Process design and scale-up of counter-current chromatography for the fractionation and recovery of polyketide antibiotics

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UCL

BIOCHEMICAL ENGINEERING

Process Design and Scale-up of

Counter-Current Chromatography for the

Fractionation and Recovery of

Polyketide Antibiotics

A thesis submitted to the University of London

for the degree of

Doctor of Philosophy

by

Andrew Jason Booth B.Sc (Hons)

Department of Biochemical Engineering, University College London,

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Abstract

R egulatory constraints on products o f the m odern biotechnology industry require final

dosage forms to exhibit high and consistent levels o f purity. A t the sam e tim e, financial

constraints on process developm ent require cost-effective purification m ethods to be

found. Counter-current chrom atography (CGC) is a liquid-liquid chrom atographic

technique, in w hich solutes are fractionated based on their selective partitioning

betw een two im m iscible liquid phases. The absence o f a solid support, as in

conventional H PLC, overcom es problem s o f irreversible adsorption and pore diffusion,

and ensures that CGC is a low pressure operation. A lthough an established analytical

scale technique, w idespread use o f GGG has been ham pered by the lack o f generic and

robust m ethod developm ent strategies and w ell-engineered industrial scale machines.

This project has exam ined the operation and scale-up o f novel J-type GGG

devices for application as a generic and scalable high-resolution purification technique.

The fractionation o f the chem ical pharm aceutical erythrom ycin A (EA) from its

structurally sim ilar analogues was used as a test system since this provides a difficult

and realistic separation challenge. Initial research addressed the need for a generic

m ethod developm ent strategy to increase the speed o f GGG phase system selection and

identification o f the optim al run mode. For the purification o f EA, a broad polarity

quaternary phase system consisting o f hexane/ethyl acetate/m ethanol/w ater was

identified. Results from a m atrix o f simple equilibrium partition experim ents w ere used

to identify a suitable solvent system for use firstly in gradient elution mode. Based on

these results, an optim ised reverse phase isocratic separation was then selected w hich

enabled the separation o f EA from all o f its closely related biosynthetic analogues.

Subsequent optim isation studies, using a m odel erythrom ycin m ixture,

addressed the im pact o f solute loading and m obile phase flow rate on EA purity, colum n

efficiency and throughput in a laboratory scale J-type GGG device. U nder optim al

conditions (8 m E.m in"'; 0.6 g solute loading) a m axim um throughput o f 0.097 kg.day'^

could be achieved, w ith an EA purity and yield o f 97% (w/w) and 100% (w/w)

respectively. Further research focused on the feasibility o f using GGG for the

purification o f EA from real Saccharopolyspora erythraea ferm entation broths. Studies

here exam ined the degree o f pre-purification required prior to GGG separation. These

used feeds consisting o f either clarified broth or solvent extracts having undergone

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rem oval required to ensure a reproducible elution profile o f EA. Further studies using a

back extracted feed stream exam ined the effects o f CCC m obile phase flow and solute

loading on the attainable EA purity and yield. The results in all cases dem onstrated a

high attainable EA purity (>97% w/w).

The results for both m odel and real systems w ere subsequently scaled-up using a

novel, pilot scale CCC machine. From an understanding o f the phase system

hydrodynam ics, a predictive scale-up m odel o f the separation was established, to

describe how solute fractionation at the pilot scale varied w ith changes in operating

variables, such as feed type, m obile phase flow rate and solute loading. Linear scale-up

was successfully dem onstrated w ith both m odel and real erythrom ycin feed stream s,

based on the param eters taken from a single laboratory scale CCC chrom atogram .

Scale-up predictions w ere accurate to w ithin 5-13% (m odel system ) and 6-10% (real

system) depending on the actual operating conditions.

Finally, this research explored the successful application o f ‘Fractionation

D iagram theory’ as a graphical tool to allow quantification o f the trad e-o ff betw een

product purity and solute yield in CCC separations. Com bined w ith a new generation o f

robust industrial scale m achines currently under construction, this w ork has

dem onstrated the potential o f CCC as a generic and flexible high-resolution separation

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Acknowledgements

First and forem ost I w ould like to express m y gratitude to m y supervisor, D r G ary Lye

for providing m e w ith this opportunity to read for a PhD in Bioehem ieal Engineering,

and for his continual and invaluable support, guidance, and encouragem ent throughout

m y studies.

I w ould like to thank Dr Les B row n o f AECS Ltd for his greatly appreciated assistance

in m ethod developm ent and initial fam iliarisation o f the CCC m achine used during this

study. M y thanks also goes out to the research team at the Brunei Institute for

Bioengineering for their continued support and expert tutoring on the operation and

m aintenance o f the Brunei CCC m achine (the fitting o f the quieter m otor couldn’t have

com e sooner! ! I) and for the loan o f the pilot scale coils.

W ithin the D epartm ent o f Biochem ical Engineering, I w ould firstly like to thank Drs

A ndreas Stein, Frank Baganz, and A ngela Scholtzovà for their support regarding H PLC

analysis, and Billy D oyle for his technical assistance w ith the ferm entations. W ith

regards the application o f the Fractionation diagram theory and subsequent result

analysis, I w ould like to express m y deepest thank to Dr Sheau-H uey N giam . To all my

friends in the departm ent and the Tads’ back hom e, especially Stu, thank you all for

your friendship and support!

Finally, to my parents, sis and Donna, thank you all so m uch for your love and support

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Table of Contents

1. IN T R O D U C T IO N ... 1

1.1. T a x o n o m y ...1

1.1.1. A ctinom ycetes... 1

1.1.2. Streptom yces... 1

1.1.3. Saccharopolyspora eryth ra ea...2

1.1.4. Biosynthesis o f both natural and recom binant polyke tid es... 3

1.1.5. M acro tide A n tib io tics... 6

1.1.6. E rythrom ycin... 6

1.1.6.1. The structure o f erythrom ycin... 6

1.1.6.2. Properties o f erythrom ycin and its mode o f action... 8

1.1.6.3. Clinical uses o f eryth rom ycin...10

1.1.6.4. Industrial production o f erythrom ycin... 10

1.1.6.4.1. F erm entation... 10

1.1.6.5. Prim ary reco ve ry... 12

1.1.6.6. P u rifica tio n...13

1.1.6.7. D ry in g...14

1.2. T heoretical aspects and the developm ent o f C C C ...14

1.2.1. Intro duction...14

1.2.2. Phase distribution within a spirally w ound coil...16

1.2.3. H ydrodynam ic phase distribution within a flo w through coil... 17

1.2.4. M echanism o f chrom atography in the J-Type CC C m a c h in e...20

1.2.3.1. D eterm ination o f optimal elution m o d e...22

1.3. H istory o f CCC d ev elop m en t... 24

1.4. A pplications of C C C ... 33

1.5. Project A im s... 35

2. M A TER IA LS AND M E T H O D S ...37

2.1. C hem icals... 37

2.1.1. F erm entation com p on ents... 37

2.1.2. Chem icals used in other unit operations and a s s a y s ...38

2.1.2.1. M icro filtra tio n...38

2.1.2.2. Solvent extraction o f clarified ferm entation broth...38

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2.1.2.5. E ryth ro m ycin...39

2.2. Production and partial purification of broth-derived eryth rom ycin ... 39

2.2.1. Strain and culture sto rage...39

2.2.2. Ferm enter design and auxiliary eq u ip m en t... 40

2.2.3. Ferm enter o p eratio n ...41

2.2.3.1. D ry cell w eight d eterm in ation... 42

2.2.4. D ownstream p ro c e ssin g ...42

2.2.4.1. Broth clarification by cross-flow m icrofiltration...42

2.2.4.2. Erythrom ycin isolation by solvent extra ctio n... 43

2.3. CCC instrum entation and auxiliary eq u ip m en t... 44

2.3.1. Laboratory scale CCC in stru m en t... 44

2.3.2. Pilot-scale CCC instrum ent...47

2.3.3. Com parison betw een laboratory and pilot scale CCC d e v ic e s ...48

2.3.4. D etection M e th o d s...49

2.3.4.1. UV-visible absorbance detector... 49

2.3.4.2. Evaporative L ight Scattering D etecto r... 50

2.3.4.3. D ata capture and construction o f CC C chrom atogram s... 50

2.3.4.4. Off-line fra ctio n a n a ly sis... 51

2.3.4.4.1. Quantitative determ ination o f total erythrom ycin concentration 51 2.3.4.4.2. H PLC analysis o f CCC fr a c tio n...51

