亲和填料ProSep Ultra Plus User Guide

Prosep? Ultra Plus Affinity Chromatography Media OPERATING INSTRUCTIONS

Millipore, Amicon and ProSep are registered trademarks of Merck KGaA, Darmstadt, Germany. The M mark is a trademark of Merck KGaA. Tween is a trademark of ICI Americas Inc.. Triton is a trademark of Union Carbide Corporation. OLI is a trademark of Wamgroup. ISO is a trademark of the International Standards Organization. ? 2008, 2012 Merck Millipore, Ltd. All rights reserved.

00104755PU Rev B, 01/2012

Notice

The information in this document is subject to change without notice and should not be construed as a commitment by Merck Millipore or an affiliate. Neither Merck Millipore nor any of its affiliates assumes responsibility for any errors that may appear in this document.

ProSep? Ultra Plus Media Operating Intructions 3 Contents

Typical Specifications (6)

Precautions and Safety Procedures (8)

Packed Column Evaluation (18)

ProSep? Ultra Plus Media Mass Balance Protocol (19)

Process Conditions (22)

Sample Preparation (23)

Cleaning (28)

Sanitization (29)

Storage and Handling (30)

Ordering Information (31)

References (32)

Appendix A (33)

Standard Warranty (35)

Technical Assistance (36)

https://www.wendangku.net/doc/1114970404.html,

ProSep? Ultra Plus Media Operating Intructions 5 Introduction

ProSep? Ultra Plus media has been developed specifically for industrial scale purification of monoclonal antibodies (MAbs). It facilitates the efficient, cost effective purification of MAbs. Its high capacity and excellent flow properties have been designed to address the needs of current and future higher titer feedstocks. Using clarified bioreactor feedstock at physiological pH and salt concentration, no pretreatment such as concentration by ultrafiltration or buffer exchange is required. This reduces time, labor and capital cost and improves retention of biological integrity of the antibody product

Designed primarily for the capture and purification of MAbs, ProSep? Ultra Plus media may also be used for the purification of fusion proteins, selective purification of antibody fragments and purification or removal of polyclonal IgG from serum.

ProSep? Ultra Plus media consists of a recombinant natural protein A immobilized on porous glass which is permeated by interconnecting pores

of uniform and precisely controlled size. ProSep? Ultra Plus media is incompressible, extremely durable, does not shrink or swell in different solutions and exhibits chemical and mechanical stability over a range of conditions such as low pH, exposure to detergents, buffers of varying ionic strengths,

and many organic solvents. The rapid mass transfer afforded by the open and uniform pore structure results in sharp breakthrough curves and high dynamic capacity being maintained over a wide range of flow rates or residence times. The proprietary chemistry used for immobilization of the protein A onto the controlled pore glass has been developed to maximize ligand distribution, orientation and stability while minimizing non-specific interactions. ProSep? Ultra Plus media, while retaining the same basic attributes of ProSep?-vA High Capacity and ProSep?-vA Ultra media, has been optimized in terms

of particle size, pore size and ligand immobilization, to provide even higher binding capacity. While specifically designed to provide high productivity and low cost of operation for today’s large volume, higher titer feedstocks it can be used for purification of MAbs from laboratory to process scale.

https://www.wendangku.net/doc/1114970404.html,

Typical Specifications

Support matrix Porous glass

Nominal particle size60 μm

Protein A ligand Recombinant protein A expressed in

E. coli

pH range 1 to 8.5

Pressure Incompressible matrix with linear

pressure/flow rate characteristics. Maximum

operating pressure< 150 psi (10 bar)

Recommended mobile

phase velocity Up to 500 cm/h at 2 bar, 20 cm bed height

Lifetime ProSep? Ultra Plus media is stable over repeated operational cycles when appro-priate regeneration and cleaning protocols are used.

Binding (Static) Capacity> 67 mg/mL ( polyclonal human IgG)

Binding (Dynamic) Capacity Typical dynamic capacities for humanized MAbs range from 45 -55 mg/mL at 3-6 min RT and 5% breakthrough.

Sh ipping buffer 0.1M Sodium Acetate, pH 5.2 + 0.5, 1%

Benzyl Alcohol

Recommended Storage0.1M Sodium Acetate, pH 5.2 + 0.5 con-

taining 1-2 % Benzyl Alcohol, +2 to +8o C

ProSep? Ultra Plus Media Operating Intructions 7 Pressure Drop and Flow Rate

The particle size range of ProSep? Ultra Plus media has been selected to provide the optimum combination of dynamic capacity and flow rate to maximize process productivity with the newer higher titer feedstocks. The rigid nature of ProSep? Ultra Plus media means that pressure/flow data exhibit a linear relationship irrespective of column diameter or bed height thus ensuring scale-up is straightforward and predictable, even when moving to very large production columns over 1 meter diameter. This predictability provides the flexibility to run longer bed heights if required, either enabling smaller diameter columns to be deployed or increasing capacity in existing columns.

