Downstream Processing and Bioseparation - American Chemical

shown by Bewley and Li (6). fl(pH) is a linear function, with formula varied for different cases. ..... density due to nonvolatiles at χ = 0 is n...
0 downloads 0 Views 2MB Size
Chapter 10

Protein Separation via Affinity-Mediated Membrane Transport Liese Dall-Bauman and Cornelius F. Ivory Downloaded by UNIV OF ARIZONA on September 10, 2015 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch010

1

2

Crew and Thermal Systems Department, Lockheed Engineering and Sciences Company, C-70, 2400 NASA Road 1, Houston, TX 77058-3711 Department of Chemical Engineering, Washington State University, Pullman, WA 99164 1

2

Affinity-mediated transport is a form of facilitated transport in which a 'switch' monoclonal antibody is used as a highly selective protein carrier. The membrane's physicochemical environment is controlled so that the antibody exhibits a high binding affinity for its antigen at the upstream boundary and a significantly lower binding affinity at the downstream boundary. Hence, complexation is favored upstream and decomplexation is favored downstream. An affinity-mediated system in which a 'switch' monoclonal antibody is used to transport its antigen, human growth hormone, has been modeled. The affinity of the antibody for the hormone is dependent on local pH. In addition to the kinetic effect, macroscopic and microscopic electrochemical effects were considered. On the larger scale, modest induced and applied electric fields were found to exert considerable influence on fluxes of antibody, hormone, and complexes. The short-range effect of Donnan potential was found to enhance the flux of hormone into the membrane.

Carrier-mediated transport provides a means of increasing the flux of a selected permeant across a liquid membrane. As shown in Figure 1, the Fickian flux of permeant is augmented by a second mechanism in which the permeant is absorbed at the membrane's upstream boundary and reacts reversibly with a carrier molecule to form a complex. The complex then diffuses across the membrane to the downstream boundary, where decomplexation occurs, the permeant is desorbed, and the carrier is free to return to the upstream boundary so that the cycle can be repeated. The carrier and complex species are generally considered to be nonvolatile in the sense of Schultz et al. (1); that is, they are assumed to be confined to the membrane phase. 0097-6156/90/0419-0188$07.00/0 © 1990 American Chemical Society

In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

DALL-BAUMAN & IVORY

Affinity-Mediated Membrane Transport

Downloaded by UNIV OF ARIZONA on September 10, 2015 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch010

10.

In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

189

Downloaded by UNIV OF ARIZONA on September 10, 2015 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch010

190

DOWNSTREAM

PROCESSING AND BIOSEPARATION

There are certain benefits associated with controlling the direction of the complexation/decomplexation reaction. If complexation can be encouraged at the upstream boundary, more permeant can be introduced into and carried across the membrane. If the equilibrium is shifted away from decomplexation throughout the membrane, the permeant w i l l be trapped i n the membrane. M o r e specifically, downstream decomplexation helps to ensure efficient use of carrier. Complexation and decomplexation can be favored i n the appropriate regions if there is some difference between the physicochemical environments i n the two regions. For example, Jain and Schultz (2) found that hemoglobin/carbon monoxide complexation was favored i n a dark environment, w h i l e decomplexation was favored i n the presence of light. A greater flux of carbon monoxide was achieved through a partially illuminated membrane w i t h darkness upstream and i l l u m i n a t i o n downstream than through totally illuminated or non-illuminated membranes. In this w o r k , the consequences of c o n t r o l l i n g the chemical environment have been considered. Specifically, the effect of p H has been examined. A number of biochemical reactions are k n o w n to be p H sensitive. For example, hemoglobin/oxygen binding tends to increase with increasing p H and most enzymes have a specific p H at w h i c h their substrate b i n d i n g affinity is maximized. Here, a mathematical model describing the effect of a transmembrane p H gradient on transport of h u m a n g r o w t h hormone (hgh) is presented. 'Switch' monoclonal antibodies are considered as carriers. 'Switch' monoclonal antibodies were developed to improve the efficiency of immunoaffinity chromatography (3). The strength of an antigen-antibody bond is described i n terms of the antibody's affinity for the antigen. In immunoaffinity chromatography, high-affinity antibodies are necessary for efficient adsorption of an antigen, particularly if the antigen is present i n limited quantities i n the feed mixture. Unfortunately, antigens are not easily desorbed from high-affinity antibodies and are sometimes denatured by the desorption process. 'Switch' antibodies can be transformed from high- to low-affinity states by relatively small changes in their environment (e.g. temperature, salt concentration, or p H ) . H i l l and coworkers discussed two pH-sensitive antibodies, one of which had a high binding affinity for hgh at p H ~ 5 and a much lower affinity at p H ~ 4. The other underwent a similar change i n the p H range 9.5-10.5. It was proposed that the p H change induced subtle changes i n structure i n the hormone or the antibody, similar to the way a change i n p H influences the activity of an enzyme by affecting the enzyme's configuration (4). L o w selectivity is another p r o b l e m that is encountered i n immunoaffinity chromatography. Obviously, if an antibody shows little preference for one antigen over another, no separation w i l l be achieved. By definition, a monoclonal antibody recognizes - and binds to - a single site and is therefore highly selective. A s implied by their name, 'switch' monoclonal antibodies are selective and, under the right circumstances, are

