrate molecule mean radius.

In conjunction with the character feature of affine filler and monoclonal antibody, (aperture of conventional affine filler is far longer than the diameter of monoclonal antibody to the present invention, and affinity chromatograph specificity is stronger, aglucon and part effect are strong, close to Irreversible Adsorption), utilize High Throughput Screening Assay research adsorption isotherm and kinetics model of biosorption, find out predominant intermolecular forces and the effect resistance of affine filler and monoclonal antibody action, and analyze static adsorption effect and the relation of dynamic adsorption effect, achieve the purpose of the dynamic carrying capacity being predicted affinity chromatograph filler by the static carrying capacity of monoclonal antibody, compare existing assay method, not only simple to operate, model convenience of calculation, required time and cost are low, and the accuracy of measurement result is higher.It addition, the present invention adopts PVP-K90 to carry out outer exclusion porosity measurement, affinity chromatograph filler will not be caused damage, therefore without the service life affecting affine filler.

Accompanying drawing explanation

Fig. 1 is the Langmuir adsorption isotherm of affine three parallel laboratory test matchings of filler used by the embodiment of the present invention 1；

Fig. 2 is the chromatography collection of illustrative plates measuring dynamic carrying capacity by traditional method on laboratory scale 3mLTricorn5/150 chromatographic column；

Fig. 3 is variable concentrations lgG4 albumen loading, predicts the dynamic carrying capacity obtained and the comparison diagram of the dynamic carrying capacity value obtained with traditional method measurement by the method for the present invention.Wherein, zero sample concentration is 0.64mg/mL, sample concentration be 0.71mg/mL, △ sample concentration be 1.34mg/mL, ◇ sample concentration is 2.33mg/mL；Abscissa is the dynamic carrying capacity value recorded by traditional method, and vertical coordinate is predict, by the method for the present invention, the dynamic carrying capacity value obtained.

Fig. 4 is in porosity measurement, and PVP-K90220nm place ultraviolet goes out peak and 1MNaCl conductance goes out peak collection of illustrative plates.Wherein, (a) figure is that ultraviolet goes out peak, and (b) figure is that conductance goes out peak.

Detailed description of the invention

Understand more specifically for the technology contents of the present invention, feature and effect are had, in conjunction with specific embodiment, technical scheme is described in further detail.

Embodiment 1

The dynamic carrying capacity assay method of affinity chromatograph filler of the present embodiment, measures static carrying capacity first with high-throughput techniques, again through the dynamic carrying capacity of the kinetics model of biosorption prediction affinity chromatograph filler optimized, specifically, comprises the following steps:

Step 1, lgG4 albumen after one step affinitive layer purification is proportionally added into in affinity chromatograph process collect fermentation broth stream wear in liquid, prepare into the sample of a series of Concentraton gradient respectively 0.5mg/mL, 1.5mg/mL, 2.5mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 8mg/mL and 10mg/mL.

Step 2, utilizes the 96 hole filler fillers required affinity chromatograph fillers (the present embodiment is EshmunoA) of preparation experiment, and be loaded into 96 hole screen plates (AcroPre^TM, lot:412803) on, every hole 20 μ L.Balancing affinity chromatograph filler with affinity chromatograph level pad (50mMTris-HAc+150mMNaCl, pH7.4), the 20%EtOH at displacement filler place preserves liquid.

Step 3, sample step 1 prepared is added to step 2 by solid-to-liquid ratio 1:20 and fills on 96 hole screen plates of affine filler, every hole 400 μ L sample.

96 hole screen plates in step 3 are carried out night incubation by step 4.Night incubation adopts the Te-Shake module on high flux platform TecanEVOFreedom150-8 (TecanSchweizAG, Switzerland), and frequency of vibration is 1100rpm, and incubation time is 24h.

Step 5, vacuum filtration, collect stream and wear liquid, and adopt the method detection stream of Titer-ProteinAHPLC to wear the concentration of lgG4 albumen in liquid.Wherein, vacuum filtration adopts the Te-Vac module on high flux platform TecanEVOFreedom150-8 (TecanSchweizAG, Switzerland), and vacuum filtration intensity is 600mbar, sucking filtration three times, each 30s.

Step 6, wears in liquid with stream that protein concentration c is for X-axis, and it is Y-axis that correspondence is adsorbed onto the carrying capacity q on filler, utilizes Langmuir adsorption isotherm (as shown in Figure 1) to be fitted, and fitting formula is:

$q=\frac{q_{m}cK}{K+c}$

Matching obtains static carrying capacity q_mAnd albumen dissociates COEFFICIENT K_dValue, respectively q_m=61.33 ± 2.233mg/mL filler, K_d=1/K=0.0806 ± 0.0171mg/mL.