2.4. CCC setup and op eration ...52

2.4.1. Laboratory seale setup and operation...52

2.4.1.1. Bobbin balancing and ro ta tio n...52

2 .4.1.2. Sam ple preparation and injection... 53

2.4.2. Pilot seale setup and op eration... 55

2.4.2.1. Bobbin balancing a nd ro ta tio n... 55

2 .4.2.2. Sample preparation and injection... 55

2.5. M ethod developm ent and CCC op eration ...55

2.5.1. M ethod developm ent...55

3. C H R O M A T O G R A PH Y AND FR A C T IO N A T IO N D IA G R A M T H E O R Y ... 59

3.1. C hrom atographic param eters for C C C ...59

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3.1.4. Separation f a e to r ... 63

3.1.5. R esolution... 63

3.1.6. Colum n efficien cy ... 64

3.1.7. T hrou gh pu t...65

3.2. CCC scale-up th eo ry ...65

3.2.1. Pilot scale predictions based on a laboratory scale separation...66

3.2.2. Prediction o f peak elution tim es w ith increased injection volum e...67

3.3. Fractionation diagram th eo ry ...68

3.3.1. Construction o f fractionation d iag ram s...69

3.3.2. C onstruction o f purification factor versus yield d ia g ra m s...69

3.4. Perform ance m easures in chrom atography (the V an deem ter equation)... 73

4. M ETH O D D E V E L O P M E N T ... 76

4.1. Solvent system developm ent... 76

4.1.1. Equilibrium distribution ratio studies...77

4.1.2. Solvent polarity indices... 78

4.1.3. D eterm ination o f optimal CCC operating m o d e ... 83

4.1.3.1. G radient elution o f erythrom ycin... 83

4.1.3.2. Isocratic elution o f erythrom ycin... 85

4.2. Sum m ary...89

5. LIQ U ID -L IQ U ID H Y D R O DY N AM IC S IN J-TY PE CCC M A C H IN E S ... 90

5.1. Introduction... 90

5.2. Phase hydrodynam ics in the laboratory scale m ach in e... 91

5.2.1. R etention data and Du p lo ts... 91

5.3. Phase hydrodynam ics in the pilot scale m a ch in e...99

5.4. Sum m ary o f retention studies...101

6. L A B O R A T O R Y SCALE O P T IM ISA T IO N ...102

6.1. Introduction...102

6.2. V ariation o f m obile phase flow ra te ... 102

6.2.1. Effect on chrom atographic retention f a c to rs ... 106

6.2.2. Effect on chrom atographic p erfo rm a n ce ... I l l 6.2.3. Effect on yield, purity and throughput...114

6.3. V ariation o f solute lo ad in g ... 114

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6.3.2. Effect on chrom atographic p erfo rm an ce... 124

6.3.3. Effect on product yield, purity and throughput...127

6.4. O ptim isation o f CCC op eration ... 128

6.5. S u m m ary... 129

7. LA B O R A T O R Y SCALE FR A CTIO N A TIO N OF FE R M E N T A T IO N BR O TH D ERIV ED ER Y T H R O M Y C IN S... 130

7.1. In trod u ction ... 130

7.2. Ferm entation characterisation... 130

7.3. A pplication o f an Evaporating L ight Scattering D e te c to r ... 132

7.4. Effect o f degree o f pre-purification on erythrom ycin sep aration 132 7.4.1. Effect on solute reten tion...132

7.4.2. Effect on chrom atographic p erfo rm an ce... 137

7.5. Influence o f repeated sam ple lo a d in g ...138

7.5.1. Effect on solute retention...138

7.5.2. Effect on chrom atographic p erfo rm an ce... 143

7.6. Effects o f operating conditions o f erythrom ycin fractionation and th ro u g h p u t... 144

7.6.1. Effect o f m obile phase flow rate on solute re te n tio n ... 144

7.6.2. Effect o f m obile phase flow rate on chrom atographic perform ance 147 7.6.3. Effect o f solute loading on solute reten tio n ...149

7.6.4. Effect on chrom atographic p erfo rm an ce... 151

7.7. EA purity, yield and th ro u g h p u t...152

7.8. S u m m ary... 154

8. PR ED IC TIVE SCALE-UP OF CCC S E P A R A T IO N S ... 155

8.1. In trodu ction ... 155

8.2. Scale-up o f m odel system sep aration... 156

8.2.1 Effect o f operating conditions on pilot scale CCC p erfo rm an ce 160 8.2.1.1. Effect on both solute retention time and fa c to r s... 163

8.2.1.2. Effect on chrom atographic p erfo rm a n ce... 164

8.2.1.3. Effect on yield, EA pu rity and throughput...165

8.3. Real system scale-up...166

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8.3.1.2. ^ ff^ c ts on chrom atographic p e rfo rm a n c e... 172

8.3.1.3. Effects on yield, EA p u rity and throughput... 173

8.4. C o m p a riso n o f m odel a n d re a l system p e r f o r m a n c e ...173

8.4.1. Com parison o f chrom atographic retention factors and perform ance 173 8.5. P red ictiv e scale-u p o f C C C s e p a r a tio n s ... 177

8.5.1. Predictive scale-up o f the m odel system ...178

8.5.1.1. Laboratory to p ilo t scale predictions... 178

8.5.1.2. Predictions at the p ilo t scale with increasing operating conditions 179 8.5.2. Predictive scale-up o f the real system ... 182

8.5.2.1. Laboratory to p ilo t scale p redictions... 183

8.5.2.2. Predictions at the p ilo t scale with increasing operating conditions 183 8.5.3. Com parison between m odel and real system s p re d ic tio n s ... 186

8.6. S u m m a r y ... 187

9. A N A L Y SIS O F P R O D U C T P U R IT Y AND Y IE L D T R A D E -O F F S F O R C C C S E P A R A T IO N S ...188

9.1. I n tr o d u c tio n ... 188

9.2. Effects o f feed ty pe a t th e la b o ra to ry C C C sc a le ...189

9.3. Effects o f o p e ra tin g v a ria b le s a t th e la b o ra to ry C C C s c a le ...195

9.3.1. Effects o f increasing m obile phase flow ra te ... 195

9.3.2. Effects o f increasing solute loading...197

9.4. Use o f PFmax v ersu s yield d ia g ra m s in o p tim isin g E A p u r it y ...199

9.5. A p p lic atio n o f fra c tio n a tio n d ia g ra m th e o ry a t th e p ilo t s c a le ...200

9.5.1. Effects o f increasing m obile phase flow ra te ...200

9.5.2. Effects o f increasing solute loadin g ... 202

9.6. C o m p a riso n b etw een la b o ra to ry a n d p ilo t scale C C C ...204

9.7. S u m m a r y ...206

10. C O M P A R IS O N O F C C C AND H P L C ...207

10.1. I n tr o d u c tio n ... 207

10.2. P rocess b en efits a n d d ra w b a c k s of C C C ... 207

10.3. E x p e rim e n ta l a n d econom ic co m p ariso n s b etw een C C C a n d H P L C ... 208

10.4. P e rfo rm a n c e co m p ariso n s betw een C C C a n d H P L C ... 209

10.5. T o w ard s process scale s e p a r a tio n s ...214

11. C O N C L U S IO N S AND F U T U R E W O R K ... 216

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11.2. Future W o r k ...218

12. R E F E R E N C E S ... 220

13. A P P E N D IC E S ... 235

13.1. C alibration cu rv es...235

13.1.1. C olorim etric calibration c u rv e s ... 23 5 13.1.2. H PLC calibration curves...236

13.2. C alculation o f R ohrschneider solvent system p o la r ity ...238

13.3. Stationary phase retention d a ta ... 239

13.4. M ass balance d a ta ... 240

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List of Figures

Figure D escription Page

num ber num ber

C H A PTER 1

1-1 M odular PKS gene cluster and enzym e assembly. 4 1 -2 PKS tem plate catalysing the biosynthesis o f the erythrom ycin 5

aglycone.