In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

10. DALL-BAUMAN & IVORY

Affinity-Mediated Membrane Transport

191

easily persuaded to release their antigens. For these reasons, they are promising candidates for use as carriers in facilitated transport of proteins.

Downloaded by UNIV OF ARIZONA on September 10, 2015 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch010

SYSTEM DESCRIPTION V i r t u a l l y no i n f o r m a t i o n o n the p h y s i c a l properties of ' s w i t c h ' monoclonal antibodies is available and their reactions w i t h antigens have not been described i n detail. For the purpose of this work, the antibody was assumed to be a gamma globulin w i t h t w o identical a n d independent binding sites for hgh. It was also assumed to have a b i n d i n g site for hydrogen that could influence both hgh binding sites — perhaps b y slowing d o w n the binding of the hormone and accelerating its release. This could occur if the bound hydrogen forced reorientation of the hgh binding sites w i t h respect to each other or to the rest of the molecule. These assumptions i m p l y the existence of several forms of complex. They, together with all other species present, are listed i n Table 1. Further assumptions were made concerning the physical parameters describing the antibody a n d the antibody-hormone complexes. The diffusion coefficients for all complexes were set equal to the value assigned to the antibody's diffusion coefficient. This is reasonable, since the molecular weight of the antibody is between 156,000 and 161,000 (5), while that of hgh is roughly 22,000 (6). The pH-dependence of the charge o n the hormone was obtained b y fitting the data read from a titration curve (6). The charge o n the antibody was assumed to be a linear function of p H , w i t h the exact formula varying from case to case. The charge o n each complex species was calculated by summing the charges of the components forming the complex. The diffusion coefficients and charge profiles for all species are listed in Table 2. The reactions occurring i n the system are shown i n Table 3. A l l of the rate a n d e q u i l i b r i u m constants are estimates. The rate constants tabulated by Steward (7) for a variety of antibody-antigen reactions range from 8.0 χ 10 1 m o l s e c to 6.2 χ 10 1 m o l " s e c for the binding reaction and from 3.4 χ 10" sec to 6.0 χ 10 s e c for the reverse reaction. These values were used as guidelines i n a very limited parameter scan done at the beginning of this study. The rate constants appearing i n the table were ultimately chosen for use throughout the remainder of the work. It must be stressed that these are not optimal values. Examination of the rate constants shows that the rate constants for the binding of the first and second permeants have identical values, as do those for the release of the two permeants. This is true whether or not the carrier is protonated and is i n accordance with the assumption of identical, independent sites. It can also be seen that the forward rate constants are lower for complexation of protonated species, w h i l e the reverse rate constants are lower for decomplexation of nonprotonated species. This is necessary, given the assumption that protonated forms are slower to bind and quicker to release hgh. 6