Step 7, the chromatographic column (Tricorn5/150column) of the Lab Scale of the affine filler of correspondence of filling 3mL, respectively with 2% column volume (V_c) PVP-K90 and the 1MNaCl loading that concentration is 0.05% (w/v), and carry out eluting with 150mMNaCl, record 220nm place ultraviolet goes out the retention volume (V) at peak and conductance goes out peak retention volume (V respectively_b), as shown in Figure 4, then by chromatographic column short circuit, record conductance goes out peak retention volume (V_v).Calculate the outer drain aperture rate ε of this affine filler according to below equation, full impregnated crosses porosity ε_bAnd the porosity ε of this affine filler_p:

ε=(V-V_v)/(V_c-V_v)

ε_b=(V_b-V_v)/(V_c-V_v)

ε_p=(ε_b-ε)/(1-ε)

Calculating obtains each porosity value and is respectively as follows: ε_p=0.820 ± 0.040, ε_b=0.892 ± 0.022, ε=0.398 ± 0.010.

Step 8, the albumen adopted in step 1 is changed liquid to affinity chromatograph level pad (50mMTris-HAc+150mMNaCl, pH7.4) in, carry out dynamic light scattering experiment (DLS) according to ZetasizerNanoZS90 (Malvern) operating process, record albumen hydrate molecule mean radius r_mFor 7nm.

Step 9, calculates the dynamic carrying capacity of affinity chromatograph filler according to below equation:

${\mathrm{\&lambda;}}_m=\frac{r_m}{r_{pore}}$

K_p=0.865 (1-2.1 λ_m+2.09λ_m ³-0.984λ_m ⁵)

τ=(2-ε_p)²/ε_p

$D_e={\mathrm{\&epsiv;}}_p{D}_p=K_p\frac{s_{p}D}{\mathrm{\&tau;}}$

$N=\frac{15(1-s)D_{e}L}{{\mathrm{ur}}_p^2}$

τ₁=1-1.03/N is (as N τ₁During > 2.5) or τ₁=0.364N-0.0612N²+0.00423N³(as N τ₁During < 2.5)

${\mathrm{DBC}}_{10\%}=\frac{q_m{c}_0}{K_d+c_0}{\mathrm{\&tau;}}_1(1-\mathrm{\&epsiv;})$

Wherein, r_poreFor affine filler aperture radius, Kp is space resistance coefficient, and De is effective diffusion cofficient, D is m protein free diffusing coefficient, L is chromatographic column post high (the present embodiment is 15.7cm), and u is linear velocity (the present embodiment is 188.4cm/h), r_pFor affine filler particles radius, the value of L/u is retention time (the present embodiment is 5min), c₀For the initial protein concentration of fermentation liquid (adopting Titer-ProteinAHPLC to measure, the present embodiment is 2.33mg/mL).

Calculating obtains: D_e=7.432 × 10^-8cm²/ s, DBC_10%=34.54 ± 1.13mg/mL filler.By this DBC_10%Be multiplied by the compressibility factor 1.15 of this affine filler, namely measurable be calibrated after dynamic carrying capacity be 39.72 ± 1.30mg/mL filler.

The actual value of dynamic carrying capacity is measured by traditional method, to verify the accuracy of dynamic carrying capacity value that above-mentioned prediction obtains, method is as follows: the chromatographic column (Tricorn5/150column) of the Lab Scale of the affine filler of correspondence of filling 3mL, adopt retention time 5min, loading protein concentration is the fermentation liquid of 2.33mg/mL, detection stream wears the concentration of albumen in liquid, calculate when stream wears that in liquid, protein concentration is the 10% of loading protein concentration, (concentration of loading albumen is multiplied by the volume of loading albumen when stream wears that in liquid, protein concentration is the 10% of loading protein concentration for the carrying capacity of affine filler, again divided by the volume loading filler in this chromatographic column), the actual value obtaining dynamic carrying capacity is 39.77 ± 1.40mg/mL filler, it is consistent with the dynamic carrying capacity numerical value of prediction.

Embodiment 2 changes retention time, it was predicted that and verify the result of dynamic carrying capacity

Retention time is changed to 3min and 7min, sample concentration c₀Constant, the value of dynamic carrying capacity is predicted according to the method for embodiment 1.When retention time is 3min, DBC_10%=34.27mg/mLresin, is multiplied by compressibility factor 1.15, it was predicted that the value obtaining dynamic carrying capacity is 39.41mg/mLresin.When retention time is 7min, DBC_10%=35.38mg/mLresin, is multiplied by compressibility factor 1.15, it was predicted that the value obtaining dynamic carrying capacity is 40.69mg/mLresin.