Figure 1: Typical Flow Rate vs. Pressure Curve

Due to the linear relationship of the pressure/flow curves the expected pressure drop for a given flow rate or bed height can be simply calculated from the following equation and is irrespective of bed height.

?P(bar)/L(cm) = 1.25x10-4 x V (cm/hr)

https://www.wendangku.net/doc/1114970404.html, Precautions and Safety Procedures 1. ProSep? Ultra Plus media is supplied in buffer containing

preservative. Do not mouth pipette. The buffer may be harmful if swallowed.

2. Do not expose ProSep? Ultra Plus media to pH outside of the

range of 1.0 - 8.5.

First Aid

Eyes Irrigate thoroughly with water. If discomfort persists obtain medical attention.

Skin Wash thoroughly with soap and water.

Mouth Rinse thoroughly with water. In severe cases, obtain medical attention.

Spills Wear appropriate protective clothing. Carefully mop-up spill and dispose of in accordance with local regulations. Large

spills of material should be contained with sand and trans-

ferred to salvage containers.

The Material Safety Datasheet for this product and all reagents used with it should be carefully reviewed prior to beginning work. Work should not start until the user is thoroughly familiar with the health and safety hazards of all reactants and the hazardous nature of their interactions.

Quality Assurance

We recognize the importance of providing regulatory support and meeting industry quality standards. ProSep? Ultra Plus media utilizes recombinant native protein A derived from E. coli. No mammalian derived materials are used to manufacture ProSep? Ultra Plus media and its components.

ProSep? Ultra Plus Media Operating Intructions 9 All ProSep? products are manufactured in a facility certified to the internationally recognized standard, ISO? 9001:2008 and subjected to routine independent surveillance audits.

Each batch of ProSep? Ultra Plus affinity media is tested prior to shipment to determine the binding capacity with standard solutions of human polyclonal IgG. The results of these tests are included in the Certificate of Analysis accompanying each pack.

A Regulatory Reference File for ProSep? Ultra Plus media is available. Please contact your local representative for details.

https://www.wendangku.net/doc/1114970404.html, Column Packing

Due to its relatively low pressure drop and incompressible nature, ProSep? Ultra Plus media can be packed to longer bed lengths than most compressible media types. Recommended bed height is 20 cm.

While ProSep? Ultra Plus media is easy to pack, it does pack differently than compressible media, such as agarose, and this needs to be taken into account when packing. A well packed column is one where the

bed is fully consolidated and homogeneous; that is the spaces between the media particles are minimized and uniform. Whereas ProSep? does not suffer the issues of bed compression experienced with agarose and other compressible media, it is important to ensure the bed is fully consolidated. The irregular shape of the resin particles can give rise to bridging, especially on small diameter columns, which hinders full bed consolidation. This is readily overcome by the application of vibrational energy during packing which disrupts the bridging and leads to a fully consolidated, stable bed.

ProSep? Ultra Plus media does not exhibit cracking of the column bed, which maybe experienced with soft matrices and is unaffected if accidentally allowed to run dry during operation. ProSep? Ultra Plus media does not expand or contract under changing conditions of ionic strength, or pH.

NOTE If the column does dry out, remove trapped air by flowing buffer in an upward direction. Some sintered bed supports may not allow

passage of air due to surface tension effects. In such cases, remov-

ing and replacing the top flow adapter should solve this. When

replacing ensure the sintered bed support is fully wetted out.

ProSep ? Ultra Plus Media Operating Intructions 11Laboratory Scale Column Packing Method - Syringe assisted tap-packing method (for use with 0.5 -5cm column diameters)

1. Buffer exchange the ProSep? Ultra Plus media by pouring or pipetting

off the storage buffer above the settled media and replace with packing buffer, i.e. purified water, to give a 50 - 60% slurry. Gently mix the slurry into a homogeneous suspension.

Allow the slurry to settle for 40 minutes. Decant or pipette off the

supernatant. Add fresh packing buffer and repeat the process two more times.