- 1

4

1

-1

8

3

1

1

1

In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

192

DOWNSTREAM

PROCESSING A N D BIOSEPARATION

Table 1. Definition of Species Vector s for H G H - Antibody System

Downloaded by UNIV OF ARIZONA on September 10, 2015 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch010

Index 1

Symbol S

2 3 4 5 6 7 8 9

M+ xH A AS AS AH AHS

10

AHS

+

2

2

Description human growth hormone (primary permeant) supporting cation supporting anion hydrogen ion antibody (carrier) single-hormone complex double-hormone complex protonated carrier protonated single-hormone complex protonated double-hormone complex

Table 2. Diffusion Coefficients and Charge Profiles for H G H - Antibody System

Species

D (ΙΟ" cm /sec)

S

0.075

fo(pH)

M+

5

ζ

2

1.35

+1

x-

1.35

-1

H+

6.75

+1

A

0.040

fl(pH)

AS

0.040

fo(pH) + fi(pH)

AS AH

0.040

2f (pH)+fi(pH)

0.040

fl(pH) +1

AHS AHS

0.040 0.040

f (pH) + fi(pH) + 1 2f (pH) + fi(pH) + 1

2

2

0

0

0

Notes: Diffusion coefficient for antibody is from Sober (5). Diffusion coefficient for hgh is an average of values given by Bewley and L i (6). A l l coefficients are for diffusion of species through water at 2 5 ° C fo(pH) is obtained by fitting titration curve shown by Bewley and L i (6). fl(pH) is a linear function, with formula varied for different cases.

In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

10. DALL-BAUMAN & IVORY

Affinity-Mediated Membrane Transport

193

The p H ranged from 5.0 at the upstream boundary to 4.2 at the downstream boundary. Hydrogen concentrations i n the reservoirs were set to fix external p H at these values. The e q u i l i b r i u m constants for the protonation reactions were chosen so that protonated and nonprotonated carrier and complex could coexist i n this p H range. The assignment of the same e q u i l i b r i u m constant to reactions 5, 6, a n d 7 implies a single hydrogen binding site which is independent of the binding of hgh. The r e m a i n i n g species present, M a n d X " , are s u p p o r t i n g electrolytes and are present as ions dissociated from the salt form of hgh, as products of the dissolved salt M X , or (in the case of X") as counterions for H . They are 'generic' ions and were given equal diffusion coefficients i n the range of the coefficients for d i f f u s i o n of ionic s o d i u m , l i t h i u m , chlorine, a n d bromine i n water (8). The permeating species are h g h (represented as S), H , M and X . The system is represented i n Figure 2. +

Downloaded by UNIV OF ARIZONA on September 10, 2015 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch010

+

+

+

-

PONNAN EQUILIBRIUM Donnan equilibrium is a well-understood phenomenon which is observed at any interface that prevents diffusion of at least one (but not all) charged species between two phases; a thin polymer membrane separating two l i q u i d mixtures provides one example. Donnan behavior also occurs at other interfaces (9) and is especially important i n ion exchange resins (10). The requirement that the electrochemical potential of permeating species i be constant across the phase-separating interface a n d certain simplifying assumptions (11, 12, 13) can be used to obtain the following relationship: Γ7F

pd)

i

where Q ( P is the concentration of species i i n phase j , z i is the electric charge, F is Faraday's constant, R is the gas constant, Τ is the absolute temperature, a n d Φ Φ is the electric potential i n phase j . This equation holds for each permeating species so that 1/Zp

1/Z2

1/2!

" p(D "

" pd) "

p(2)

p(2) 2

" pd) "

...

4

= exp

2)

•ρ Φ (

( 2 )



( 1 )

)'

RT

=p

In Downstream Processing and Bioseparation; Hamel, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

(2)

194

DOWNSTREAM

PROCESSING A N D BIOSEPARATION

Table 3. Reaction Network for H G H - Antibody System

RATE CONSTANTS

Downloaded by UNIV OF ARIZONA on September 10, 2015 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch010

Forward

Reverse

1. A + S