Protein Purification - American Chemical Society

Chapter 14

Purification Alternatives for IgM (Human) Monoclonal Antibodies G. B. Dove, G. Mitra, G. Roldan, M. A. Shearer, and M.-S. Cho

Downloaded by UNIV OF NEW ENGLAND on February 23, 2017 | http://pubs.acs.org Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch014

Cutter Biological Laboratory, Miles, Inc., Berkeley,CA94704

Methods suitable for purification of IgM monoclonal antibodies from serum-free tissue culture supernatants are described in this case study. We review techniques found to be useful in purifying proteins and the techniques applied to establish u t i l i t y . Partitioning techniques include polyethylene glycol (PEG) precipitation, size exclusion chromatography, anion and cation exchange chromatography, hydroxylapatite chromatography, and immunoaffinity. Modification techniques include the use of enzymes (e.g. DNAse). Protein purity is achieved primarily with precipitation, size exclusion chromatography, and immunoaffinity. DNA removal is greatest with anion exchange, immunoaffinity,and a combination of DNAse and size exclusion chromatography. Virus is partitioned most effectively through hydroxylapatite and immunoaffinity. A cascade of several appropriate steps provides contaminant protein clearance of >100x (purity greater than 99%), DNA clearance of >l,000,000x, and virus clearance of >100,000x. This paper presents a case study of the definition of purification processes for monoclonal IgMs produced by tissue culture fermentation. We review techniques that we have found to be useful and the approaches taken to establish their utility. The monoclonal antibodies are specific to various bacterial antigens for use as a therapeutic product. Serum-containing and serum-free (supplemented with proteins) broths are purified, although the purification methods are optimized for serum-free media. Purification is necessary to remove contaminants introduced by the media and the cells. Contaminants include media components (albumin, transferrin, insulin, and many serum components), nucleic acids, viruses, and other cellular products. 0097-^156790/D427-O194$06.00A) © 1990 American Chemical Society

Ladisch et al.; Protein Purification ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

14. DOVE ET AL.

Purification Alternatives for IgM Monoclonal Antibodies


Polymers, high molecular weight aggregates and fragments of IgM and albumin must be removed as well. There a r e s e v e r a l approaches t o p u r i f i c a t i o n i n terms o f p r o p e r t i e s (Table 1). The common p r o p e r t i e s are s i z e , charge and i o n i c i n t e r a c t i o n , and a f f i n i t y . Separation i s optimized by ex p l o i t i n g (e.g. size between IgM and albumin) or creating differences (e.g. DNAse) between the product IgM and contaminants. Techniques discussed i n t h i s study include f i l t r a t i o n s , pre c i p i t a t i o n with PEG and s a l t s (1^. 2^ 3), size exclusion chromatogra phy (41. 5. 6), anion (4^. 5)and c a t i o n exchange (7), contact with hydroxylapatite (8_«_ 9) , immunoaff i n i t y , a d d i t i o n of reagents (e.g. DNAse (10)) and combinations of these techniques (11).

