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Optimization of a vancomycin purification process: or - how to develop a purification process in three easy steps.

With the introduction of penicillin during World War II, the use of antimicrobial agents has greatly reduced the death and suffering caused by infectious diseases around the world. However, the recent rise of resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), demonstrates the need for newer and more effective antibiotics. (1) The purpose of this work was to present a simple, economical process for the purification of an example antibiotic, vancomycin. Vancomycin is a branched tricyclic glycosylated nonribosomal peptide produced by the fermentation of Amycolatopsis orientalis, and it used in the treatment of infections caused by Gram-positive bacteria.

OBJECTIVES

The goal of this study was to develop a simple purification process consisting of two to three chromatography steps. Starting with a crude vancomycin broth that had an initial purity of 34%, the objective was to deliver a final vancomycin purity of at least 85% with greater than a 60% overall process yield. The first portion of the study examined the effect of mobile phase conditions on a reversed phase chromatography step. Capacity, yield and purity were used to measure the effectiveness of this step. The second portion of the study examined the use of a low cost, strongly basic anion exchange resin for decolorization of the crude vancomycin broth. Various resins were screened for their effectiveness in color removal as well as vancomycin recovery. The final portion of the study examined the combination of an initial color removal step with reversed phase chromatography purification. Overall process yield and purity were used to assess the effectiveness of this two-step process.

[ILLUSTRATION OMITTED]

LOW-LOAD REVERSED PHASE PURIFICATIONS

Low-load experiments were conducted using columns with two different reversed phase packing materials. One was a polystyrenic resin with a 35 micron particle size and a 150A mean pore size. The other column contained a silica packing with a 10 micron particle size and a 300A mean pore size. Both materials are intended for use in peptide purifications. Silica packings are generally operated under acidic conditions, and are not chemically stable under basic pH conditions. Polymeric packings provide the capability to operate within a much wider pH operating window.

As shown in Table 1, mobile phase conditions and pH had a dramatic effect on the yield and purity of vancomycin. The silica packing performed better than the polystyrenic resin at low pH. At basic pH, however, the polystyrenic resin performed better than the silica packing even with a larger particle size. Operating the purifications with ammonium acetate provided the additional benefit of a volatile salt and left the peptide in the desirable acetate form. Finally, dynamic capacities were highest at basic pH values. Therefore, due to the stability of the polystyrenic resins at high pH, all further work was performed with these resins.
Table 1 -Yield/Purity Result Under Low-load Conditions

Resin Conditions Total 90% 95%
 Recovery Purity Purity
 Recovery Recovery

35 micron 0.1% TFA, pH 2.0 100% 56% 0%
Polystyrenic Resin

35 micron 50mM tris, pH 96% 86% 69%
Polystyrenic Resin 8.0

35 micron 50mM acetate, pH 91% 80% 69%
Polystyrenic Resin 8.5

10 micron C4 Silica 0.1% TFA, pH 2.0 100% 75% 45%

10 micron C4 Silica 50mM tris, pH 98% 70% 26%
 8.0

10 micron C4 Silica 50mM acetate, pH 100% 76% 38%
 8.5

Sample: vancomycin broth, expressed from yeast and filtered with
0.7[micro]m filter

Loading: 2.5mg of vancomycin, influent purity of 34%

Column: 1.0cm I.D. x 25cm L, packing material listed in Table 1

Mobile Phase: A: listed in Table 1; B: acetonitrile

Gradient: 7.5% to 30% B in 150 minutes

Flow rate: 1mL/min

Detection: UV @ 280nm

Fractions: 1 mL


DYNAMIC CAPACITY MEASUREMENTS

Dynamic capacities for vancomycin were determined under various conditions using the 35 micron polystyrenic resin packed into lcm ID x 25cm L stainless steel columns. Capacity was determined using the 1% breakthrough point on the chromatogram and the total saturation capacity which was determined by comparing the UV adsorption of the original influent solution to the UV adsorption of the collected column effluent.