1-3 Recom binant polyketide (triketide lactone). 5

1 -4 Structure o f erythrom ycin. 7

1-5 Biosynthesis o f various form s o f erythrom ycin analogues. 9 1 -6 A rchim edean screw principle in a helical coil. 16 1-7 V ariations in the distribution o f heavy and light phases w ithin 17

a helically w ound coil over a range o f rotational speeds.

1 -8 V ariation in coil orientations using the J-type configuration. 18 1-9 Synchronous planetary m otion o f the I and J-type CCC 19

configurations.

1-10 V ector m aps o f generated centrifugal force fields during coil 19 rotation in the I and J-type CCC configurations.

1-11 Coil m otion in the J-type CCC machine. 21 ] -12 M ixing and settling zones in a spirally w ound coil m ounted in 21

the J-type configuration.

1-13 (A) Jantzen CCD apparatus (B) C ountercurrent distribution 24 centrifuge.

] -14 The counter current extractor (CCE). 25 1-15 The rotating locular CCC (RLCCC) device. 25 1-16 D roplet CCC device illustrating configuration for using lighter 27

and heavier phase as m obile phase.

1-17 The vertical flow through CPC device (VFTCPC). 29 1-18 The horizontal flow through CPC device (HFTCPC). 30

1-19 The toroidal CCC device. 31

1-19 The m ultilayer CCC device. 32

C H A PTER 2

2-1 The Brunei CCC instrum ent illustrating the m ounting position 44 o f the two bobbins.

2-2 Coil w inding illustrating the entrance and exit o f the flying 45 lead arrangem ents.

2-3 B obbin m ounting betw een two rotor plates together w ith the 46 novel flying lead arrangem ent.

2-4 Photograph o f the B runei pilot-scale CCC m achine. 47 2-5 CCC operating conditions for reverse phase m ethanol 58

gradient.

C H A PTER 3

3-1 Schem atic representation o f a CCC chrom atogram illustrating 61 the relationship betw een the solvent front, elution o f a sample

and the system volum e.

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num ber 3-3 4-1 4-2 4-3 4-4 4-5

Schem atic representation o f a PF versus yield diagram.

CH A PTER 4

R elationship betw een percentage aqueous m ethanol content in the CCC m obile phase and erythrom ycin distribution ratio. G radient elution CCC chrom atogram

A nalytical reverse phase HPLC chrom atogram o f a com m ercial erythrom ycin preparation.

Isocratic CCC chromatogram .

H PLC chrom atogram s o f fractions collected at 2 m inute intervals from isocratic CCC run.

num ber 72 83 84 86 87

CH A PTER 5

5-1 D u plot o f percentage stationary phase retention against 94 square root o f m obile phase flow rate at the laboratory CCC

scale.

5-2 D u plot o f percentage stationary phase retention against 96 square root o f m obile phase flow for range o f PTFE coils at

the laboratory CCC scale.

5-3 D u plot o f percentage stationary phase retention against 97 square root o f m obile phase flow for range o f stainless steel

coils at the laboratory CCC scale.

5-4 D u plot o f percentage stationary phase retention against 100 square root o f m obile phase flow at the pilot CCC scale.

CHA PTER 6

6-1 CCC chrom atogram s from experim ents perform ed at m obile 103 phase flow rates 4 to 10 m L .m in '\

6-2 Effect o f m obile phase flow rate on EA distribution ratio. 107 6-3 R elationship betw een linear m obile phase flow rate and 108

erythrom ycin distribution ratios.

6-4 R elationship between stationary phase retention and 110 erythrom ycin retention factors.

6-5 R elationship betw een m obile phase flow rate and colum n 111 efficiency.

6-6 Relationship betw een stationary phase retention and colum n 112 efficiency.

6-7 CCC chrom atogram s from solute loading experim ents w ith a 116 1 mL injection volume.

6-8 CCC chrom atogram s from solute loading experim ents w ith a 117 2 m L injection volume.

6-9 Effect o f solute loading on distribution ratios o f 122 erythrom ycins.

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number number

6-13 CCC chrom atogram o f optim ised erythrom ycin fractionation. 129

CH A PTER 7

7-1 G rowth profiles from S.erythraea ferm entation on a soluble 131 com plex medium.

7-2 Laboratory scale CCC chrom atogram o f a crude erythrom ycin 133 fractionation using the ELSD.

7-3 Laboratory scale CCC chrom atogram s o f erythrom ycin w ith 134 (a) clarified broth, (b) forw ard extract, (c) back extract.

7-4 CCC chrom atogram s showing the effect o f repeated feed 139 injections using the forward extract feed on erythrom ycin A

retention.

7-5 CCC chrom atogram s showing the effect o f repeated feed 141 injections using the back extract feed on erythrom ycin A

retention.

7-6 CCC chrom atogram s from increased m obile phase flow rate 145 experim ents.

7-7 CCC chrom atogram s from increased solute loading m ass 150 experim ents.

C H A PTER 8

8-1 Linear scale-up o f the laboratory scale separation (Figure 4-4) 158 to the pilot scale CCC machine.

8-2 A nalytical HPLC chrom atogram s o f selected fractions from 159 the pilot scale CCC fractionation o f erythrom ycin.

8-3 (a) Pilot scale CCC chrom atogram o f erythrom ycin 161 fractionation at 20 m L.m in’’; 1 gram loading, (b) P ilot scale

CCC chrom atogram o f erythrom ycin fractionation at 20 m L .m in '’; 10 gram loading.

8-4 (a) Pilot scale CCC chrom atogram o f erythrom ycin 162 fractionation at 10 m L .m in ''; 1 gram loading (co = 1200 rpm),

(b) Pilot scale CCC chrom atogram o f erythrom ycin fractionation at 40 m L .m in '\ 1 gram loading (m = 1200 rpm)

8-5 Pilot scale CCC chrom atogram o f erythrom ycin fractionation 168 w ith back extracted feed at 10 m L.m in’^

8-6 (a) Pilot scale CCC chrom atogram o f erythrom ycin 170 fractionation at 20 mL.min*’, 1 gram solute loading mass, (b)

Pilot scale CCC chrom atogram o f erythrom ycin fractionation at 40 mL.min'% 1 gram solute loading mass.

8-7 Pilot scale CCC chrom atogram o f erythrom ycin fractionation 171 at 20 m L .m in '\ 10 grams solute loading mass.

8-8 (a) C om parison o f a betw een pilot scale studies w ith the 176 m odel and real systems, (b) Com parison o f Rg betw een m odel

and real system pilot scale studies.

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number number

8-10 Laboratory scale CCC chrom atogram illustrating the KA-start 178 and KA-end points used for scale-up predictions.

8-11 Pilot scale CCC chrom atogram illustrating the effects o f 181 increased injection volum e (V,) on m odel erythrom ycin

separation.

8-12 Pilot seale CCC chrom atogram illustrating the effects o f 185 increased injection volum e (V,) on erythrom ycin separation

w ith the back extract feed.

8-13 Parity plot o f m easured and predicted pilot seale elution tim es 186 for both m odel and real system experim ents.

CH A PTER 9

9-1 Fractionation diagrams illustrating the purification o f 189 Erythrom ycin A w ith spiked clarified broth, forw ard and back

extract feeds.

9-2 M atLab generated PF versus yield diagram for Erythrom ycin 191 A purification from a back extract feed at the laboratory CCC

scale

9-3 M axim um PF versus yield diagram illustrating the 191 com parisons betw een spiked clarified broth, back and forw ard

extract feeds for Erythrom ycin A purification using the laboratory scale CCC.

9-4 Laboratory scale CCC chrom atogram from back extract feed 194 separation illustrating the cut points to achieve required

Erythrom ycin A purity and yield.

9-5 Fractionation diagrams illustrating the purification o f 196 Erythrom ycin A from the 2 and 5 mL.min"^ m obile phase flow

rate experim ents w ith the back extract feed.

9-6 M axim um PF versus yield diagram illustrating the com parison 196 betw een 2 and 5 m L .m in'’ for Erythrom ycin A purification

using the laboratory scale CCC.