2. To determine the volume of slurry needed to give the final required

bed height, first determine the gravity settled concentration by

allowing the slurry to settle under gravity for 40 minutes on a surface free from vibration. In either a 15 or 50 mL graduated centrifuge tube depending on the slurry volume.

3. The volume of slurry required to pack a given bed height can be

estimated using the following formula:

This will provide a target bed height +/-10%. (accuracy can be

further increased, once the column has been packed once or twice,

by adjusting the consolidation factor to reflect results of the specific column and packing set up).

4. Mark the given bed height on the column tube.

5. Assemble and mount the column vertically.

6. Fill a syringe with 1-2 mL of water and connect to the bottom of the

column.

7. Use the syringe to fill bottom of column with 1-2 cm of water to wet

bottom bed support. Leave the syringe connected to the column.

8. Mix the slurry into a homogeneous suspension. Ensure there are no

clumps of media at the bottom of the container.

V olume slurry = V olume packed bed x 100 Slurry %

x 1.3 (consolidation factor)

https://www.wendangku.net/doc/1114970404.html, 9. Add the slurry to the column. Avoid air entrapment by pouring the

slurry down the column wall using a funnel or a glass rod. If the slurry cannot be added to the column in a single addition, add as much

slurry as possible to fill the column.

Note: Alternatively, to ensure a homogeneous slurry is present in the column prior to the packing, add the slurry up to 2-3 cm below the

top of the column. Then, cover the column tube with parafilm and

mix the slurry in the column ensuring the resin above the bot-

tom frit has not formed any clumps. For larger diameter columns,

e.g. 4.4 or 5 cm, a pipet can also be used to mix the slurry in the

column. Once the slurry is mixed into a homogeneous suspension

set the column vertically and proceed to step 10.

10. Using the syringe slowly pull liquid through the bottom of the column.

As the liquid is removed the formation of the bed will be observed. 11. Stop drawing liquid when the bed is not moving any further. Leave at

least 5 mm of liquid on top of the bed.

12. Add the remaining slurry to fill the column again and repeat steps

10 and 11 (Mix to ensure the remaining slurry is an homogeneous

suspension before addition).

Note: If the syringe fills up it can be disconnected, emptied and quickly reconnected to the bottom of the column. Ensure there is liquid on

top of the bed before disconnecting the syringe. Air entrapment

may be seen at the base of the column during this stage, but this

will be removed during step 19 and will not cause any performance

issues.

13. If the column is full and there is remaining slurry to be added tap the

sides of the column vigorously along the length of the settled bed

with a hard object (e.g. a hard plastic rod or a metal bar covered with rubber tubing) for 20-30 sec.

14. Using the syringe slowly draw liquid from the bottom of the column. The bed

will consolidate to a lower bed height. Stop drawing liquid when the bed is

ProSep? Ultra Plus Media Operating Intructions 13

not moving any further. Leave at least 5 mm of liquid on top of the bed. 15. Repeat steps 11-14 until all the slurry has been added to the column

and it is possible to connect the top adapter.

16. Disconnect the syringe from the bottom of the column and connect to

the packing system.

17. Ensure the column outlet is closed and connect the top flow adapter

while venting air out of the inlet tube.

18. Start flow with the pump at a low flow rate (e.g. 2 mL/min), prime the

column inlet line, open the bottom outlet and connect the outlet of the pump to the inlet connector of the column, without entrapping air. 19. Pump packing buffer through the column at a linear flow of 1000cm/

hr (if 1000cm/hr cannot be achieved, use at least 500 cm/hr or

maximum operational flow rate depending on the capability of the

packing system) for approximately 5 seconds until the bed does not consolidate further.

20. Stop the flow and tap the sides of the column along the full length

of the packed bed for 30 seconds. Note that the bed will usually not consolidate during this step but only when flow is reapplied.

21. Repeat steps 19-20 until the bed has not consolidated further over 3

consecutive tapping/flow cycles.

22. Move down the top flow adapter to contact the bed and repeat steps

19-20 at least twice more to ensure the bed does not consolidate any further. Check for the formation of a liquid space above the packed

bed. If a space forms, turn off the pump and move the top flow adapter until it re-contacts the top of the bed. Repeat steps 19-20 at least twice more to ensure the bed does not consolidate any further. At this point further consolidation of the bed may only be noticed by checking the tightness of the bed support onto the actual bed.

23. Condition the packed bed by flowing 1 column volume of water at

1000 cm/hr (if 1000cm/hr cannot be achieved, use at least 500 cm/hr

https://www.wendangku.net/doc/1114970404.html, or maximum operational flow rate depending on the capability of the

packing system).