As shown in Table 2, pH has a profound impact on the dynamic binding of vancomycin. Basic pH conditions improve capacity by a factor of three- to four-fold over acidic conditions. Although not shown, capacity measurements with other packing materials, whether silica or polystyrenic, indicated the same binding differences. In addition, there does appear to be a slight effect of the mobile phase itself. Capacities obtained at pH 8 using Tris-HCl were slightly higher than those obtained using ammonium acetate at pH 8. This may be a function of the salt form of the vancomycin. Ammonium acetate may still be preferable as a buffering agent since a number of peptides are more stable in the acetate form and the volatility of the acetate buffer makes downstream processing easier. Finally, high concentrations of ammonium acetate do not appear to be necessary, as increasing the concentration of buffering salt did not appear to affect on the binding capacity.
TABLE 2 - Vancomycin Dynamic Binding Capacities

 Conditions 1% Capacity Total Capacity
 (mg/mL) (mg/mL)

 0.1% TFA, pH 2.0 20 21

50 mM Ammonium Acetate, pH 25 29
 5.0

50 mM Ammonium Acetate, pH 41 56
 7.0

50 mM Ammonium Acetate, pH 89 93
 8.0

 50mM Tris-HCI, pH 8.0 107 110

50mM Ammonium Acetate, pH 85 93
 8.5

100mM Ammonium Acetate, pH 91 94
 8.5

Sample: vancomycin hydrochloride, Sigma Aldrich V2002

Column: 35 micron polystyrenic resin, 1.0cm I.D. x 25cm L

Flow rate: 2 mL/min

Mobile Phase: listed on table + 7.5% acetonitrile

Detection: UV @ 280nm


PREPARATIVE-LOAD REVERSED PHASE PURIFICATIONS

A two-step preparative process using two different polystyrenic reversed phase resins was employed for the preparative-load purifications. The initial purification was performed on the 35 micron, 150A pore size resin, and this was followed by a polishing step using a reversed phase polystyrenic resin with a mean pore size of approximately 300A and a particle size of 10 micron.

The loading on the first column was 18.6mg/mL and the yield and purity were 79% and 82%, respectively. The initial purity of the feed was 34%. The effluent pool from the first column was then diluted 50/50 (v/v) with 50mM ammonium acetate in order to adjust the feed concentration so that the acetonitrile concentration was approximately 7.5%. This diluted pool was then loaded onto the second reversed phase column. This second step gave a 61% yield of 90% pure vancomycin for a total overall yield of 48%.

DECOLORIZATION EXPERIMENTS

Batch decolorization experiments were conducted with the crude filtered vancomycin broth by adding 1g (based on dry weight) of various anion exchange resins to 20mL of broth, and shaking the contents for four hours. The pH of the broth was adjusted by titrating with ammonium hydroxide. Color removal was assessed spectrophotometrically at 410nm, and vancomycin recovery was assayed via reversed phase HPLC.

Table 3 presents the percent color removal for the different resins and pH values, and Table 4 presents the corresponding vancomycin recovery for the different resins and pH values. All of the weak base anion exchange resins provided excellent decolorization. However, vancomycin recovery at all pH values with these resins was poor. One of the strong base anion exchange resins, strong base anion exchange resin #3, was determined to be the best candidates at near-neutral or neutral pHs for vancomycin broth decolorization
Table 3-Effect of pH on Percent Color Removal