9-7 Fractionation diagram s illustrating the purification o f 197 Erythrom ycin A from the 100, 500 and 1000 m g experim ents

w ith the back extract feed.

9-8 M axim um PF versus yield diagram s illustrating the 198 com parisons betw een the 100, 500 and 1000 m g experim ents

for the Erythrom ycin A purification using the laboratory scale

CCC.

9-9 Fractionation diagram s illustrating the purification o f 200 Erythrom ycin A for the 10, 20 and 40 m E .m in’’ experim ents

w ith the back extract feed using the pilot scale CCC.

9-10 M axim um PF versus yield diagram s illustrating the 201 com parisons betw een the 10, 20 and 40 mL.min'* for

Erythrom ycin A purification using the pilot scale CCC.

9-11 Fractionation diagram s illustrating the purification o f 202 Erythrom ycin A for the 10 and 100 m L solute loading volum e

experim ents w ith the back extract feed using the pilot seale

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number number

9-12 M axim um PF versus yield diagram s illustrating the 203 com parisons between 10 and 100 m L solute loading volum e

experim ents using the back extract feed for Erythrom ycin A purification using the pilot scale CCC.

9-13 (a) Fractionation diagrams, (b) M axim um PF versus yield 205 diagrams; illustrating the com parisons betw een a laboratory

and pilot scale CCC for purification o f Erythrom ycin A using back extract feed.

C H A PTER 10

10-1 V an deem ter plots for determ ining optim al m obile phase flow 213 rate.

CHA PTER 13

13-1 Typical calibration curve for quantification o f total 235 erythrom ycin using the colorim etric sulphuric acid assay for

use in equilibrium distribution studies.

13-2 Typical calibration curve for quantification o f total 236 erythrom ycin using the colorim etric sulphuric acid assay for

use in CCC studies.

13-3 Typical calibration curves for quantification and identification 237 o f the various erythrom ycins using the H PLC assay.

13-4 Representative laboratory scale CCC chrom atogram o f a 244 crude erythrom ycin fractionation using the ELSD for use in

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List of Tables

T able D escription Page

num ber num ber

C H A PTER 1

1-1 A ntibiotics produced by Streptomyces. 2 1-2 Structural variations o f the erythrom ycins. 8 1-3 Sources o f nutrient requirem ents for the ferm entation. 11 1-4 Typical constituents o f a harvested antibiotic ferm entation

broth.

11

1-5 A ntibiotics fractionated by CCC. 34

CHA PTER 2

2-1 Com position o f the soluble com plex m edium (SCM ) used. 37 2-2 D im ensions o f the 20 L ferm enter used during this study. 40 2-3 D im ensions o f both the inner and outer PTFE coils on bobbins 45

7/1 and 7/2 together with their respective B eta ratio ranges.

2-4 D im ensions o f both the inner and outer stainless steel coils on 46 bobbins 6/1 and 6/2 together w ith their respective B eta ratio

ranges.

2-5 D irect com parison between laboratory and pilot scale CCC 48 devices used.

2-6 Step-wise procedure for bobbin balancing and determ ination 54 o f S /in the Brunei J-type counter-current chrom atograph.

2-7 D istribution ratio experiments perform ed w ith the biphasic 56 solvent system used.

2-8 N orm al and reverse phase CCC gradient options. 57 2-9 Solvent phase com positions used to perform a reverse phase 58

m ethanol gradient.

CHA PTER 4

4-1 V ariation in equilibrium erythrom ycin distribution ratio w ith 78 phase system com position and pH.

4-2 Rohrschneider solvent polarity param eters (P ) for the pure 79 solvents used in this study.

4-3 V ariation in quaternary solvent system polarity (P ') w ith 80 variation in volum etric fraction o f hexane, ethyl acetate,

m ethanol and water.

4-4 Stage 2 screening study results. 80 4-5 Stage 2 screening study results presented in pseudo-tw o- 82

com ponent notation, signifying the percentage change in aqueous methanol.

C HA PTER 5

5 -1 List o f hydrodynam ic experiments perform ed. 91 5-2 Exam ple o f raw data from a retention study perform ed on the 92

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5-4 Corrected m obile phase flow rates together w ith the square 95 root o f m obile phase flow and S/

5-5 Regression analysis between S /a n d the square root o f m obile 98 phase flow rate.

5-6 Corrected m obile phase flow rates together w ith the square 100 root o f m obile phase flow and S/ for the pilot scale CCC

machine.

5-7 Regression analysis between S /a n d the square root o f m obile 101 phase flow rate for the pilot scale CCC machine.

C H A PTER 6

6-1 S /results for flow rate experim ents perform ed betw een 2 and 104 10 m L .m in ''.

6-2 Identification and quantification o f the various forms o f 104 erythrom ycins from isocratic CCC experim ents.

6-3 Com parison o f EA elution tim es determ ined from off-line 105 HPLC and directly from CCC chromatogram s.

6-4 V ariation in linear m obile phase velocity w ith m obile phase 106 flow rate and m obile phase volum e at hydrodynam ic

equilibrium.

6-5 V ariation in distribution ratio (K) and retention factor (k) o f 107 Erythrom ycin A w ith m obile phase flow rate.

6-6 V ariation in K and k o f Erythrom ycins C/D and B/psu-EEA 108 w ith mobile phase flow rate.

6-7 Regression analysis betw een distribution ratio o f the 109 erythrom ycins and linear m obile phase velocity.

6-8 Regression analysis betw een the retention factor o f the 110 erythrom ycins and S/.

6-9 V ariation in the separation factor w ith m obile phase flow rate. 113 6-10 V ariation in perform ance param eters w ith m obile phase flow 114

rate.

6-11 Identification and quantification o f various form s o f the 118 erythrom ycins from isocratic CCC experim ents perform ed

w ith a 1 mL injection volume.

6-12 Identification and quantification o f various form s o f the 118 erythrom ycins from isocratic CCC experim ents perform ed

with a 2 mL injection volume.

6-13 Com parison o f EA elution tim es determ ined from off-line 119 HPLC analysis and directly from CCC chrom atogram s.

6-14 V ariation in K o f EA, EC/ED and EB/psu-EEA w ith solute 120 loading.

6-15 V ariation in k o f EA, EC/ED and EB/psu-EEA w ith solute 121 loading.

6-16 V ariation in the k o f EA w ith solute loading based on the 123 retention tim es determ ined directly from the corresponding

CCC chrom atogram s and off-line HPLC analysis.

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CH A PTER 7

7-1 V ariation in the K and k factor o f EA w ith feed type. 136 7-2 V ariation in K and k o f EC/ED and EB/psu-EEA w ith feed 136

type.

7-3 V ariation in the separation factors w ith feed type. 138 7-4 Identification and quantification o f fractions corresponding to 140

the various forms o f erythrom ycins from repeated feed injection experim ents using forw ard extract feed.

7-5 Identification and quantification o f fractions corresponding to 142 the various forms o f erythrom ycins from repeated feed

injection experim ents using back extract feed.

7-6 V ariation in K and k o f EA, EC/ED and EB /psu-EEA w ith 142 repeated injections o f the forward and back extract feeds.

7-7 V ariation in the separation factor and resolution w ith repeated 143 feed injections o f the forward and back extract feeds.

7-8 V ariation in K and k o f EA, EC/ED and EB /psu-EEA w ith 146 increasing m obile phase flow rate using the back extract feed.

7-9 V ariation in the separation factor and resolution w ith 148 increasing m obile phase flow rate using the back extract feed.

7-10 Identification and quantification o f fractions corresponding to 148 the various forms o f erythrom ycins from corresponding flow

rate experim ents using the back extract feed.

7-11 V ariation in K and k o f EA, EC/ED and EB /psu-EEA w ith 149 increasing solute loading using the back extract feed.

7-12 V ariation in the separation factor and resolution w ith solute 152 loading using the back extract feed.

7-13 V ariation in the perform ance param eters w ith operational 153 variables.

C H A PTER 8

8-] Operating conditions for optim isation experim ents perform ed 160 on the pilot scale CCC machine.

8-2 V ariation in the K and k o f EA, EC/ED and EB /psu-EEA at 163 the pilot scale.