According to the method for embodiment 1, the accuracy of above-mentioned predicted dynamic carrying capacity value being verified, retention time is 3min and 7min respectively, and the result is: when retention time is 3min, and dynamic carrying capacity actual value is 37.41mg/mLresin；When retention time is 7min, dynamic carrying capacity actual value is 44.44mg/mLresin, is substantially consistent with the dynamic carrying capacity numerical value of prediction.

Embodiment 3 changes sample concentration, it was predicted that and verify the result of dynamic carrying capacity

By sample concentration c₀Changing to 0.64mg/mL, 0.71mg/mL and 1.34mg/mL respectively, retention time is 5min, according to the method for embodiment 1, it was predicted that obtain the value respectively 36.50mg/mL filler of dynamic carrying capacity, 36.91mg/mL filler and 38.77mg/mL filler.

According to the dynamic carrying capacity value of the above-mentioned prediction of the method validation of embodiment 1, sample concentration c₀Being respectively adopted 0.64mg/mL, 0.71mg/mL and 1.34mg/mL, retention time is 5min, records dynamic carrying capacity actual value respectively 35.92mg/mL filler, 34.29mg/mL filler and 39.81mg/mL filler, is substantially consistent with the numerical value of the dynamic carrying capacity predicted.

Claims (5)

1. the dynamic carrying capacity assay method of affinity chromatograph filler, it is characterised in that step includes:

1) albumen after affinitive layer purification is proportionally added into in affinity chromatograph process collect fermentation broth stream wear in liquid, be configured to the sample of variable concentrations gradient；

2) prepare affinity chromatograph filler and be loaded on screen plate, with affinity chromatograph equilibration buffer affinity chromatograph filler, the preservation liquid in displacement filler；

3) by step 1) sample prepared is added to step 2) loaded on the screen plate of affinity chromatograph filler, and hatch；

4) vacuum filtration, collects stream and wears liquid, and detect the protein concentration that stream is worn in liquid, and then with protein concentration for X-axis, the corresponding protein load being adsorbed onto on affinity chromatograph filler is Y-axis, matching adsorption isotherm；

5) chromatographic column of the Lab Scale of the corresponding affinity chromatograph filler of filling, respectively by PVP-K90 and NaCl loading, eluting, record ultraviolet goes out the retention volume at peak and conductance goes out peak retention volume, again by chromatographic column short circuit, record conductance goes out peak retention volume, and the calculating outer drain aperture rate of affinity chromatograph filler, full impregnated cross porosity and the porosity of this affine filler；

6) albumen after affinitive layer purification is changed liquid in affinity chromatograph level pad, carry out dynamic light scattering experiment, measure albumen hydrate molecule mean radius；

7) the dynamic carrying capacity of affinity chromatograph filler is calculated.

2. method according to claim 1, it is characterised in that step 4), utilize Langmuir adsorption isotherm to be fitted, fitting formula is:

Wherein, c is that stream wears protein concentration in liquid, and q is the protein load being adsorbed onto on affinity chromatograph filler, q_mFor maximum static carrying capacity, K is dissociation constant.

3. method according to claim 2, it is characterised in that step 5), the computing formula of the porosity that the outer drain aperture rate of affinity chromatograph filler, full impregnated cross porosity and this affine filler is:

ε=(V-V_v)/(V_c-V_v)

ε_b=(V_b-V_v)/(V_c-V_v)

ε_p=(ε_b-ε)/(1-ε)

Wherein, V_cFor chromatographic column volume, V is the retention volume that ultraviolet goes out peak, V_bPeak retention volume, V is gone out for conductance_vDuring for short circuit chromatographic column, conductance goes out peak retention volume, and ε is the outer drain aperture rate of affine filler, ε_bFull impregnated for affine filler crosses porosity, ε_πPorosity for affine filler.

4. method according to claim 1, it is characterised in that step 5), the sample concentration of PVP-K90 and NaCl is 0.05% (w/v) and 1M respectively, and applied sample amount is 2% chromatographic column volume, and ultraviolet detection wavelength is 220nm.

5. method according to claim 3, it is characterised in that step 7), the dynamic carrying capacity of affinity chromatograph filler is calculated according to below equation:

K_p=0.865 (1-2.1 λ_m+2.09λ_m ³-0.984λ_m ⁵)

τ=(2-ε_p)²/ε_p

As N τ₁During > 2.5, τ₁=1-1.03/N

As N τ₁During < 2.5, τ₁=0.364N-0.0612N²+0.00423N³

K_d=1/K

Wherein, r_poreFor affine filler aperture radius, Kp is space resistance coefficient, and De is effective diffusion cofficient, and D is m protein free diffusing coefficient, and L is that chromatographic column post is high, and u is linear velocity, r_pFor affine filler particles radius, the value of L/u is retention time, c₀For the initial protein concentration of fermentation liquid, r_mFor albumen hydrate molecule mean radius.