Resin pH 2.5 pH 5.0 pH 7.0 pH 8.0 pH 8.5

Strong Base Anion Exchange 40% 68% 85% 85% 85%
Resin #1

Strong Base Anion Exchange 28% 58% 79% 80% 83%
Resin #2

Strong Base Anion Exchange 23% 48% 71% 72% 75%
Resin #3

Waek Base Anion Exchange 75% 83% 85% 84% 84%
Resin #1

Waek Base Anion Exchange 68% 71% 68% 65% 64%
Resin #2

Waek Base Anion Exchange 71% 75% 74% 70% 69%
Resin #3

Waek Base Anion Exchange 70% 76% 74% 71% 72%
Resin #4

Table 4 -Effect of pH on Vancomycin Recovery

Resin pH 2.5 pH 5.0 pH 7.0 pH 8.0 pH 8.5

Strong Base Anion Exchange 84% 79% 52% 23% 14%
Resin #1

Strong Base Anion Exchange 93% 91% 77% 57% 39%
Resin #2

Strong Base Anion Exchange 94% 92% 83% 70% 59%
Resin #3

Weak Base Anion Exchange 31% 26% 21% 20% 19%
Resin #1

Weak Base Anion Exchange 21% 30% 35% 36% 39%
Resin #2

Weak Base Anion Exchange 59% 53% 48% 48% 47%
Resin #3

Weak Base Anion Exchange 23% 26% 28% 30% 30%
Resin #4


DECOLORIZATION CYCLING EXPERIMENTS

A sample of the strong base anion exchange resin #3 was subjected to ten decolorization cycles by weighing out 10g (dry weight) of resin into 200mL of crude filtered vancomycin broth and shaking the contents for two hours on an orbital shaker. The resin was then recovered from the treated broth, and the treated broth was assayed at 410nm for color removal determination and by reversed phase HPLC to determine vancomycin recovery. The recovered resin was then weighed back into a bottle and a fresh 200mL of crude filtered vancomycin broth was added and shaken for two hours. This was repeated for a total of ten cycles. The color removal was still fairly effective after ten cycles, dropping from an initial value of 66% removal to 46% removal at the tenth cycle. Vancomycin recovery remained good throughout the ten cycles, starting at 86% and ending at 90% on the tenth cycle. It must be emphasized that the resin was not regenerated in any way during this cycling study. However, this strong base anion exchange resin can be effectively regenerated with 0.1N HCl and then re-equilibrated with a 10% NaCl solution in 0.5% NaOH.

OPTIMIZED PREPARATIVE-LOAD TWO-STEP PURIFICATIONS

Based on the decolorization results, several further preparative purifications were conducted at loading levels of 20-25g/L. with the incorporation of a decolorization step using the strong base anion exchange resin selected in the last section. Initially the purifications were done with no further pH adjustment of the vancomycin broth after decolorization. The results indicated that reversed purifications conducted at pH 5.0 and pH 7.0 provided relatively low purities (<75%). However, if the pH of the broth was adjusted to a more basic pH of 8.5, the purity levels improved (>85%). During the course of these purifications, it also became apparent that the use of an upstream decolorization step was improving the column lifetime of the reversed phase resins. There was less column fouling, as evidenced by diminished increases in column backpressure over subsequent purifications. Therefore, column cleaning cycles could be reduced. Additionally, due to the improvement in broth color and purity from the decolorization step, a more economical reversed phase resin could be substituted for 35 micron resin. Target purity levels could be still be achieved with a single reversed step using a larger particle size resin with the same porosity. Results are summarized in Table 5.
Table 5 - Optimized Preparative-Load Purifications

Initial Reversed Final RPC Overall Process Yield
Decolorization Phase Purity Yield

Step Purification
Broth decolored 35 micron 70% 68% 62%
with strong Polystyrenic
base anion Resin,
exchange resin purification
at pH 5.0 RPC done at pH
purification 5.0
run at pH 5.0
Broth decolored 75 micron 87% 71% 65%
with strong Polystyrenic
base anion Resin,
exchange resin purification
at pH 5.0 then done at pH
adjusted to 8.5
8.5


CONCLUSIONS

* High pH mobile phase conditions improved the yield and purity of vancomycin, particularly on the polystyrenic resins. Additionally, high pH conditions increased the binding capacity of vancomycin to reversed phase packing materials.

* An effective, two-step reversed process using polystyrenic resins was developed for the purification of crude vancomycin. An overall yield of 48% with 90% purity was achieved.

* Decolorization of crude filtered vancomycin using a strong base anion exchange resin improved the purification of the vancomycin with subsequent reversed phase chromatography. The decolorization also diminished fouling of the reversed phase packing material, and allowed an effective single reversed phase step to achieve purity levels of 87% with an overall process yield of 65%.

REFERENCES

(1.) WHO, Fact Sheet Number 194, "Antimicrobial Resistance" (January 2002).

* By J. Fisher, Dow Chemical Company, Spring House, PA
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Title Annotation:PURIFICATION
Author:Fisher, J.
Publication:Pharmaceutical Processing
Date:Jun 1, 2010
Words:2065
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