8-3 V ariation in the separation factor and resolution w ith m obile 165 phase flow rate and solute loading.

8-4 V ariation in perform ance param eters w ith operational 166 variables.

8-5 V ariation in K and k o f EA, EC/ED and EB /psu-EEA at the 167 pilot scale w ith a m obile phase flow rate o f 10 m L .m in’’.

8-6 O perating conditions for experim ents perform ed on the pilot 169 scale CCC machine using a back extract feed.

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Table D escription Page

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8-8 V ariation in the separation factor, resolution and colum n 172 efficiency w ith m obile phase flow rate and solute loading at

the pilot scale using the back extract feed.

8-9 V ariation in perform ance param eters w ith operational 173 variables.

8-10 Com parison o f predicted and experim ental erythrom ycin A 179 elution times for m odel system CCC scale-up from laboratory

to pilot scale.

8-11 Com parison o f predicted and experim ental erythrom ycin A 180 elution times for m odel system CCC scale-up at the increased

m obile phase flow rate o f 20 m L .m in"\

8-12 Com parison o f predicted and experim ental erythrom ycin A 181 elution tim es for different injection volum es.

8-13 Com parison o f predicted and experim ental erythrom ycin A 182 elution times for different m obile phase flow rates at a

rotational speed o f 1200 rpm.

8-14 Com parison o f predicted and experim ental erythrom ycin A 183 elution times for real system CCC scale-up from laboratory to

pilot scale.

8-15 Com parison o f predicted and experim ental erythrom ycin A 184 elution times for real system scale-up w ith increased m obile

phase flow rate.

8-16 Comparison o f predicted and experim ental erythrom ycin A 185 elution times for different injection volumes.

C H A PTER 9

9-1 Variation in the cum ulative fraction o f total erythrom ycins 190 eluted.

9-2 Exam ple o f start (ti) and end (t2) sample collection tim es (cut- 192

points) corresponding to different PF versus yield values.

9-3 Summary o f attainable PFmax values and final EA purities for 193 the different feed types together w ith corresponding out­

points.

9-4 Summary o f fraction cut-points for the range o f m obile phase 198 flow rate and solute loading experim ents perform ed.

C H A PTER 10

10-1 Com parison betw een Lab prep CCC and range o f H PLC 209 scales o f operation in term s o f m obile phase flows, processing

yields and initial hardw are costs.

C H A PTER 13

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T able Description Page

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13-2 Raw data o f volum etric com position o f individual solvents in 239 the quaternary phase system together w ith calculated pure

solvent polarities and the resulting solvent m ixture polarity.

13-3 D ata collection chart illustrating m easure volum es o f 240 stationary and m obile phases eluting from the CCC colum n

during the establishm ent o f hydrodynam ic equilibrium .

13-4 Q uantitative mass balance data for the 2 mL.min'* laboratory 241 scale CCC separation using the m odel system.

13-5 Q uantitative mass balance data for the 2 mL.min'* laboratory 242 scale CCC separation using the back extract feed.

13-6 Q uantitative mass balance data for the 10 m L .m in * pilot scale 243 CCC separation using the model feed.

(22)

List of Abbreviations

A Anstrom

ACP Acyl carrier protein Abs Absorbance (nm)

A ECS A gricultural and Environmental Services AT A cyl-transferase

ATT A nti-trypanosom al factor

BIB Brunei Institute for Bioengineering CCC C ounter-current chrom atography CCD Countercurrent distribution CCE C ountercurrent extractor CER Carbon dioxide evolution rate CPC Coil planet centrifuge

Da Daltons

DAP D iam inopim elic acid

DCCC D roplet counter-current Chrom atography DEBS 6-deoxyerythonylide B synthase

DMA D eoxyribonucleic acid DOT D issolved oxygen tension DW C Dry cell w eight

EA Erythrom ycin A

EB Erythrom ycin B

EC Erythrom ycin C

ED Erythrom ycin D

ED TA Ethylenediam inetetraacetic acid EEA Enol ether o f erythrom ycin A

EF Erythrom ycin F

ELSD Evaporative light scattering detector EtOA c Ethyl acetate

G LCCC Gyration locular countercurrent chrom atography

H2O W ater

Hex Hexane

H FTCPC Horizontal flow -through coil planet centrifuge HIV H um an im m unodeficiency virus

HPLC H igh perform ance liquid chrom atography

KS Ketosynthase

LCCC Locular countercurrent chrom atography LLC Liquid-Liquid Chrom atography

M eO H M ethanol

M LCPC M ultilayer coil planet centrifuge M PBV M obile phase bed volum e OBM Oil based m edia

OLCCC O scillation locular countercurrent chrom atography O UR Oxygen uptake rate

pH H ydrogen ion concentration pKa -log 10 (acid dissociation constant)

PKS Polyketide Synthase

(23)

Psu-EEA Ring-contracted enol ether o f erythrom ycin A PTFE Polytetrafluoroethyiene

RLCCC Rotating locular countercurrent chrom atography rpm Revolutions per minute

RQ Respiratory quotient SLPM Standard litres per m inute SS Stainless steel

TM IF Total M ass In Fraction (mg)

u v U ltra V iolet

(24)

Nomenclature

A

C ; i q C n , Corg

C s

d

EApiax F

F m - a c tiia l

H

H m i 11

i.d. K

K e a

K eB /p s u- E E A

K e c v e d

k

kE A ^EB/psu-EEA Fe c/e d

L Mai Mbi M o Mpj Ms M il N

N e f f N o p

o.d. P ' PF

P F m a x

PurEAfinai Q m Q . Q s l'i l'o R

Cross-sectional area o f the CCC coil

Solute concentration in the aqueous phase (mg.mL'^) Solute concentration in the lower m obile phase (mg.mL'^) Solute concentration in the organic phase (m g.m L’^)

Solute concentration in the upper stationary phase (mg.mL'^) Internal coil (tubing) diam eter (mm)

V ariation in tm between two CCC runs perform ed at the same m obile phase flow rate (minutes)

M axim um EA concentration corresponding to the peak m axim a (mg.mL'^) M obile phase flow rate (m L.m in’')

Square root o f actual m easured m obile phase flow rate H eight equivalent to a theoretical plate (cm/plate)

M inim um height equivalent to a theoretical plate (cm /plate) Internal coil (tubing) diam eter (mm)

D istribution ratio

D istribution ratio o f erythrom ycin A

D istribution ratio o f erythrom ycin B/psu-EEA D istribution ratio o f erythrom ycin C/D

Retention factor

Retention factor o f erythrom ycin A

Retention factor o f erythrom ycin B/psu-EEA R etention factor o f erythrom ycin C/D

Coil length (cm)

Concentration o f impurity, A, in a specific collected fraction at the z-th time interval (m g.m L’')

Concentration o f impurity, B, in a specific collected fraction at the z-th time interval (m g.m L’’)

Initial target solute concentration in the feed (m g.m L’')

Concentration o f target product, P, in a specific collected fraction at the z- th tim e interval (m g.m L’')

A m ount o f sample target solute injected (mg.mL"')

Total concentration o f all solutes in a particular fraction at the z-th tim e interval (m g.m L’*)

Colum n efficiency (theoretical plate num ber) Effective theoretical plate num ber

N um ber o f operational CCC runs achievable per day (24 hours) O uter coil (tubing) diam eter (mm)

R ohrschneider solvent polarity param eter Purification factor

M axim um purification factor

Purity o f EA in fractions collected after processing by CCC

N um ber o f m olecules o f a solute in the m obile phase per unit volum e (mg) M ass o f recovered erythrom ycin (mg)

N um ber o f m olecules o f a solute in the stationary phase per unit volum e

(mg)

(25)