24. Assess the quality of the packed bed ( See ‘Packed Bed Evaluation) Pilot Scale Column Packing Method - Hybrid Flow/Vibration Method (for use with >5 cm column diameters)

1. Ensure that the column is mechanically integral by performing a

hydrostatic hold test at the column’s maximum pressure rating. The liquid-filled column should be held at the maximum pressure for 15 minutes.

2. Ensure that the bed supports are either new or clean.

3. Fill the column to the target bed height with RO water/WFI and

generate a pressure-flow curve. The flow rate should span 200 cm/hr –

1.5x operational linear velocity.

4. If the resin is new and stored in storage buffer, the following

procedure is recommended for exchanging into packing buffer. The recommended packing buffer is purified water.

a. Slurry resin completely.

b. Allow the resin to settle under gravity for 40 min.

c. Decant supernatant and add packing buffer to replenish decanted

volume to approximately 50% slurry.

d. Repeat steps a-c at least two more times.

Note: It may be more effective to exchange the solutions by forming a packed bed and flowing WFI (200-300 cm/h) for 3 CV or until the

inlet and outlet column conductivities are equal. However, may

affect the cleanliness of the bed supports if there are fines present

(only in the case of used resin). Once the resin is in the packing

buffer, slurry it completely and allow to gravity settle to determine

slurry concentration and total slurry volume required (step 9).

5. If the resin has been used, de-fining at least twice is recommended.

Each defining step involves slurrying the resin completely, allowing

ProSep? Ultra Plus Media Operating Intructions 15 resin to settle under gravity for approximately 40 minutes and

decanting the supernatant.

6. Prepare 1M NaCl solution in the packing buffer for qualifying the

packed bed. Alternatively, a 1 to 2% acetone solution in RO water

could also be used to qualify the packed bed.

7. Clamp the appropriate vibrator(s) to bottom flange of column. If the

vibrator(s) cannot be clamped to the bottom flange due to spatial

constraints, clamp it to the underside of the bottom flange. Table 1

lists the appropriate vibrator(s) for various column sizes

Note: Vibration devices are sized using Millipore QuikScale columns with acrylic tubes. Differing column material or design may require fur-

ther evaluation of device needs). When using more than one vibra-

tor, place the vibrators equidistant from each other. It is strongly

recommended that a separate air source be employed for each vi-

brator. Ensure the air supply used delivers the flow rate indicated

in Table 1 at 50 psi. If needed, adjust the pressure to meet the flow

rate recommendations.

8. Ensure the column is leveled.

9. When using resin from a new carboy or bottle, the ratio of new resin

volume to final packed bed volume is approximately 1:1 when using the packing method described in this document. For example, 10 L

of media from a new carboy results in a final packed volume of 10 L.

If the target bed height/volume requires a fraction of a new container then mix the resin thoroughly in the carboy or bottle and remove

the appropriate amount of slurry needed noting that new containers are calibrated to a 50% slurry volume. For example if 3 L of media are required, remove 6 L of slurry (= 3 L ÷ 50% slurry). If using resin from a container with an unknown volume refer to the Mass Balance Protocol in the next section to determine the slurry volume needed to achieve the target volume.

Table 1: Vibrator recommendation for various column sizes (acrylic tubes)

10. Pump packing buffer through the bottom port to remove air from

under the bottom bed support. Stop flow when the water level is ~1 cm above the bottom bed support.

11. Prepare ~ 50% slurry of the resin in packing buffer (RO/WFI Water)

and transfer the slurry into column. The transfer can occur either using

a diaphragm/peristaltic pump or manually. The slurry percent can be

in the range 40-60%, though 50% is recommended.

12. Manually slurry the resin thoroughly in the column. Slurry the resin

by gently stirring the supernatant until resin is fully slurried. Avoid

contact with settled resin as much as possible as this may generate

fines.

13. Lower the top adjuster to the surface of the slurry, purging the top

lines of air. Execute this step as quickly as possible to minimize resin settling.

14. Apply downward flow at 300 cm/hr for 1 minute. After 1 minute of

flow, stop pump and put column on bypass.

15. Apply vibration at the required pressure to deliver the air flow listed

on Table 1 (Item 7 in Pre-pack Activities) for 1 minute. After 1 minute vibration, put column online.

16. Repeat steps 5-6 for 29 more cycles (1 minute flow and 1 minute

vibration is 1 cycle) for 30 total cycles (60 minutes).