Materials and Methods. Monoclonal (human) antibodies of c l a s s M (m-IgM) were derived from human Β lymphocyte c e l l l i n e s , designated A, B, C, D, and E. A n t i body from each l i n e was directed toward a d i f f e r e n t , s p e c i f i c bacte r i a l antigen. C e l l s were grown i n suspension c u l t u r e o r hollow f i b e r . P o l y c l o n a l plasma-derived IgM was obtained from Cohn f r a c t i o n I I I (ethanol f r a c t i o n a t i o n of human plasma (12). Chemicals were reagent-grade. S a l t s were obtained from Mallinckrodt, Paris, Ky. Enzymes were obtained from Sigma, St. Louis, Mo. Chromatography r e s i n s and equipment, unless noted otherwise, were obtained from Pharmacia, Uppsala, Sweden. Hydroxylapatite (DNA-Grade Bio-Gel HTP) was obtained from Bio-Rad L a b o r a t o r i e s , Richmond, Ca. Proteins were characterized by SDS-polyacrylamide gels using a 2-10% agarose gradient, stained with either Coomasie Blue or s i l v e r . General product and contaminant analysis were quantitated by Pharma c i a FPLC (Fast Protein L i q u i d Chromatography) system with a column matrix o f Superose 6, u t i l i z i n g s i z e e x c l u s i o n chromatography. Buffers defined the state as native, reduced, or denatured. Absorbance at 280 nm. was used f o r approximate measurements. Various ELISA were developed to provide a consistent basis f o r loss of IgM as w e l l as denaturation during processing. An antigen ELISA using anti-u chain IgG detected the Fc region and i s u s e f u l i n monitoring y i e l d . A f u n c t i o n a l ELISA i n d i c a t e d antibody binding e f f i c i e n c y to antigen. Epstein-Barr v i r u s (EBV) was derived from B95-8 c e l l s (13). EBV-specific nuclear antigen (EBNA) was demonstrated by an a n t i complementary immunofluorescence (ACIF) assay with m o d i f i c a t i o n s (14). Residual native DNA was assayed by dot b l o t h y b r i d i z a t i o n analysis (15. 16). P32-labeled DNA was prepared by n i c k - t r a n s l a t i o n of host c e l l DNA isolated from culture harvests of c e l l l i n e C. The DNA was s p i k e d i n t o v a r i o u s p r o c e s s steps and r e c o v e r e d ( 1 7 ) . Samples were spotted to f i l t e r paper, p r e c i p i t a t e d and washed with 10% t r i c h l o r o a c e t i c acid (TCA) and measured by s c i n t i l l a t i o n coun ter (LKB, model #1217 Rack Beta).


195

PROTEIN PURIFICATION


196

Table 1.

Component

IgM

I s o l a t i o n Parameters f o r P u r i f i c a t i o n

Size (xlOOO MW)

800

I s o e l e c t r i c Point (Neutral Charge)

Other Characteristics

pH 6-6.5

Contaminants : Albumin DNA

69 variable

pH 5 pH 5

Viruses

>1000

pH 4-6

HAPT DNAse

Basis of Separation: Albumin DNA

SEC

Viruses All

SEC

IEC

HAPT DNAse Inactivation Immunoaffinity

SEC: size exclusion chromatography IEC: ion exchange chromatography HAPT: hydroxylapatite DNAse: degradation by DNAse Inactivation: elimination of b i o l o g i c a l a c t i v i t y


14. DOVE ET A L


Clearance values were calculated by a r a t i o of concentrations: (Contaminants) p u r i f i c a t i o n step (IgM) = Clearance b

e

f

o

r

e

(Contaminants) i (IgM) For example, 99% removal of contaminants with 100% y i e l d generates a factor of lOOx. Contaminants included proteins, DNA, and viruses. a