Rs R esolution (measure o f zone overlap betw een two adjacent solute peaks) Sy Stationary phase retention w ithin the CCC coil (% v/v)

c Time from sample injection to elution o f the K=1 peak (m inutes) c' Time from solvent front to elution o f the K = 1 peak (m inutes)

k R etention tim e o f peak (K) o f interest from sample injection (m inutes) k ' Time from solvent front to elution o f K peak (m inutes)

k-aiiai Elution tim e o f the peak o f interest from an analytical scale CCC sample volum e experim ent (minutes)

k-start Beginning o f elution o f a peak w ith a distribution ratio o f K (m inutes) k-end End o f elution o f a peak w ith a distribution ratio o f K (minutes)

i Time for injection o f solute o f know n volum e at a set m obile phase flow rate (m L .m in ’)

m Time from sample injection to the solvent front (K = 0) (m inutes) TMIF Total m ass o f erythrom ycins in a collected fraction (mg)

R Retention tim e for a peak o f interest from sample injection Ü A verage linear m obile phase velocity (cm .s'')

Vc System volum e (mL)

Vc V olum e o f eluted stationary phase volum e originally in the coil that has been displaced by the m obile phase during phase equilibrium (mL) Vc-corrected Corrected volum e o f eluted stationary phase obtained by subtracting the

volum es o f the inlet (Vjn) and outlet (Vout) flying leads (mL) V, Solute injection volum e (mL)

Yin V olume o f the coil inlet flying lead (mL)

Vpi M obile phase volum e w ithin the CCC coil (mL) Vout V olum e o f the coil outlet flying lead (mL) VR Retention volum e for a peak

Vs Stationary phase volum e w ithin the CCC coil (mL)

Vs-start Recorded volum e o f stationary phase in the graduated m easuring cylinder prior to hydrodynam ic equilibrium (mL).

Vs-end Recorded volum e o f stationary phase in the graduated m easuring cylinder after hydrodynam ic equilibrium (mL).

wb, wa Baseline bandw idth o f two adjacent peaks, B and A (m inutes or cm) Wii/2 Peak w idth at one h alf the peak height

Wk W idth o f a peak w ith a distribution ratio K (m inutes)

G reek Svmbols

a Separation factor

f3-ratio Ratio o f inner (r;) or outer (ro) coil radius to the rotor (R) radius Pu Pi V iscosity o f upper and low er phases (cP)

pu pi D ensity o f upper and lower phases (kg.m'^) (js Interfacial tension (mN.m"’)

a Standard deviation o f the Gaussian peak O V olum e fraction o f pure solvent

(26)

1. Introduction

1.1. Taxonom y

1.1.1. Actinom ycetes

A ctinom ycetes are am ong the m ost w idely distributed groups o f m icro­

organism s, w ith the m em bers o f this extensive class considered as chem o-organotrophs,

requiring a form o f organic carbon for growth. The extent o f their grow th on an

artificial m edium is regulated by the availability o f oxygen, inorganic nitrogen source,

other additional nutrients and an approxim ately neutral pH (G usek and K insella, 1992).

U ntil thirty years ago, there was not a clear consensus as to the classification o f

actinom ycetes. Bacteriologists considered them to be bacteria due to the presence o f

elongated cells or filaments exhibiting some degree o f branching (G oodfellow et al.,

1983), while mycologists classified them as fungi due to characteristics such as their

grow th on a solid substrate. N owadays, actinom ycetes are generally accepted as

bacteria, more specifically gram positive, sporalating, saprohytic bacteria (G oodfellow

et al., 1988). A com mon feature o f this class is hyphae form ation during growth. These

hyphae, consisting o f num erous tubular cells, obtain nutrients required for grow th and

replication by solubilising the organic m edia via the secretion o f hydrolytic enzym es. It

is this m orphology that plays a crucial role in antibiotic production, w ith m any factors,

such as the species strain, grow th m edia and conditions influencing product titers.

A ctinom ycetes, therefore potentially exhibit a wide range o f m orphological structures

from simple rods and cocci to com plex m ycelia (G oodfellow et al., 1983), w ith the

strictures changing due to variations in their environm ent, from cell grow th, through to

prim ary and secondary m etabolism , where the production o f bioactive m etabolites

occurs.

1.1.2. Streptomyces

Streptomyces are m em bers o f the family streptom ycetaceaes and are considered

to be the m ost com m on Gram -positive bacteria, belonging to the order o f

(27)

They are abundant in soil, degrading a variety o f organic m aterials w ith the ability to

produce num erous bioactive products, such as antibiotics, vitam ins, enzym es and

im m uno-suppressors (G ilbert et al., 1995). This classifies them am ong the m ost

industrially im portant m icro-organism s (G ilbert et al., 1995). Specifically, in the

context o f this study, 60% o f all m icrobially produced antibiotics (Table 1-1) are

produced from Streptom yces (Queener et al., 1986).

Strain Antibiotic A ntibiotic group

Streptom yces aureofaciens Chlorotetracycline

Tetracyclines

Streptom yces rim osus O xytetracycline

Streptom yces griseus Streptomycins

A m inoglycosides

Streptom yces fra d ia e N eom ycin & Tylosin

Streptom yces kanam ycetius Kanam ycin

Streptom yces clavuligerus Clavams p-lactam s

Streptom yces venezuelae Chloramphenicol C hloram phenicols

Streptom yces am bofaciens Spiram cin

M acrolides

Streptom yces narbonensis N arbom ycin

T able 1-1 A ntibiotics produced by Streptom yces (from Q ueener et a/., 1986).

1.1.3. Saccharopolyspora erythraea

Saccharopolyspora erythraea, was first discovered in 1952 in a soil sam ple from

lloiol City in the Philippines, w ith the w ild type strain know n as N R R L 2338 (N orthern

U tilisation R esearch and D evelopm ent Division, US D epartm ent o f A griculture). Prior

to its reclassification (Labeda, 1987), it was originally know n as Streptom yces

erythreus. The reason for its reclassification was due to the differences in the cell wall

structure. The Streptom yces possess a type-I cell wall (H olt et al., 1994), containing L-

diam inopim elic acid (D A P) and m ycolic acids, whereas the Saccharopolyspora genus

possess a type IV cell w all (H olt et al., 1994) containing m eso-D A P and no m ycolic

acids. One o f the m ost abundant secondary m icrobial products o f the

(28)

1.1.4. Biosynthesis o f both natural and recom binant polyketides

The form ation o f polyketides, such as erythrom ycin, occurs by the sequential

activity o f a num ber o f enzym es and carrier proteins, collectively term ed polyketide

synthase or PKSs. These giant (> 300kDa) m odular m ulti-enzym e com plexes are

m ultifunctional proteins, possessing different active sites, or m odules for each

successive step o f polyketide chain assembly. The PK Ss contains six o f these m odules,

w hich can be segregated from the amino to the carboxylic term ini into a loading

domain, four m odules, and finally a releasing dom ain, as illustrated in

Figure 1-1.

The loading dom ain o f the PKS consists o f the enzym e acyl-transferase (AT)

and an acyl carrier protein (ACP). The interm ediate m odules are responsible for the

extension o f the polyketide chain and contain a ketosynthase (KS), A T and ACP.

Finally the off-loading dom ain contains a thioesterase that also possess a degree o f

cyclase activity and acts by thiolysis or acyltransfer. In this biosynthetic process, the

PKSs not only contain the num erous required active sites for synthesis, but also acts as

the platform for it. A s shown in Figure 1-2, the polyketide chain traverses each m odule,

w here it increases in length by two carbon atoms w hilst being covalently attached to the

enzym e as a thiol ester. Each module contains the m inim um set o f dom ains required for

the form ation o f carbon-carbon bonds, and therefore the num ber o f m odules present

dictates the size o f the polyketide.

In addition to the presence o f dom ains dedicated to the form ation o f the carbon-

carbon bonds, additional domains are present, perform ing the function o f encoding the

oxidation state o f the p-carbon o f each tw o-carbon unit. Together w ith this, m odules

can contain up to three addition enzym es, m odifying the resulting P-keto group

following each cycle o f chain elongation prior to its transfer to the next m odule. From

this arises the concept that the structure o f the polyketide is dependent on the order in

w hich the various m odules are arranged along the PKS. Together w ith the specificity o f

the enzym e, AT, and the presence o f these p-m odified enzym es, the order o f these

dom ains w ithin m odules, m odifying the structure o f the polyketide produces a diverse

(29)

L oa d in g M o d u le 2 M o d u le 4

l ' K S

Ui-iie

c l u s t e r

M o d u le I M o d u le 3 R e le a se

l’KS

Aevl C o A

V

V

P oly k e tid e

I i^ui c 1-1 Illustration of the modular PKS gene cluster and enzym e assem bly

(redrawn from Carreras and Santi, 1998).