ProSep? Ultra Plus Media Operating Intructions 17

NOTE The total packing time may be shorter depending on the bed height and/or column diameter. If the bed height does not change after

three consecutive cycles proceed to step 8. Close attention should

be put when measuring the bed height since the consolidation of

the bed may be only ~1 mm after each of the last cycles. For ex-

ample, a 10 cm diameter column may require 20 to 25 cycles, but

a 45 cm column may require only 10 cycles due to differences in

wall support.

17. Lower top flow adapter onto the settled bed. During lowering, the excess

liquid will be exhausted through the top process port. Record final bed height. (Note: This is only practical on glass or acrylic columns).

18. The final packed bed height should be within a target bed height ± 10%.

19. Condition packed bed by applying the highest operational flow rate for

1 CV. Note: If the system pump can deliver a higher flow rate and the

pressure does not exceed the rating of the column execute this step at

a flow rate of >= 500 cm/hr.

20. At the end of Step 10, if there is further bed consolidation, repeat step

8.

NOTE This is only practical on glass or acrylic columns.

21. Assess the quality of the packed bed.

https://www.wendangku.net/doc/1114970404.html, Packed Column Evaluation

Before use the quality of the packing should be checked by measuring

the packed column efficiency. This can also be repeated during column operation prior to re-use after storage and/or if deterioration in separation performance is observed.

The commonly used method for assessing column efficiency is in terms of the height equivalent to a theoretical plate (HETP) and asymmetry factor (As). These values are determined by applying either a sample such as 1% acetone or 0.5M – 1M NaCl in water or buffer.

The values for HETP and As will depend on the specific test conditions e.g. sample concentration and volume, flow rate and system tubing/pipework. It should, therefore, be used only as a reference and these conditions maintained the same when directly comparing specific values.

Typical sample volumes would be 1-2 % of column volume or equivalent to a bandwidth of 5 mm and a flow rate of 100-200 cm/hr.

Acceptable guideline values for ProSep? Ultra Plus media are HETP <0.1 cm and As 0.8 – 1.8.

ProSep? Ultra Plus Media Operating Intructions 19 ProSep? Ultra Plus Media Mass Balance Protocol

The objective of this protocol is to estimate the slurry volume of ProSep? Ultra Plus media needed to pack a column when the volume of resin in

a container is not accurately known. If the column is packed using the hybrid flow/vibration method the expected accuracy of this method is approximately +/- 5% of the target bed height.

In cases where new containers of resin will be used to pack a column the recommended approaches are:

? If using resin from a new carboy or bottle, there is approximately

a 1:1 ratio between new resin volume and final packed bed volume

when using the hybrid flow/vibration packing method. For example,

10 L of media from a new carboy results in a final packed volume of

approximately 10 L.

? If the target bed volume requires a fraction of a new container then mix the resin thoroughly in the carboy or bottle and remove the

appropriate amount of slurry needed noting that new containers are calibrated to a 50% slurry volume. For example if 3 L of media are required, remove 6 L of slurry (= 3 L ÷ 50% slurry).

Materials

? Balance

? Centrifuge – Swinging-bucket rotor for 50mL centrifuge tubes (minimum speed 3700 g)

? ProSep? Ultra Plus media slurry

? 4 x Amicon? Ultra-15 Centrifugal Filter Units,Ultracel-100 membrane (Catalogue No. UFC910024)

? 25 mL Pipets and pipet gun

https://www.wendangku.net/doc/1114970404.html, Method

1. Remove the filter insert from each Amicon? Ultra-15 unit. Label,

weigh and record the mass of each insert. Place back the insert into the centrifugal unit.

2. Mix thoroughly the resin slurry until it is a homogeneous suspension.

If possible visually check for sedimented resin at the bottom of the container.

3. Remove accurately 10 mL from the center of the slurry using a 25 mL

pipet. Transfer this slurry sample into one centrifugal filter unit and close the device.

4. Repeat steps 2 and 3 three more times.

5. Centrifuge the filter units for 25 min at 3700 g. A swinging-bucket

rotor is recommended.

6. Remove the insert, weigh and record the mass of each insert.

7. Calculate the mass of resin in each sample by subtracting the value

recorded in step 1 from the corresponding value recorded in step 6. 8. Calculate the average of the values calculated in step 7. Record this

value as A.