f

t

e

r

p

u

r

i

f

i

c

a

t

o

n

s

t

e

p


Results and Discussion. P r e c i p i t a t i o n . C l a r i f i e d harvests were concentrated on 100,000 MW membrane, adjusted to s p e c i f i c pH and p r e c i p i t a t e d with PEG (Table 2, c e l l l i n e B). Low pH and high PEG concentration resulted i n the highest y i e l d o f IgM and the lowest p u r i t y . I t should be noted that, as the p u r i t y of the i n i t i a l m a t e r i a l increased, the y i e l d from p r e c i p i t a t i o n with PEG increased. S t a b i l i t y data substantiate t h i s observation; IgM Is s t a b i l i z e d i n s o l u t i o n by proteins (e.g. albumin). Further, IgM at a concentration of l e s s than 50 ug/ml p r e c i p i t a t e d poorly. P r e c i p i t a t i o n of high molecular weight aggregates with low concentrations of PEG were studied. 1% and 2% PEG produced only s l i g h t reductions of aggregates, as measured by FPLC-Superose 6. P r e c i p i t a t i o n with other agents and r e p r e c i p i t a t i o n o f an i n i t i a l p r e c i p i t a t i o n were examined, u t i l i z i n g ammmonium s u l f a t e , dextran sulfate, ethanol, and boric acid (Table 2). With the excep t i o n of ammonium s u l f a t e , these agents d i d not p r e c i p i t a t e IgM at the c o n c e n t r a t i o n s t e s t e d . R e p r e c i p i t a t i o n gave low y i e l d s and unremarkable p u r i t y with a l l agents. Size Exclusion Chromatography (Gel F i l t r a t i o n ) . I n i t i a l experiments with plasma-derived IgM demonstrated good separation of IgM from contaminants on Sephacryl S-300, an a c r y l i c based g e l . However, separation and y i e l d were poor with monoclonal IgMs. Sepharose CL6B, an agarose based g e l , produced e x c e l l e n t r e s o l u t i o n from albu min. A t y p i c a l a n a l y t i c a l chromatogram o f a PEG p r e c i p i t a t e on FPLC-Superose 6 (30 χ 1 cm. dia.) i s shown i n Figure 1, compared to a t y p i c a l l a r g e - s c a l e run (80 χ 37 cm. d i a . ) . The leading ( l e f t side) peak consisted of aggregates of IgM and albumin, the second ( l a r g e s t ) peak was IgM, and the t h i r d peak was p r i m a r i l y albumin with minor low molecular weight fragments. Clearance of DNA was approximately lOx. Yields were improved by high s a l t concentration, which reduced non-specific binding and increased s t a b i l i t y . Howev er, several workers have demonstrated improved separation with the use of both low and high ionic strength buffers (4j_ 18) . Degradation of DNA by DNAse. Endogenous or exogenous DNAses degrade the contaminant DNA by enzymatic cleavage, changing the s i z e and charge of the DNA and thereby a l t e r i n g the e f f i c i e n c y of the separa t i o n cascade between the product (IgM) and contaminant (DNA).


197


198

Table 2.


Agent

P r e c i p i t a t i o n by Various Agents

Concentration of agent (%)

pH

Yield (%)

Purity (% IgM)

Samples and agent were mixed f o r 1 hr. at 4 C. Line B, 200 ug/ml. PEG 10 PEG 8 PEG 5

7.4 7.4 7.4

84 75 20

80-95 95+ 95+

PEG PEG PEG PEG

5 5 10 10

5.5 7.4 5.5 7.4

66 10 95 86

95+ 98+ 80-95 80-95

E, 100 ug/ml, Sulfate 18 Sulfate 18 Sulfate 24 Sulfate 31

6.5 7.2 7.2 7.2

7 0 49 57

45

Line C, 100 ug/ml, Amm. Sulfate 24 PEG 12

7.2 5.5

79 96

80 60

39 80 0 71 2 0 0

85 80

Line Amm. Amm. Amm. Amm.

The PEG p r e c i p i t a t e of l i n e C was reprecipitated by each of the following: PEG 10 PEG 12 Amm. Sulfate 11 Amm. Sulfate 24 Dextran Sulfate 10 Ethanol 25 Boric Acid 5 5.0

Amm:

90 38

90

Ammonium




14. DOVE ET AL.

Fraction Figure 1. A. B.

Chromatograms of size exclusion (SEC). FPLC-Superose 6, 30 χ 1.0 cm. d i a . Sepharose CL-6B, 80 χ 37 cm. d i a . Load: p r e c i p i t a t e by PEG.