M anipulation o f the polyketide chain length, as developed by Kao et al. (1994),

uses a plasm id-based expression system for 6-deoxyerythonylide B synthase (D E B S) in

Streptoniyces coelicolor. As a result o f this research, truncated PK Ss w ere generated by

the deletion o f m odules late in the biosynthetic pathw ay by the use o f the natural

thioesterase. resulting in the production o f novel triketide, tetraketide and hexaketide

lactones Ifom 2-, 3- and 5-m odule derivatives o f D E B S, respectively. A n exam ple o f

the structural form o f the triketide lactone can be seen in Figure 1-3, w here truncation

(30)

D EB SI 0 E B S 2 D E Ô S 3

nnnliik*

■i / ---moduk', 3

m o d u l e 2 " " " " " " " " m o d u l e 4

m o d u l e 5

m o d u le 6

\ % A C r KS A T KR 4C P K!) AT KK \ ( : P > K.S A r A CP k S A I UH KR KM A (l y KS AT KR ACM KS A T KR A C f l E

: I I I 1 , 1

rv'N

S

l=<

/

f ) =

0 =

H O .

/

H O ^

?

O H

OH

UH

Fiiîurc 1-2 Illustration of the PKS template which catalyses the biosynthesis

of the erythromycin aglycone (Carreras and Santi, 1998).

OH

H3Cv / \ ^ C H 3

o

o

CHs

(31)

1.1.5. M acrolide Antibiotics

The macrolide antibiotics are a diverse range o f com pounds w hich include

erythrom ycin, oleandom ycin, leucom ycin (kitasam ycin), spiram ycin, m idecam ycin and

tylosin. The first reported macrolide antibiotic was pikrom ycin (B rockm ann and

H enkel, 1950), then erythrom ycin (M cG uire et al., 1952), leucom ycin (H ata et ah,

1953), spiram ycin (Pinnert-Sindico et ah, 1954), and oleandom ycin (Sobin et ah, 1955).

These m acrolides are m em bers o f a large and diverse range o f natural com pounds called

polyketides, w hich include aromatic, polyether and m acrolactone com pounds. O f these

antibiotics, erythrom ycin was the first com m ercially produced antibiotic, w ith annual

figures for the production o f polyketides in the region o f 2000 tonnes (Schuler, 1987).

The m arket value o f these m icrobial secondary m etabolites has exceeded annual sales o f

$10 billion (Carreras & Santi, 1998). The production o f erythrom ycin, its structure and

function is described in the sections to follow.

1.1.6. Erythrom ycin

J. 1.6.1. The structure o f erythromycin

Clinically useful m acrolide antibiotics are classified into three distinct groups

based on the size o f the m acrocyclic lactone ring, i.e. containing 14, 15, or 16

m em bered rings w ith sugars linked via glycosidic bonds (Elks and G anellin, 1991).

W ithin the 14-membered group, erythrom ycin is one o f the m ost industrially im portant

com pounds. Structurally, this class o f antibiotic can be characterised by 1) large lactone

(32)

A

H,C.

/ R 5

CH,R,

^

3

/

B

0

Figure 1-4 Structure o f erythrom ycin. Section (A) corresponds to the

14-m e14-m bered lactone ring. (B) corresponds to the am ino sugar

m oiety (D-desosamine). (C) corresponds to the secondary sugar A-caladinose. R% represents specific points on the m olecule w here the erythrom ycin analogues vary, as illustrated in T able 1-2.

The large polyhydroxylactone ring o f erythrom ycin, illustrated in Figure 1-4 (A)

has, in addition to it one or m ore sugar residues substituted into the ring (Figure 1-4 (B)

and 1-4 (C)). These sugar residues can also, w ith the addition o f a nitrogen atom, take

the form o f an am ino sugar m oiety (D-desosamine), as illustrated in Figure 1-4 (B). The

ferm entation process for erythrom ycin m anufacture results in the form ation o f small

quantities o f erythrom ycins B (EB), C (EC), D (ED), E (EE) and F (EF) in addition to

EA (K anfer et al., 1998). In com m ercially produced erythrom ycin preparations there

are also small quantities o f both acid and basic degradation products derived from

erythrom ycin A, nam ely the enol ether o f erythrom ycin A (EEA) and the ring-

contracted enol ether o f erythrom ycin A (Psu-EEA) respectively (K anfer et al., 1998).

All o f these analogues o f erythrom ycin possess the D-desosamine ring, but vary

at specific points on the erythrom ycin molecule, labelled as Rj to in Figure 1-4.

Table 1-2 illustrates the stm ctural variations that exist betw een the different form s o f

(33)

As can be seen from Table 1-2, the erythrom ycin analogues are structurally and

chem ically very similar, differing in some cases by only a single hydroxyl or m ethyl

group. The biosynthetic pathw ay leading to the production o f erythrom ycin A and its

analogues is shown in Figure 1-5.

Ri Rz R3 R4 Rs Molecular

formula

Mw

Erythrom ycin A (E A )

O H H H O C H3 C H3 C37H67NO13 734

Erythrom ycin B (E B )

H H H O C H3 C H3 C37H67N0]2 718

Erythrom ycin C (E C )

O H H H O H C H3 C37H65NO13 720

Erythrom ycin D (E D )

H H H O H C H3 C37H67NO14 750

T able 1-2 Structural variations o f the erythrom ycins (from K anfer et al.,

1998).

1.1.6.2. Properties o f erythrom ycin and its mode o f action.

Erythrom ycin A, with the em pirical formula: C3 7H67N O13 has a m olecular

w eight o f 734 Da, and a m axim um UV absorbance at 278 nm. It is slightly soluble in

water, but m ore soluble in organic solvents and has a pKa o f 8.8, w ith its basic character

conferred through the D-desosamine sugar m oiety (K anfer et a l , 1998). M axim um

stability is in the pH range 6-9.5 (K avanagh and D ennin, 1963), w ith it losing its

biological activity at a pH<5 (Hahn, 1967). The erythrom ycins are bacteriostatic agents

w hose biological activity is dependent on intracellular accretion and ribosom e binding.

M ore specifically, the erythrom ycins act by binding to the 50S ribosom al subunit and

stim ulate dissociation o f peptidyl-tRN A from the ribosom e during the translocation

process (Brisson-N oel et a l , 1988). G ram -positive bacteria are m ore sensitive to

erythrom ycin A than G ram -negative forms. This is due to the G ram -positive forms

(34)

P ropionyl C oA

2-M ethy] m alon yl C oA

C ondensation

6 C O .

6-D eoxyeryth ron olid e B

^ 6-D eo x y ery th ro n o lid e B

Erythronolide B

^ M ycarsylation at C-3

3 -a -L -m y ca ro sy l erythronolide B

D esoam in ylation at C-5

E r y th r o m y c in D

iiyci

1

H ydroxylation _ . M éthylation

- ■ ■ - at C -3 ”

y \

E r y th r o m y c in C E r y th r o m y c in B

■\ y

M éthylation

^

H ydroxylation

at C -3 ” a tC -1 2

E r y th r o m y c in A

I

Esterificaton

betw een C -2 and C - 1 ’

E r y th r o m y c in E

(35)

1.1.6.3. Cl inical uses o f erythromycin

Erythrom ycin, as w ith other m acrolides is very effective against G ram -positive

bacteria, in particular staphylococci, and m ycoplasm as. O ther clinical applications o f

erythrom ycin include:

1) A ctive against L egionella species.

2) A ctive against Cam pylobacter species.

3) A useful substitute for penicillin and flucloxacillin in hypersensitive patients

(e.g. upper respiratory, skin and soft tissue infections).

4) Clinically useful against atypical pneum onia, severe Cam pylobacter infections,

non-gonococcal genital infections.

5) Erythrom ycin is the antibiotic o f choice for diphtheria.

6) As a com bined treatm ent for HIV.