9. Calculate the target packed column volume (CV) in milliliters (mL):

CV = ∏ x r2 x h

where:

r = column radius (cm)

h = target column bed height (cm)

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孤岛余?——任务攻略 游戏介绍： 在公元2009年，你作为?名医?在?个未知的岛屿上进?科研活动。这?将发?什么事情呢?这和那个奇怪的梦又有什么联系?未来和过去的谜团，等待你来揭开!最新?机游戏|?机游戏下载|?机?户交流 操作?法： 游戏中对应按键分别为： 上下左右分别为2，4，6，8。同时你可以使?PA D键辅助移动，功能和数字键盘?样。5键或者中间键为确定键，?于与?物对话或者进?活动。如攻击或者拾取物品。 左软键:打开?囊。 右软件:打开游戏菜单 当你进??个新的区域，游戏将?动保存。或者你也可以回到休息室，睡觉保存。 游戏界?介绍： 在N P C的头顶出现??感叹号时，代表TA有任务给给你。 绿?感叹号，代表你已经完成TA的任务， 红?感叹号，代表你还未完成TA的任务。 常?物品介绍以及获得办法： 急救包(?，?)，?于恢复?命，可有散落在各地的箱?得到，也可以购买 铁铲：码头的商?或者园丁处购买，?于挖掘?蚁巢?，得到鱼饵来复枪：教授给与，?于配合飞镖使?。对付恐龙的利器。 斧头：教授给与，?于看到树?得到时光穿梭机的燃料 锋利的斧头：后期可在码头商?处购买，?于劈开某些难缠的树?。。。。。 主线任务： 第?章 1.从梦中醒来，听到外?嘈杂的吵闹声。我迷迷糊糊?出休息间。想去看看外?发?了什么事。 下楼梯看见我亲爱的格蕾丝，过去得到消息说吵闹声来?搬运?批货物。我很好奇，格蕾丝建议我去找索恩将军了解更多。我往北去。前往索恩将军的办公室。

2.来到索恩的办公室。索恩要我去【西边的码头】接收货物，经过?番争吵。我还是不得不接受这个命令。前往南边的码头接收货物。2 3.来到西边码头，和研究?员对话，得知货物和教授都已经在【实验室】了，前往实验室. 4.从实验室出来，拿着教授给的【安全卡】，来到仓库查看。 5.我被仓库中的卫兵赶了出来，他们说这?是禁区?回到教授的研究室，教授告诉我那个神秘的事物其实是他的交通?具-时光穿梭机。并告诉我他因为穿越时空，造成了?体D N A的损坏。我提出帮忙恢复他的健康。然后回到休息区休息。 第?章完成。 第?章 1我在床上听到爆炸声，赶快出来查找原因。好像是从仓库传来，于是前往仓库。 2.在仓库看到倒地的警卫和被破坏的时光机，我得赶快去通知教授。前往研究室。 3得到消息的教授很惊讶，急忙和我来到仓库查看。教授说道时光机还可以修复，我想起我之前的研究可能对教授有所帮助，于是前往实验室去收集【D N A样本】 4.实验室【D N A样本】(也就是红?的?瓶?)分别是左边两个(内部房间?个)，右边?个。收集完毕后将实验后的【时空旅?配?】交给教授。 5.来到仓库，教授提议我??试验【时光旅?配?】，并告诉我，我暂时只能去?个时代。同时给我【?醉来福枪】【斧头】和【**x0】 6去园丁那使?【斧?】砍倒树?，得到时光机的燃料【?头】，(每次时光穿梭消耗?头1，请多备些?头)回到仓库，乘坐时光机，探索恐龙时代。 7，穿越时空，来到恐龙时代，时光机却出现故障，我不得不收集散落的【时光机零件】，以便?后修复它。(收集完毕后，此时可收集?些恐龙的遗物如【??】，后?实验室要使?。并尽可能的带回?个【琥珀】(看树有?定?率得到)。但是不建议?量收集植物种?和恐龙蛋，因为收集带回去的都是损坏的。等到后?就可以收集了) T I P S：各种恐龙出现的时间是不同的，可以在以后实验室中?查看。如果此时你带有【铁锹】，可以去偷窃?草龙的蛋。注意：偷窃是会被?草龙发现并追杀的。只要是你偷窃过?种恐龙的蛋，以后它都会主动攻击你 第?章结束 第三章 1与教授对话，得知【化学同位素】可以修复时光机，建议我去找东码头的J商了解情况。2.找到东码头的J商，在这?可以买卖弹药，急救包和?头。?300元买了【同位素】，回去交给教授。教授完成时光机的修复，此时可以前往未来探索。3.来到未来2011年，发现惊天秘密。?岛的?都

《孤岛余生》攻略-绝境求生手册

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