199


200


Exogenous DNAses were added to the p u r i f i c a t i o n processes to accelerate the e f f e c t of the endogenous enzymes, and degraded v i r t u a l l y a l l of the DNA q u i c k l y to allow separation. S p e c i f i c a l l y , i n p r e c i p i t a t i o n s or s i z e exclusion chromatography, reduction of the molecular weight of the contaminanting DNA improved separation from a high molecular weight product (e.g. IgM). Bovine pancreas DNAse was immobilized on an agarose matrix to characterize and demonstrate controlled degradation of a p u r i f i e d DNA preparation passed through a column of the matrix. The p u r i f i e d DNA p r e p a r a t i o n c o n s i s t e d i n i t i a l l y of molecular weight 1,000,000. Two column passes resulted in an approximate b e l l d i s t r i b u t i o n with a broad range of 1,000,000 to 10,000 daltons and median at molecular weight 100,000. Many passes r e s u l t e d i n a narrow range of approximately 10,000 daltons. DNA degradation was monitored by s i z e exclusion chromatography and SDS-PAGE (Figure 2). In a separate experiment, a harvest of IgM was passed through the DNAse column and then fractionated on FPLC-Superose 6. DNA clearance was 10,000x, compared to lOx without DNAse digestion (10). Anion Exchange. Anion exchange on DEAE-Sepharose was optimized f o r IgM p u r i f i c a t i o n from DNA. The f o l l o w i n g b u f f e r conditions were studied: T r i s , pH 8.0; phosphate, pH 6.5; sodium acetate, pH 6.5; and no buffer. E l u t i o n was achieved by a l i n e a r gradient of sodium chloride. Separation from other proteins was marginal, but removal of albumin was s l i g h t l y superior i n the phosphate buffer. The s a l t buffers gave comparable reduction i n DNA (10,000x), with the unbuffered system giving l,000x. To demonstrate removal of DNA more c l e a r l y , several preparations were bound to DEAE-Sepharose: a) a p a r t i a l l y p u r i f i e d IgM preparation, b) a p u r i f i e d preparation of DNA, and c) a combination of both. Preparations were bound i n 0.05 M T r i s , 0.05 M NaCl, pH 8.0 and eluted by l i n e a r gradient of sodium chloride. Referring to Figure 3A, IgM was recovered i n the f i r s t e l u t i o n peak at 0.15 M NaCl. The second and t h i r d e l u t i o n peaks at 0.2 and 0.34 M NaCl contained native DNA. Figure 3B shows e l u t i o n of the DNA preparat i o n at 0.3 and 0.35 M NaCl. Mixing the p u r i f i e d IgM and DNA preparations and repeating the elution yielded a precise superimposition of the two chromatograms (Figure 3C), i n d i c a t i n g the two e n t i t i e s eluted independently. T y p i c a l chromatograms are shown i n Figure 4, with e l u t i o n by l i n e a r gradient and step elution. A small amount of protein did not bind, i n d i c a t e d by the peak on the l e f t . A high s a l t s t r i p a f t e r e l u t i o n of the IgM produced a peak of s i m i l a r magnitude, which contained IgM, DNA, and albumin. A step e l u t i o n at 0.15 M NaCl gave DNA clearances l i s t e d i n Table 3. Native DNA was assayed by dot blot hybridization. Clearance studies with P32 labeled DNA gave s u b s t a n t i a l l y lower clearance factors. P32 labeled DNA derived from harvests of c e l l l i n e C gave approximately twice the clearance of v i r a l DNA. Other anion exchange resins gave comparable or poorer r e s u l t s for IgM. Use of Q-Sepharose resulted i n s l i g h t l y t i g h t e r binding of both IgM and DNA. Repetitive chromatography on DEAE did not r e s u l t in increased removal of DNA from IgM.


Purification Alternativesfor IgM Monoclonal Antibodies


DOVE ET AL.

1

5

1

5

Molecular Height (logs)


2

'3

1

. Si .


k

)

1

Figure 2. DNA degradation by immobilized DNAse. Chromatograms (FPLC-Superose 6) of: A. 0 passes across DNAse column(initial DNA prep.). B. 2 passes. C. 14 passes.



202


2

*

«H

Ί

s CO

«4

Ξ »•Ζ3 — g

30· 10Θ '

Figure 3. A.

Ιο h U Fraction

le 1β ίο k i

Protein Purification - American Chemical Society

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