1.1.6.4. Industrial production o f erythromycin.

This section aim s to outline the industrial production by ferm entation and the

dow nstream recovery and purification operations o f the target product. The industrial

production o f natural products, such as antibiotics incorporates a num ber o f unit

operations, from the ferm entation, w ith its continual optim isation, through to recovery

from initial clarification o f the w hole broth, prim ary extraction/concentration by the

technique o f liquid-liquid partitioning, and finally drying and form ulation.

1.1.6.4.1. Ferm entation

In the production o f secondary m etabolites the first stage involves the grow th o f

the selected strain o f m icro-organism , specifically Sacchropolyspora erythraea. A t this

point, upon the addition o f the inoculum to the ferm enter, the process can last for

anything up to seven days, w ith the continuous or interm ittent addition o f extra

nutrients, chem ical precursors, acid or alkali to m aintain a successful level o f m icrobial

(36)

tem perature. As already discussed in Section 1.1, Sacchropolyspora erythraea

possesses a distinctive m orphological feature o f form ing branched filam ents, or hyphae.

R equirem ent Source

Carbon Glucose, sucrose, starch, m olasses, soya bean oil, rapeseed oil, anim al fats

N itrogen Soya meal, blood meal, com steep liquor, casein, yeast extract, m eat extract

Inorganics Calcium salts, am m onium salts. Sulphides, phosphorus salts, trace elem ents

A ntifoam Polypropylene glycol (PPG)

T able 1-3 Source(s) o f nutrient and antifoam requirem ents for the

ferm entation

Research within our laboratories studies the optim isation o f erythrom ycin

production using both soluble com plex m edia (SCM ) and a m ore industrially relevant

oil based m edia (OBM), in addition to the interactions o f the ferm entation w ith initial

product recovery by m icrofiltration (Davies et al., 2000). The filtered ferm entation

broth used during this study was produced using the soluble com plex m edium

form ulation (Table 2-1).

D uring the final ferm entation phase, the biosynthesised product w ill either be

associated w ith the microbial cells, or in solution. In the case o f erythrom ycin, the latter

process occurs. The resultant ferm entation broth, the ‘whole broth’, is com posed o f the

biom ass in a dilute aqueous solution containing unused nutrients, undefined im purities

such as unw anted m etabolites, surfactants (antifoam ing agents) and the target product.

The typical constituents o f an antibiotic ferm entation broth are show n below in Table

1-4.

Broth constituent % by w eight

W ater 75-95

Cells 2-6

Product 0 - 1 0

Residual raw m aterials 1-5

Proteins /polysaccharides 0-5

(37)

During the course o f process developm ent and continual process optim isation,

w ith the aim being to increase the product titers and reduce m edia costs, the

ferm entation scientist will be continually m odifying the ferm entation m edia

form ulations. These m odifications can be in the form o f the chosen carbon or nitrogen

sources (Table 1-3) and their relative amounts. However, the effects o f these m edia

changes on subsequent dow nstream processing operations are rarely considered, w ith

the result o f this being that any increase in product titre can be com prom ised by product

loss during recovery operations (Davies et ah, 2000). The m ain objective o f

dow nstream processing is to successfully recovery the target product from the initial

ferm entation broth at an acceptable purity and yield. This objective can be considered

as a num ber o f stages; cell removal, product concentration, purification and finally

isolation.

1.1.6.5. Prim ary recovery

This section aims to give an overview o f the prim ary recovery steps em ployed

by industry and their relative merits. The harvested broth from a ferm entation process

can be considered to consist o f a num ber o f phases, nam ely the continuous-aqueous and

solid-m edium phases, consisting o f the m icrobial cells, residual raw m aterials such as

soya bean flour and insoluble organic elem ents e.g. oil. A s the erythrom ycin is

produced extracellularly, there is no need for prior cell disruption techniques such as

hom ogenisation to be employed. The first step is to condition the broth by either

acidification or the addition o f flocculation aids to rem ove cells. A cidification further

assists in preparing the product in a form suitable for solvent extraction. There are then

a num ber o f unit operations for m ycelia removal, such as:

1) Filtration

a. Rotary drum vacuum filters - robust, reliable and can handle a w ide

range o f broths. However, the addition o f the filter aid m ay cause

problem s w ith dow nstream equipment, e.g. pum ps.

Figure

Table Description

Table Description

p.18
Figure 1-4Structure of erythromycin.

Figure 1-4Structure

of erythromycin. p.32
Figure 1-5 Biosysnthesis of the various forms of erythromycin analogues.

Figure 1-5

Biosysnthesis of the various forms of erythromycin analogues. p.34
Figure 1-8 Schematic representation of various coil orientations investigated

Figure 1-8

Schematic representation of various coil orientations investigated p.43
figure 1-9 Representation of the synchronous planetary motion of the I and

figure 1-9

Representation of the synchronous planetary motion of the I and p.44
Figure 1-12

Figure 1-12

p.46
Figure 1-20

Figure 1-20

p.57
Table 1-5 Antibiotics fractionated by CCC.

Table 1-5

Antibiotics fractionated by CCC. p.59
Table 2-2 Dimensions of the 20 L fermenter used during this study.

Table 2-2

Dimensions of the 20 L fermenter used during this study. p.65
Figure 2-1 The Brunei CCC instrument illustrating the mounting

Figure 2-1

The Brunei CCC instrument illustrating the mounting p.69
Figure 2-2 ( oil winding illustrating the entrance and exit of the two sets

Figure 2-2

( oil winding illustrating the entrance and exit of the two sets p.70
Figure 2-3 Bobbin mounting between two rotor plates together with the

Figure 2-3

Bobbin mounting between two rotor plates together with the p.71
Table 2-4 Dimensions of both inner and outer stainless steel coils on bobbins

Table 2-4

Dimensions of both inner and outer stainless steel coils on bobbins p.71
Table 2-5 Comparison between the two scales of CCC.

Table 2-5

Comparison between the two scales of CCC. p.73
Table 2-7 Distribution ratio experiments performed with the hexane/ethyl

Table 2-7

Distribution ratio experiments performed with the hexane/ethyl p.81
Table 2-8 Normal and reverse phase CCC gradient options.

Table 2-8

Normal and reverse phase CCC gradient options. p.82
Table 2-9 Solvent phase compositions used to perform a reverse phase

Table 2-9

Solvent phase compositions used to perform a reverse phase p.83
Figure 2-5 CCC operating conditions for a reverse phase methanol

Figure 2-5

CCC operating conditions for a reverse phase methanol p.83
Figure 3-1 Schematic represention of a CCC chromatogram illustrating the

Figure 3-1

Schematic represention of a CCC chromatogram illustrating the p.86
Figure 3-2 Stages involved in the construction of fractionation and PFmax

Figure 3-2

Stages involved in the construction of fractionation and PFmax p.96
Figure 3-3 Schematic represention of a PF versus product yield diagram. For

Figure 3-3

Schematic represention of a PF versus product yield diagram. For p.97
Table 4-1 Variation in equilibrium erythromycin distribution ratio with

Table 4-1

Variation in equilibrium erythromycin distribution ratio with p.103
Table 4-4 Stage 2 screening study results - Variation in individual

Table 4-4

Stage 2 screening study results - Variation in individual p.105
Table 4-3 Variation in quaternary solvent system polarity (P') with

Table 4-3

Variation in quaternary solvent system polarity (P') with p.105
Table 4-5

Table 4-5

p.107
Figure 4-1 Relationship between percentage aqueous methanol content in the

Figure 4-1

Relationship between percentage aqueous methanol content in the p.108
Figure 4-2 Gradient elution CCC chromatogram. (---- ) Relative signal from

Figure 4-2

Gradient elution CCC chromatogram. (---- ) Relative signal from p.109
Figure 4-3 Analytical reverse phase HPLC chromatogram of a commercial

Figure 4-3

Analytical reverse phase HPLC chromatogram of a commercial p.111
Figure 4-4 Isocratic CCC chromatogram . ( ----- ) absorbance. ( g g )  results

Figure 4-4

Isocratic CCC chromatogram . ( ----- ) absorbance. ( g g ) results p.112
Figure 4-5 HPLC chromatograms of fractions collected at 2 minute intervals

Figure 4-5

HPLC chromatograms of fractions collected at 2 minute intervals p.113

References